Review Article Vacuolar H -ATPase: An Essential...

Review ArticleVacuolar H+-ATPase An Essential Multitasking Enzyme inPhysiology and Pathophysiology

L Shannon Holliday12

1 Department of Orthodontics University of Florida College of Dentistry 1600 SW Archer Road Dental Tower D7-18 CB 100444Gainesville FL 32610 USA

2Department of Anatomy amp Cell Biology University of Florida College of Medicine Gainesville FL 32610 USA

Correspondence should be addressed to L Shannon Holliday shollidaydentalufledu

Received 8 September 2013 Accepted 7 November 2013 Published 23 January 2014

Academic Editor Dzung H Dinh

Copyright copy 2014 L Shannon HollidayThis is an open access article distributed under the Creative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Vacuolar H+-ATPases (V-ATPases) are large multisubunit proton pumps that are required for housekeeping acidification ofmembrane-bound compartments in eukaryotic cells Mammalian V-ATPases are composed of 13 different subunits Theirhousekeeping functions include acidifying endosomes lysosomes phagosomes compartments for uncoupling receptors andligands autophagosomes and elements of the Golgi apparatus Specialized cells including osteoclasts intercalated cells in thekidney and pancreatic beta cells contain both the housekeeping V-ATPases and an additional subset of V-ATPases which playsa cell type specific role The specialized V-ATPases are typically marked by the inclusion of cell type specific isoforms of one ormore of the subunits Three human diseases caused by mutations of isoforms of subunits have been identified Cancer cells utilizeV-ATPases in unusual ways characterization of V-ATPases may lead to new therapeutic modalities for the treatment of cancer Twoaccessory proteins to the V-ATPase have been identified that regulate the proton pump One is the (pro)renin receptor and datais emerging that indicates that V-ATPase may be intimately linked to reninangiotensin signaling both systemically and locally Insummary V-ATPases play vital housekeeping roles in eukaryotic cells Specialized versions of the pump are required by specificorgan systems and are involved in diseases

1 The Importance of lsquolsquoHousekeepingrsquorsquoAcidification in Eukaryotic Cells

Eukaryotic cells use the localized concentration of protonsin vesicles powered by ATP hydrolysis by the vacuolarH+-ATPase (V-ATPase) for essential purposes [1] Acidicproteases in the lysosome are converted by changes in pH toactive forms that can degrade other proteins as in the caseof acid cysteine proteinases of the cathepsin family [2] Thelow pH also triggers changes in the conformation of proteinsthat make them more susceptible to proteolytic degradationIn a more selective sense many proteins are processed byproteolytic enzymes from a pro form and this is often linkedto cycling of the proteins through acidic compartmentson their way to their final destination (Figure 1) [3] Incompartments for uncoupling receptors and ligands (CURLcompartments) receptors make use of V-ATPase-dependent

acidification to release their ligand in order to allow recyclingof the receptor to the plasma membrane and reuse [1]

Equally important V-ATPases energize membranes andthis can be used to power the movement of molecules acrossmembranes by coupled transporters [4] For example theuptake of glutamate a neurotransmitter into presynapticvesicles is coupled to proton secretion by V-ATPases

Accumulating evidence shows that the pH of the lumenof vesicles can be sensed by transmembrane proteins orcomplexes and that information can lead to changes in thedestiny of the vesicle The V-ATPase has been reported tohave such acid sensing ability [5ndash7] As will be describedin greater detail below the V-ATPase senses the pH of thelumen of vesicles into which it is embedded and based on thatinformation recruits factors that regulate vesicular traffickingand cytoskeletal reorganizations In a recently identified vari-ation of this theme it was reported that sensing of aminoacids

Hindawi Publishing CorporationNew Journal of ScienceVolume 2014 Article ID 675430 21 pageshttpdxdoiorg1011552014675430

2 New Journal of Science

ATPADP

VGLUT

Glutamate release

Clathrin-mediatedendocytosis

Secretoryvesicles

Endocytosis

Coated vesicle

Sorting endosome

Recycling vesicle

Recyclingendosome

MultivesicularbodyLysosome

Late endosome

rec return to GolgiMannose-6-phospahate

Mannose-6-phospahate recwith bound cathepsinto late endosome

ATPADP

ATP

ADP

ATPADP

ATPADP

ATPADP

Role of Vsecretory

-ATPase in vesicle fusion

GlutamateH+

H+

Figure 1 Roles of V-ATPases in cell physiology V-ATPases have vital roles in theGolgi apparatusMutations in the a2-subunit which is foundin the Golgi result in a form of cutis laxa due to impaired glycosylation In secretory pathways energy from a proton gradient generated byV-ATPases may be used to load molecules like glutamate V-ATPases in endosomal compartments are involved in disassociating complexeslike the mannose-6 phosphate receptor from cathepsins Acid-dependent enzymes may also become active due to V-ATPase acidification tomodify proteins in the lumen of the vesiclesThere is also increasing evidence that the V-ATPase may be able to recruit regulators of vesiculartrafficking like ARF6 V-ATPases in the lysosome provide an acidic environment for the degradation of proteins by acidic proteases

by mTORC1 requires ATPase activity by the V-ATPase [8ndash11] This central nutrient sensing apparatus is a vital nodein physiological signaling but also represents a potentialldquoAchilles heelrdquo of cancer cells which may be made vulnerableas a more sophisticated understanding of the role of theV-ATPase in the process emerges [9]

Given the importance of acidification it is not surprisingthat most eukaryotic cells cannot survive if V-ATPase activityis lost either due to the administration of a selective inhibitoror molecular genetic loss of a ubiquitous subunit [12ndash14]An important exception is the yeast Saccharomyces cerevisiaewhich is able to take up protons from acidic media tocompensate for lack of V-ATPase activity V-ATPase-deficientyeast thrive in media at pH 5 but fail to grow in alkalinemedia This allows knockouts and replacement knockouts tobe performed to analyze functions of specific subdomains ofV-ATPase subunits [15] Yeast has become one of the mostpowerful model systems for studying the V-ATPase [16] Thefact that the V-ATPase is highly conserved also makes theyeast V-ATPase a semiuniversal model for understanding thebasic mechanisms of the pump

2 Structure and EnzymaticFunction of V-ATPases

The mammalian V-ATPase is composed of 13 subunits thatcan be divided into 8 peripheral proteins which is also called

the V1 and 5 membrane intrinsic proteins are called V0[17 18] (Figure 2) The V-ATPase is referred to formally asATP6 [19] V1 subunits are ATP6V1A (A-subunit) ATP6V1B(B-subunit) ATP6V1C (C-subunit) ATP6V1D (D-subunit)ATP6V1E (E-subunit) ATP6V1F (F-subunit) ATP6V1G (G-subunit) and ATP6V1H (H-subunit) The V0 domains areATP6V0a (a-subunit) ATP6V0b (crdquo-subunit) ATP6V0c (c-subunit) ATP6V0d (d-subunit) and ATP6V0e (e-subunit)As indicated in Figure 2 many of the subunits have isoformsIn higher organisms there are ubiquitous isoforms andisoforms that are selectively expressed in specific cell typesThese are associated with subsets of V-ATPases that per-form specialized functions However specialized V-ATPasesrepresent a mixture of cell type selective isoforms andubiquitous isoforms Indeed some specialized functions maybe carried out by V-ATPases that are indistinguishable fromthe ubiquitous enzyme

As an example in mammals there are 4 isoforms of the a-subunit ATP6V0a1 (a1) and ATP6V0a2 (a2) are ubiquitouslyexpressed and are associated with subsets of housekeepingV-ATPases ATP6V0a3 (also referred to as TCIRG1 Atp6iTIRC7 and a3) is found at high levels in osteoclasts [20]microglia [21] gastric parietal cells [22] pancreatic beta cells[23] and perhaps a few other cell types and associates with aspecialized subset of V-ATPases that perform nonhousekeep-ing functions Osteoclasts for instance express a1 a2 anda3 [24] The first two are thought to perform housekeeping

New Journal of Science 3

d

c

ddd

c

A

D

c

B

F

EG

a

e

ATP

ADP-Pi

ADP

Cytoplasm

H

Lumenextracellular

dd

Vacuolar H+-ATPase

V1

V0c998400998400

H+

H+

C

Isoforms

B1-kidney epididymis

B2-ubiquitous osteoclasts

C1-ubiquitous

C2a-lung

C2b-kidney

E1-acrosome

E2-ubiquitous

G1-ubiquitous

G2-synaptic vesicles

G3-kidney

a1-endomembranes

a2-endomembranes

a3-osteoclasts pancreatic beta cells

a4-kidney epididymis

d1-ubiquitous

d2-kideny osteoclasts dendritic cells

Figure 2 Structure and subunit composition of V-ATPases Themodel represents recent proposals regarding the subunit compo-sition stoichiometry and organization of V-ATPases A list ofsubunits with isoforms is provided ATP hydrolysis occurs on the A-subunit and it is thought that the three subunits have at any specifictime ATP ADP-Pi or ADP bound The conformational changes inthe AB heterohexagon promote turning of the central rotor and thering of c and c10158401015840 (b) subunits The pathway of protons through themembranes includes both the rotating cb-ring and the a-subunit

functionsThe last is a component of V-ATPases targeted to aplasma membrane domain and is crucial to osteoclasts boneresorptive function Knockout of a3 in transgenic mice ormutations that reduce or eliminate its expression or abilityto form functional pumps in humans leads to autosomalmalignant osteopetrosis [20 25ndash27] Thus even thougha3-knockout osteoclasts contain functional V-ATPases thehousekeeping V-ATPases are unable to compensate for theloss of a3This appears to be a general themeThe a4-subunitis restricted to kidney intercalated cells proximal tubules anda few other types of epithelium [28 29]There is evidence thata4may be upregulated andmay be able to compensate for lackof a3 in some situations [30] As with a3 V-ATPases with a1or a2 do not compensate for the lack of a4 [31]

TheV-ATPase can be divided into functional subdomains[1 18 32] (Figure 2)TheATP catalytic domain is composed ofa heterohexamer of alternatingA- and B-subunits Hydrolysis

of ATP on the A-subunit drives a conformational changein the heterohexamer which powers rotation of a centralrotor The rotor contains an evolutionarily conserved D-subunit which serves as the main axle of the rotor the F-subunit which is associated with the axle and may serve aregulatory function and d-subunit which couples the axleto the ion pump components of the rotor The ion pumpcomponent is composed of a rotating ring of b- and c-subunits which along with the a-subunit create the channelthrough which protons move across the membrane as thebc-subunit ring rotates A collar domain containing C-subunit and H-subunit is thought to wrap around the statorand link to the transmembrane a-subunit Three stator armscomposed of dimers of E- and G-subunits emanate fromthree nonequivalent attachment sites [33] Two attach to theaminoterminal domain of the a-subunit This large domain(500 aminoacids or so) varies amongst a-subunit isoformsand almost certainly contains information that is involved indifferently regulating isoforms of a-subunit The three statorsare known to interact with H- C- and a-subunits althoughthe exact binding sites have not yet been determined [34ndash38]The stators terminate at and interact with the aminoterminaldomain of the B-subunit and the a-subunit interacts directlywith the B-subunit [39ndash41]

V-ATPases contain two coupled rotary motors onedriven by ATP hydrolysis the other by a proton gradient[42] Typically V-ATPases pump protons powered by ATPhydrolysis with the ion motor resisting the pumping activitybased on the size of the electrochemical gradient whichconsists of both the proton gradient and the membranepotential against which the V-ATPase is working The pH ofthe lumen of vesicles is thought to be ldquofine tunedrdquo throughthe actions of voltage-gated chloride channels which have theability to dissipate membrane potential by allowing entranceof chloride ions negatively charged counter ions to protons[43ndash45]

3 Isoforms and Splice Variants ofV-ATPase Subunits

Numerous V-ATPase subunits are present as isoforms Inaddition splice variants have been identified for a numberof subunits although little is known about the functionalsignificance of these variations Here is a brief summation ofthe current knowledge of the subunits and their isoforms

31 A-Subunit (70 kDa) The A-subunit contains a Walkerconsensus sequence which binds ATP ATP hydrolysis occursat the interface of the A- and B-subunits [46 47] Most ofthe catalytic site residues are found in the A-subunit [48] Inmammals only one isoform of A-subunit is present A splicevariant of A-subunit was reported in chickens that lackedthe Walker consensus ATP binding sequence and whenexpressed it prevented assembly of theV-ATPase [49 50]Thephysiologic significance of this splice variant is not knownand no mammalian version has yet been identified

A number of studies suggest that phosphorylation ofthe A-subunit by cyclic-AMP-dependent kinase (PKA) may


play a vital role in regulating V-ATPase [51ndash54] The keyphosphorylation site was identified as serine-175 Whiledata show that A-subunit is phosphorylated and that PKAphosphorylation regulates V-ATPase activity the underlyingmolecular mechanism remains unresolved

32 B-Subunit (B2 56 kDaB1 58 kDa) Two isoforms of B-subunit are expressed in mammals [55] B2 is expressedubiquitously and is an element of the ldquohousekeepingrdquo V-ATPase subsets The B2 mRNA was originally isolated fromhuman brain and it was initially called the brain isoform[56] It is also present in V-ATPases performing specializedfunctions including those osteoclasts and in presynapticneurons [57] The mRNA for the B1 isoform was first isolatedand sequenced from human kidney [58] It is expressed intype A intercalated cells in the kidney as well as cochleaand the endolymphatic sac of the inner ear and in epithelialplasma membranes of the epididymis [59 60]

The B1-isoform has a PDZ-binding domain present at theextreme C-terminus that is missing in B2 [61] Both B1 andB2 have a high affinity (100ndash200 nM) microfilament bindingdomain in the aminoterminus [62] Binding to microfila-ments can be achieved by 44 aminoacids 23ndash67 in B1 and29ndash73 in B2 [63] Within the overall microfilament bindingdomain a subdomain called the ldquoprofilin-likerdquo domain ispresent that has sequence similarity to a portion of theactin binding domain of mammalian profilin 1 [63] Thirteenaminoacid peptides derived from B1 or B2 that included theprofilin-like domain boundmonomeric actinwith an affinityof 20120583Mandmicrofilaments with an affinity of about 200120583M[63] Other features in the overall binding domain conferredspecificity for microfilaments over monomeric actin and thehigh affinity A key residue in the overall sequence wasphenylalanine 59 in B1 or 65 in B2 [63 64] Change ofthat residue to an alanine or glycine reduced the affinity ofthe binding interaction by an order of magnitude Althoughthe sequence of the actin binding domain of B1 and B2varies substantially both bound microfilaments with similaraffinities [62] This implies that specialized epithelial cellsexpressing B1 may make use of microfilament binding Thecurrent understanding of the function of actin binding willbe described in a subsequent section

Yeast only express one isoform of B which displaysmicrofilament-binding activity [64] However the actinbinding domain starts at aminoacid 10 rather than 23 or29 The functions of the aminoterminal additions to the B-subunit in B1 and B2 are not known Figure 3 shows anoverall comparison of B2 B1 yeast B and a B-subunit froman Archaean

33 C-Subunit (C1 42 kDa C2 48 kDa) Two isoforms of C-subunit are present in mammals [65] C1 is ubiquitous andC2 is found in the kidney and lungThe C-subunit is thoughtto wrap the rotor structure and supply an attachment site forthe EG stator arm [35ndash37] The C-subunit is elongated anddumbbell shaped [66] It is present in the V1-V0 interface andis thought to contact two of the three EG stator arms [33]An EG contact site in the head domain (aminoacids 158ndash277)

Sequence of ldquoprofilin-likerdquo domain

B2

B1

Yeast B

Pyrococcus B

Actin binding domain

G P L V I L D H V K F

G P L V V L D R V K F

B2

B1

Yeast B G P L V I L E K V K F

Figure 3 Location of the actin-binding site on mammalian B2 andB1-subunits and in yeast B compared with an Archaean B-subunitthat does not bind microfilaments Identical colors indicate highconservation The position of the minimal actin binding domainis indicated by the blue transparent square As depicted the C-terminus of the actin binding domain is highly conserved betweenB2 B1 and yeast but the N-terminal portion is less well conservedPart of the sequence that makes up the actin binding domain ismissing in the Archaean and the rest is only modestly conservedThe sequence of the ldquoprofilin-like domainrdquo which is in the redconserved domain is provided belowMutation of the phenylalanineto a glycine is sufficient to reduce the binding affinity for actin about10-fold

holds a high affinity binding site for an EG stator [36 37]Interaction with that binding site stabilizes the EG dimerThe second site is thought to be of lower affinity and maycoordinate with one of the a-subunit binding sites

One mode of regulation of the V-ATPase is the reversibledisassembly of the V1 from the V0 leaving a membranebound V0 and cytosolic V1 [67 68] The C-subunit hasbeen suggested to be involved in this reversible disassemblyIt was proposed that an unknown environmental stimulileads to a conformational change in the C-subunit (twodifferent conformations of C-subunit have been observedin different crystals) This change breaks the low affinityEG stator interaction with C-subunit This is followed byrotation driven by ATP hydrolysis that breaks the highaffinity interaction It was noted that the bond energy ofthe high affinity C-subunit interaction with EG is roughlyequivalent to the amount of energy released by the hydrolysisof 1 ATP molecule This idea may be oversimplified inthat there is certainly more bonds than those between EGand C-subunit that must be broken in order to releaseV1

Interestingly like the B-subunit the C-subunit has beenshown to bind actin [69 70] Unlike B-subunit C-subunitbinds both monomeric and filamentous actin and C-subunitholds two actin binding domains one in either end of thebarbell Because of this evidence was presented showing thatC-subunit could crosslink microfilaments into higher orderstructures [70]


ATP6V1C1

(a)

ATP6V1C2

(b)

Figure 4 Models of C1 and C2a produced by Swiss-Model areshown Note the insertion in C2a that is derived from the differentialsplicing (arrow) C2b like C1 lacks that interruption of the helixfound in the splice variant

It is not clear that the C-subunit while wrapped aroundthe rotor near the membrane surface can interact with therelatively massive microfilament It has been proposed thatinteractions with actin occur after V-ATPase disassembly[71] C-Subunit interaction with microfilaments could main-tain the location of C-subunit to the area of the disassembledV0 or could be involved in organizing the microfilamentnetwork in the area in which the V-ATPase has disassembledFurther studies that precisely identify the actin binding sitesand mutate the sites so that actin binding activity is lost willbe required to answer these questions

The C2-subunit has two splice variants one containinga 48 aminoacid insert not present in C1 [72] Modeling C1and C2 using Swiss-Model suggests that the insert breaks along helical domain and certainly is positioned to have animportant regulatory role (Figure 4) The C2 splice variantwith the insert (C2a) is located in the lamellar bodies of thelung whilst the other splice variant (C2b) is found in theplasma membrane of intercalated cells in the kidney Onepossibility is that the insertmay extend toward themembraneraising the possibility it may interact with membrane lipidsor proteins that are tightly associated with the membraneThere are relatively low levels of sequence homology betweenyeast C-subunit and the human C-subunits (30 identity)Caution must be exercised in interpreting results fromstudies of yeast or Manduca to humans Given the poten-tial regulatory role suggested for C-subunit examinationsof differences in isoforms and splice variants may provecrucial to a general understanding of how the V-ATPase isregulated

34 D-Subunit (34 kDa) Only one isoform of D-subunitexists in mammals and the D-subunit has been highlyconserved (99 identity betweenmouse and human and 52identity between human and yeast) Like the A- and B-subunits the pressure tomaintain the structure for its key roleas the rotor likely explains the conservation In addition itsposition and function would suggest that it is unlikely to bedirectly interacting with elements outside of the pump

35 E-Subunit (31 kDa) There are two isoforms of E-subunitexpressed inmammals E1 is ubiquitous andE2 is found in thetestis The two isoforms are the same length and share over80 identity

In recent years it has become well established that Esubunit forms a dimer with G-subunit and the two intertwineto make a long extended helical structure that projects fromthe collar of the V-ATPase to the top of the AB heterohexagon[36] Although for many years it was accepted that eachV-ATPase only contained 1 E-subunit and 1 G-subunit it isnow thought that fully assembled and functional V-ATPaseshave 3 of each [73]

TheEG-dimers represent possible regulatory targets totaldisassembly of the V-ATPases into V1 and V0 sections wouldseem to require disruption of the connections mediated byall three stator arms This could be accomplished either byregulation at the sites from which the stators emanate fromthe collar for example at subunit C as described above Thiswould seem to require three different regulatory signals todisrupt the three at the collar Alternatively regulation of EGdimerization overall conformation or at the interaction siteat the top of the B-subunit could provide the opportunity toregulate all three stators at once

The E-subunit has been identified as the binding siteof the glycolytic enzyme aldolase both in mammals and inyeast and a variety of evidence suggests that this interactionis a crucial element of a more extensive set of interactionsbetween V-ATPase subunits and glycolytic enzymes [7 74ndash82] These have been proposed to represent a metabolon inwhich physical interactions between V-ATPase subunits andglycolytic enzymes create functional relationships allowingrapid access for the V-ATPase to ATP and protons bothbyproducts of glycolysis [75 80]

36 F-Subunit (14 kDa) Only one isoform of the F-subunitexists in mammals The F-subunit is required for assembly ofthe V-ATPase [83ndash86] It is not required for rotation of therotor but is involved in stimulating ATPase activity perhapssimply by being required for assembly of the pump

37 G-Subunit (13 kDa) Three isoforms of G-subunit arepresent in mammals G1 is ubiquitous G2 is found in thebrain and G3 is expressed in the kidney [65] As describedpreviously the G-subunit forms a heterodimer with the E-subunit and the dimers form the three statorsThe importantrole of these stators the fact that they are exposed onthe outside of the V-ATPase and the fact that disassemblyof the stators from the pump must occur for disassemblyof the V1 from the V0 and the reverse must occur for


assembly make G-subunit an attractive potential regulatorysubunit

38 H-Subunit (5057 kDa) TheH-subunit is known to acti-vate ATP-powered proton pumping in intact V-ATPases andblockATPase activity in freeV1 sectors after disassembly [87]The H-subunit (then called SFD) was originally identified asa component of the bovine V-ATPase that had the capacityto activate isolated pumps in an in vitro system [88] Workin yeast then confirmed the original observation and alsoshowed the ability of the H-subunit to inhibit the activityof disassembled V1 This prevents hydrolysis of ATP whenproton pumping is not possible [87]

H-subunit is located like C-subunit at the base of the V1it interacts with the E-subunit of at least one stator armThuslike the C-subunit the H-subunit is positioned to be involvedin regulatory functionsThe H-subunit has armadillo-likerepeat domains that bind themedium chain (mu2) of adapterprotein complex-2 [89] This was shown to interact with theNef protein from HIV providing a mechanistic basis forearlier studies that documented an association between V-ATPase and Nef [90 91] It was proposed that this interactionmight play a vital role in HIV infections However afteran initial series of articles nothing further has emergedregarding this intriguing interaction

39 a-Subunit (115 kDa) The a-subunit is of critical impor-tance to V-ATPases As described previously mammals havefour isoforms [31 92] Indeed multicellular organisms assimple as C elegans have four isoforms [93] and yeast hastwo [94] The a1-subunit is ubiquitously expressed and likelyplays the role of a-subunit for many of the housekeeping V-ATPases In addition to its role in housekeeping V-ATPasesevidence has emerged that it may be involved in the fusion ofsynaptic vesicles in presynaptic neurons [95] It is expressedat high levels in the brain which may reflect a specialized rolein neural tissues beyond its normal housekeeping functions

The a2-subunit was recently shown to be involved in thepathology of a form of autosomal recessive cutis laxa [96ndash98]This a2 mutation results in severe skin wrinkling it inducesgeneralized connective tissue weakness leading to herniasand hip dislocations and triggers osteopenia or osteoporosiswith increased fracture risk cardiovascular and pulmonarydysfunction and in some cases mental retardation thatmay be associated with brain malformations [96 99ndash101]This syndrome is associated with alterations in patterns ofglycosylation and defects in vesicular trafficking in the Golgiapparatus [97 98 102] These results are consistent withprevious studies that indicated that a2 is localized to theGolgi[24 103]

Subunit a3 was first identified as required for osteoclastfunction in studies of a transgenic knockout mouse [20] Aspontaneousmutation that triggered osteopetrosis in an oftenstudied mouse model proved also to be a mutation in a3 [25]Soon thereafter it was shown that about half of the humanpatients that suffer from autosomal malignant osteopetrosishave mutations in the a3-subunit [27 104] The a3-subunitis expressed at high levels in osteoclasts in pancreatic beta

Actin V-ATPaseConfocal images of resorbing osteoclast

(a)

Side view of resorbing osteoclast

Resorption compartment Ruffled membrane

Actin ring

(b)

Figure 5 Location of the a3-containing V-ATPases in resorbingosteoclasts Osteoclasts require expression of functional a3-subuniteven though they also express a1 and a2 The top panels showconfocal images of a resorbing osteoclast on a bone slice stainedwith phalloidin-to detectmicrofilaments andwith an anti-E-subunitantibody to detect V-ATPases Notice that the microfilaments areconcentrated in a structure called the actin ring The actin ringsurrounds the V-ATPases in the ruffled plasma membrane wheremost V-ATPases are concentrated Below is a schematic showinga side view of a resorbing osteoclasts with actin filaments greenand V-ATPases red The resorption compartment is the site of bonedegradation

cells in microglia in the brain in gastric parietal cells anda few other cell types [21ndash23 105ndash107] It was found in avariety of tissues in early screens but it is not clear whichcell types [106] it was derived from There is evidence that itmight be linked to protection from pathogen infection [108]Recently evidence was presented that a3 is expressed in thestomach and that its lack of expression in the stomach mightbe linked to osteomalacia [22 109] It has also been shownto be expressed in cancer cells [110] and to play an importantrole in cancer growth and metastasis [111]

The a3-subunit has been studied most extensively inosteoclasts (Figure 5) It is required for the transport ofV-ATPases to the ruffled plasma membrane of osteoclasts[20] Until recently little was known regarding the mecha-nism by which a3-conferred its ability to target V-ATPasesto the plasma membrane A recent study showed that inosteoclasts the V-ATPases containing the a3-subunit boundmicrofilaments whereas the housekeeping V-ATPases in thesame osteoclasts did not [112] Evidence has been presentedsuggesting that the interaction between the B-subunit andmicrofilaments is also necessary for plasma membrane tar-geting [113] It is possible that exposure of the actin binding


sites in the B-subunit to allow microfilament binding may beregulated through the a3-subunit by displacing one or moreEG-stators that normally block access [114]

The gene for the a4-subunit was identified as the cause fora form of distal renal tubular acidosis [115] It is found at highlevels in intercalated cells and proximal tubules of kidneys[115] Like the a3-subunit the a4-subunit is present in V-ATPases that are targeted to the plasma membrane Becausethe B1-subunit the specialized isoform of B found in thesecells bindsmicrofilaments (at least in vitro) it is possible thata4 also modulates microfilament bindingThemechanism bywhich V-ATPase binding to microfilaments may modulatetrafficking of V-ATPases and other proteins will be discussedbelow

310 b-Subunit (21 kDa) Mammals contain two varieties ofthe proteolipid subunits (b and c) that make up the spinningtransmembrane ring that is directly involved in protontransport [116] The b-subunit is the less abundant of the twoand probably only 1 is present in each V-ATPase [117 118] Ofthe two the b-subunit is larger (21 kD compared with 16 kD)and has 5 membrane spanning domains compared with 4 inthe c-subunit In yeast there are 3 proteolipid subunits andall three are required [1]

311 c-Subunit (16 kDa) Multiple c-subunits together witha b-subunit compose a rotating membrane-embedded ringThis ring along with the a-subunit forms half channels thatallow protons to move across the membrane in conjunctionwith the rotation of the ring [1] In addition to its vital rolein the machinery of the pump the c-subunit has been shownto interact with Arf6 a small GTPase that directs membranetrafficking and cytoskeletal dynamics [6]

312 d-Subunit (38 kDa) There are two isoforms of the d-subunit The d1-subunit is ubiquitously expressed while theexpression of d2 is restricted In mice mutation of d2 leadsto a mild form of osteopetrosis [119] To date no humanpathology resulting from d2 mutation has been identifiedMutation of d2 in mice reduces the fusion of osteoclastprecursors to form the characteristic giant cells This isassociated with reductions in resorption capacity It has beenproposed that d2may function independently from its role inV-ATPases as a fusogenic factor [119] Alternatively d2 mayplay a role in osteoclasts that allows the sorting of fusogenicfactors to the plasma membrane [114]

Knockout of d2 in mice led to both decreased osteoclastbone resorption and increased rates of bone formation[119] This suggested that agents might be identified thatwere directed against d2 and were bone anabolic Initialenthusiasm has been somewhat tempered by the finding thatthese mice lose bone at the same rate as wild type rats in amodel of post menopausal osteoporosis [120]

313 e-Subunit (9 kDa) The e-subunit is probably associatedwith the a-subunit and is highly hydrophobic Its position hasnot yet been definitively reported It is essential in yeast [121]and is present inManduca [122] and mammals [123]

4 V-ATPase Accessory Proteins

Two accessory proteins to the V-ATPase have been identifiedATP6AP1 also known as Ac45 and ATP6AP2 also knownas the (pro)renin receptor The precise relationship betweenthese accessory proteins andV-ATPase is at best tenuous andnumerous questions remain unanswered Are these proteinsassociated only with certain specialized V-ATPases How dothey interact with V-ATPases What subunits if any bindthe accessory proteins How do they affect and regulate V-ATPase activity

41 ATP6AP1 ATP6AP1 (known initially as Ac45) was firstfound in V-ATPase preparations from bovine chromaffingranules [124 125] It can be separated with the V0 complexIt contains an internalization sequence in a 26 aminoacidsequence in the C-terminus [126] Knockout of ATP6AP1in transgenic mice is lethal in embryos [127] ATP6AP1 isupregulated during osteoclastogenesis [128] When knockeddown by RNA interference (RNAi) in osteoclast-precursorsless fusion was detected the osteoclast cytoskeleton wasaltered and less bone resorption occurred in in vitro assays[128 129] Overexpression of a form of ATP6AP1 that lackedthe internalization domain also impaired bone resorption[130]

In osteoclasts ATP6AP1 interacts with the small GTPaseRab7 [128] which regulates vesicular trafficking However itremains to be demonstrated whether the effects of ATP6AP1require its association with V-ATPase and its hypothesizedability to regulate V-ATPase activity

42 ATP6AP2 It was a surprise that the V-ATPase accessoryprotein ATP6AP2 originally referred to as M89 proved tobe the long sought (pro)renin receptor [131] This protein hasthe capacity to stimulate angiotensin signaling by activating(pro)renin and to directly stimulate ERK12 MAPK andPI 3-kinase signaling pathways [132ndash135] ATP6AP2 was alsoshown to serve as a scaffolding protein linking V-ATPase tothe WNT-signaling pathway [136]

ATP6AP2 was originally detected as a 10 kD peptide inV-ATPase isolates prior to the identification of the (pro)reninreceptor [123] Full length ATP6AP2 is 38 kD and the 10 kDportion proved to be the membrane spanning domain leftafter the (pro)renin receptor portion was cleaved ATP6AP2is cleaved by furin perhaps in a regulated manner in somecell types to produce 28KD and 10 kD (M89) fragments[137] Cleavage of furin may be a cell type specific processRecent genetic data supports ATP6AP2 as a protein inti-mately linked to V-ATPase [138 139]

The renin-angiotensin system (RAS) is a systemic hor-mone system that regulates blood pressure and fluid balance[140] Hypertension is commonly treated using drugs thatblock the RAS [141] Emerging data suggest that local RASregulation occurs in various tissues including bone [142ndash145]In rodents stimulation of the RAS leads to decreases inbone mass and inhibition of RAS increases bone mass[143 146ndash148] It is possible that both systemic and localRAS regulationmay affect bone remodeling and bone quality


Bone cells express elements of the RAS signaling makinglocal RAS regulation of bone remodeling plausible [147] RASregulation of bone remodeling may play a role in integratingbone remodeling with systemic calcium and phosphate regu-lation

Binding of either renin or (pro)renin to ATP6AP2 stim-ulates the enzymatic activity of renin (4-fold) or (pro)renin[131] ATP6AP2 forms a necessary renin-independent linkbetween LRP56 and Frizzled (Fz) [136] and acidification ofendosomes by V-ATPase is required for LRP56 phosphory-lation and activation of the WNT120573-catenin pathway

Full length ATP6AP2 might serve both as the (pro)reninreceptor and as a V-ATPase-associated scaffolding proteinbut this has not yet been demonstrated [135] Furin-cleavageof ATP6AP2 yields a soluble 28KD and an integral 10 kD(M89) fragment [137] It was reported that full lengthATP6AP2 represses WNT signaling perhaps by blockingproton pumping activity Cleavage of ATP6AP2 by furinreleased this inhibition [149]

5 Coordinate Expression andAssembly of V-ATPases

V-ATPases are large multisubunit complexes For them tofunction the constituent proteins must be expressed in cor-rect quantities and the subunits must be assembled into theactive enzymesThis is particularly challenging in that the V-ATPases contains both peripheral and integral componentsIn addition there is coordinate regulation required thatincludes both ubiquitous and cell-specific isoforms Evidencehas accumulated to suggest that both transcriptional and posttranscriptionalmechanisms are in play to produce the correctamount of V-ATPase proteins in a specific cell at a particulartime

51 Transcriptional Regulation Transcriptional regulation ofthe ubiquitousV-ATPase isoforms involvesTATA-lessG+C-rich regulatory regions containing multiple Sp1 andor AP-2-like binding sites [150] This type of promoter is found inother housekeeping enzymes and contains promoters withCpG islands areas rich in C next to G [151 152] The pin CpG refers to the phosphodiester bond joining the Cand G residues CpG islands are usually defined as a regionwith at least 200 base pairs that is greater than 50 C + Gand which has an observed to expected CpG ratio ((Numof CpG(Num of C times Num of G)) greater than 60 CpGislands are relatively rare in vertebrate genomes Methylationof cytosines in CpG islands is established as a methodfor regulation of transcriptional activity it seems likelythat expression of housekeeping V-ATPases may be subjectto this form of epigenetic regulation [152ndash156] Moreoverbecause the specialized V-ATPases in cells like osteoclastsrequire both specialized isoforms and over expression ofhousekeeping isoforms methylation of CpG islands may beof particular importance

mRNAs of genes expressing V-ATPase isoforms thatare selectively expressed in the kidney (B1 and a4) areregulated by the forkhead transcription factor Foxi1 [157]

This transcription factor is expressed in relevant tissues in thekidney and is also found in cochlea and the endolymphaticsac of the inner ear and in epithelial plasmamembranes of theepididymis where V-ATPases are required for maintainingspermatozoa in a quiescent state [158]

The expression of a3 is induced by a factor termed thereceptor activator of NF-120581B ligand or RANKL which isessential for osteoclast differentiation [159] RANKL alsoupregulates a3 in microglia [160] The sequence responsiblefor RANKL sensitivity is located about 16 kb upstream ofthe a3 coding region Basal transcriptional activity of a3 issuppressed by the binding of poly (ADP-ribose) polymerase-1 (PARP-1) to this sequence and RANKL treatment causesdegradation of PARP-1 thereby resulting in increased expres-sion of a3 [161 162] PARP-1 is a highly conserved nuclearprotein that modulates chromatin structure in some genescausing transcriptional inhibition [163] A second PARP-1binding site may similarly regulate binding of a JunDFra2heterodimer which is the most abundant form of AP-1in mature osteoclasts It is reasonable to hypothesize thatubiquitously expressed PARP-1 may continually keep theexpression of a3 at low levels in cells that are not respondingto RANKL signaling but this remains to be confirmedexperimentally [161 162]

Mechanisms must be in place to coordinate expressionof ubiquitous isoforms and cell type specific isoforms Aswill be described below posttranscriptional regulation of thestability and translation of mRNAs likely has a role It ishowever intriguing that PARP-1 regulation is known to beintegrated with DNAmethylation [164 165] Indeed PARP-1has been shown to regulate expression and activity of DnmtDNAmethyltransferase activity PARP-1 interacts with newlysynthesized polymers of ADP-ribose which inhibits Dnmtmethyltransferase activity [166 167] Satisfyingly degrada-tion of PARP-1 downstream of RANKL activity could bothallow a3 transcription and block methylation which mighttrigger increased expression of housekeeping subunits

52 Posttranscriptional RegulatoryMechanisms Recent stud-ies point to a key role for V-ATPase 31015840 untranslated regionsin regulating posttranscriptional processing For examplepromoter activity of the c-subunit in RAW 2647 cells andNIH3T3 fibroblasts was similar despite RAW 2647 cellsexpressing 6ndash8-fold more mRNA [168] Likewise mRNAstability of the ubiquitous B2- E1- F- c- and a1-subunitsbetween the same two cell lines showed that all but B2were more stable in RAW 2647 cells These studies suggestthat expression of housekeeping V-ATPases is regulated atboth transcriptional and posttranscriptional levels Molecu-lar genetic analysis of the ubiquitously expressed E1-subunitmRNA showed that an AU-rich element (ARE) near thepolyadenylation site of the 31015840 untranslated region was crucialfor regulation of the mRNArsquos stability AREs associate withRNA binding proteins and regulate the stability of mRNAtranscripts Pull-down assays showed interaction of the E1and c-subunit mRNAs with regulatory proteins called HuRand hnRNP D [169 170] A similar result was obtainedin studies of colorectal carcinoma cells [171] These studies


suggest that regulators of mRNA stability play a vital role inmodulating V-ATPase expression levels

HuR has antiapoptotic functions in mammalian cells Itis usually found in the nucleus but it shifts to the cytoplasmin response to various types of stress In the cytosol HuR iscapable of binding its mRNA targets and thereby stabilizingthe mRNA HuR can be activated by hypoxia [172] UVirradiation [173] heat shock [174] and nutrient and energydepletion [169 170]

Recent studies have identified a second mechanism bywhich the 31015840 untranslated regions are involved in regulatingV-ATPase subunit expression Large numbers of microRNAs(miRNAs) and small (22 nucleotides) noncoding RNAs areexpressed in higher vertebrates [175] These miRNAs bindby base pairing with complementary sequences in mRNAsusually resulting in gene repression via translational suppres-sion or mRNA degradation A genome-wide linkage analysisof twins in the US and Australia uncovered a role for thea1-subunit in influencing hypertension and this was linkedto miRNA regulation A single nucleotide polymorphismidentified in approximately 5 of the population substitutesa cytosine (C) for a thymidine (T) at position 3246 resultingin lowered systolic blood pressure [176] Detailed analysisof the a1 mRNA demonstrated that lowered expression wasfound to be due to the creation of a higher efficiencybinding site for miRNA miR-637 in the C-allele variantThismiRNA then is predicted to reduce a1 translation resultingin alteration of chromagranin A processing and impairmentof its progress into the regulated secretary pathway which hasa prohypertension effect This identifies a functional miRNAbinding site within a V-ATPase subunit with human health

6 V-ATPase Inhibitors

The V-ATPase is important in diseases and disease statesand for that reason considerable efforts have been madeto identify V-ATPase inhibitors and to develop them fortherapeutic purposes However to date V-ATPase inhibitorshave not been employed in the clinic Although specializedV-ATPases (like those in the ruffled plasma membrane ofosteoclasts) differ in the inclusion of specific isoforms (ie thea3-subunit) for most other V-ATPases this does not seem toalter them so that specific or sufficiently selective inhibitors ofthose V-ATPases are easily identified In addition the molec-ularmechanisms underlying the selective activities associatedwith the isoforms (eg altered vesicular trafficking associatedwith a3) are not understood Finally the inhibitors identifiedhave been from natural sources and are produced as toxinsit may be unlikely that a natural product would develop toinhibit osteoclasts It is possible that as the structure andmechanisms of the V-ATPase become better known reversechemical genetic approaches can be utilized to identify novelselective inhibitors of the specialized subset of pumps Cancerin particular has shown selective susceptibility to V-ATPaseinhibitors and is sufficiently grave to warrant the risk ofoff target effects V-ATPases inhibitors have been recentlyreviewed by several groups [177ndash179] A brief summary willbe provided below

61 Plecomacrolides Bafilomycins A1 B1 C1 and D

1were

isolated from Streptomyces [180] These are macrolide antibi-otics with a 16-membered lactone ring Bafilomycin A

1

was identified as the first selective inhibitor of V-ATPases[181] Concanamycin was isolated in the mid-1980s fromStreptomyces [182 183] Studies from various groups haveidentified the c-subunit as being the primary binding sitefor plecomacrolides [184ndash186] There is some evidence alsofor a-subunit contributing to binding of plecomacrolidesCurrently it is thought that these inhibitors function bymechanically perturbing rotation of the bc-ringwith relationto a-subunit

62 Archazolid Archozolid is produced by themyxobacteriaArchangium gephyra and Cystobacter violaceus and blocksgrowth of mammalian cells [187] and inhibits mammalian V-ATPases in the nanomolar range [188] Archazolid competeswith concanamycin for a binding site on the c-subunit Effortsto improve the pharmaceutical properties of archazolidsby structure function analysis and characterization of thebinding site and mechanism of action are underway [189190]

63 Benzolactone Enamides These inhibitors have beenextracted from a variety of sources These molecules haveIC50s in the nanomolar range against mammalian V-ATPases

[188 191] but surprisingly do not affect fungal V-ATPases[191]

64 Pea Inhibitor Recently a new V-ATPase inhibitor wasidentified from peas (pea albumin 1 subunit b) that is activeagainst the V-ATPases from several types of insects that feedon peas but does not interact with mammalian V-ATPasesThis inhibitor is unique for two reasons It is the first peptideinhibitor of V-ATPases It is also selective for certain types ofinsects and does not affect mammalian V-ATPases [192]

7 Rational Approaches to the Identification ofNew V-ATPase-Directed Therapeutic Agents

To date inhibitors of the enzymatic or proton pumping activ-ity of V-ATPases that are selective for specialized V-ATPaseslike those found in the plasma membrane of osteoclasts orintercalated cells of the kidney have not been identified It isnot clear that a natural product that has such specificitywouldbe selected for by evolution However it seems likely thatsuch cell type specific inhibitors exist or can be synthesizedSuch inhibitors that are useful for application like blockingthe plasma membrane V-ATPases of osteoclasts will likelyrequire either isolation of osteoclasts V-ATPases intact andlarge scale assay of small molecules or rational structuralbased approaches Recently two groups have pioneered suchrational approaches

Manolson and colleagues used yeast two-hybrid screen-ing to identify an interaction between the a3-subunit andB2-subunit of V-ATPase and this interaction was con-firmed using fusion proteins [40] Small molecules thatdisrupt binding between a3 and B2 but not B2 with other


c cc

d

A

D

B

C

F

H

G

a

e c998400998400

(a)

c c

d

c

F

D

A B G

E

ae c998400998400

C

H

(b)

Figure 6 Identification of new antiresorptives by disrupting subunit-subunit interactions unique for the a3-containing V-ATPases inosteoclasts (a) A binding interaction between a3 and B2 was identified and a high throughput screen was developed to identify smallmolecules that blocked the interaction Such molecules would be expected to block V-ATPase assembly (b) A binding interaction betweena3 and d2 was detected and small molecules were sought to block this interaction In this case exclusion of d2 from an otherwise assembledpump might be expected Mice in which d2 was knocked out suggested that this might both disrupt osteoclast resorption and also lead tostimulation of bone formation by osteoblasts

a-subunit isoforms and thus might represent a means toselectively target the a3-containing ruffled plasmamembraneV-ATPases were sought (Figure 6(a)) An ELISA system wasutilized to determine that in fact B2 binds all isoforms of a-subunit but with different affinities The basic system wasadapted to use to screen of 10000 small molecules seekingselective inhibitors of the a3-B2 interaction This screenresulted in the identification of the molecule 34-dihydroxy-N1015840-(2-hydroxybenzyilidine) benzohydrazide This moleculeinhibited the a3-B2 interaction and the ability of osteoclaststo resorb bone in vitro with an IC

50of 12 120583M [40]

The osteoclasts ruffled membrane V-ATPases containboth the a3 and d2 isoforms and interaction was identifiedbetween these two subunits as well A similar strategywas employed to identify luteolin as an inhibitor of a3-d2 interaction [193] (Figure 6(b)) Luteolin inhibited boneresorption but did not perturb fusion of osteoclast precursorsto form giant cells or formation of V-ATPases The IC

50in

vitro for inhibiting bone resorption was 25 120583Mand it did notaffect viability of osteoclasts or other cells at concentrations ashigh as 40 120583M Other biological activities of luteolin includeinhibitor of phosphodiesterase [194] tumor necrosis factor120572 and interleukin 6 activities [195] as well as a stimulatorof heme oxygenase expression [196] Luteolin was recentlyshown to reduce wear particle osteolysis in a mouse model[195]

The fact that these strategies have identified fairly potentand selective osteoclast inhibitors froma screen of only 10000small molecules is encouraging It is reasonable to propose

that large scale screens of 100000rsquos of small molecules mayyield very potent and selective osteoclast inhibitors

Data from our group suggested that direct bindingbetween the B2-subunit of V-ATPase and microfilamentsis vital for V-ATPase function in osteoclasts [114] Thesedata led to the hypothesis that a small molecule thatbound the microfilament-binding site on the B2-subunuitand sterically-inhibited the interaction between B2 andmicrofilaments would be a new type of osteoclast inhibitor(Figure 7) A reverse chemical genetic approach makinguse of supercomputer-based virtual screen initially identified100 candidates predicted to bind that microfilament bindingdomain of B2-subunit A number of the candidates alsoblocked binding between B2-subunit and microfilamentsin test tube assays [197] The lead molecule chosen fromthat screen enoxacin inhibited osteoclastogenesis and boneresorption by osteoclasts without affecting osteoblasts [197]Detailed characterization of enoxacin showed that it likelyfunctions by disrupting vesicular trafficking [112] Morerecent data showed that a bone-targeted version of enoxacininhibits orthodontic tooth movement in a rat model [198]

8 Regulation of V-ATPase

81 Reversible Assembly into V1 and V0 Components (Figure8) This process has been described and characterized indetail in yeast [32 199] and in Manduca sexta [71 200] Thesame mechanism has been reported in mammalian dendriticcells [201] and kidney cells [202] but characterization in


c c

d

A

D

BG

E

a

c998400998400c

dda FdC

e

H

(a)

c

d

A

D

BG

F

E

a

c998400998400

dC

e

H

(b)

Figure 7 Identification of new antiresorptives by disrupting binding between microfilaments and the B2-subunit (a) V-ATPases bindmicrofilaments through an actin binding site in the B-subunit Here we have depicted a conformational change in an EG stator arm toallow access to the actin binding site Alternatively the EGmay disassemble (b) Experimental evidence suggested that a molecule that boundthe B-subunit and prevented interaction with microfilaments might prevent trafficking of V-ATPases to the ruffled plasma membrane andthereby block bone resorption

Protein kinase APhosphoinositide

Signal

c

A

D

B G

E

c998400998400

FC

c

a

c

A

D

B G

HE

c998400998400

FC

e cea

H

Figure 8 Regulation of V-ATPase by reversible disassembly of V1 and V0 components Reversible disassembly might be triggered byphosphorylation by kinases like protein kinase A or this may be regulated by signaling lipids The H-subunit blocks ATP hydrolysis bythe free V1 and the V0 does not serve as a proton channel without the presence of V1

mammalian systems has not been as detailed Disassemblyin yeast andManduca were both shown to be fully reversible[67 199 203]

Initial studies showed that reversible disassembly of V-ATPase could be triggered by glucose deprivation A num-ber of regulatory pathways have now been described that

modulate this process These include RAVErabconnectin[204] aldolase [75 76] and other glycolytic enzymes phos-phatidylinositol 3-kinase [202] and protein kinase A [205206]

When V1 and V0 are disassembled both the ATPaseactivity ofV1 and the ion transport capacity ofV0 are inactive


In the case of V1 inhibition of ATPase activity has beenshown to be an activity of the H-subunit [87]

Although the reversible assembly hypothesis is well sup-ported by studies performed by a number of groups indifferent systems some vital questions remain unresolvedPerhaps the most perplexing is how once the V-ATPase isdisassembled the V1 component finds its way back to the V0for reassembly It is known that initial V-ATPase assemblyin yeasts requires assembly factors that dwell in the endo-plasmic reticulum [207] and similar factors are present inmammals

9 V-ATPase-Binding Proteins

91 Neuronal Proteins Neurotransmitter release and mem-brane fusion has been tied to activities of the V0 domainTheprotein components of the Torpedo marmorata electric organsynaptosomesldquomediatophorerdquo a pore-forming protein com-plex capable of Ca2+-dependent secretion of acetylcholine[42] was shown to include the c-subunit [208] Evidencefrom studies on yeast vacuole [209 210] fly neurons [211] andmouse pancreatic 120573-cells [23] supports the hypothesis thatthe V0 domain plays a role in Ca2+-dependent exocytosisEvidence from developing zebrafish suggest that V-ATPaseis involved in brain lysosome-phagosome fusion duringphagocytosis [95]

The V-ATPase V0 domain has been shown to interactwith the SNARE system (synaptobrevin and synaptophysin)[212] involving Ca2+calmodulin binding to synaptobrevin[213]This finding together with the fact that inTorpedomar-morata nerve terminals the a1-subunit interacts with synap-tobrevin [214] provides a possiblemolecular link betweenV0and Ca2+-dependent membrane fusion Disruption of the a1-subunit gene in Drosophila inhibited vesicle fusion with thepresynaptic membrane further implicating a post-SNARErole for a1 and V0 in synaptic membrane fusion [211]

92 Microfilaments It was first suggested that V-ATPasemight associate with the cytoskeleton in 1997 by Sudarsquoslab [215] Study of osteoclasts in osteosclerotic mice whichproved to harbor a mutation in the a3-subunit of V-ATPase[25] showed that V-ATPases did not interact with thedetergent-insoluble cytoskeleton of osteoclasts This sug-gested that a3 might be crucial for a linkage of the V-ATPase to the cytoskeleton Because the osteoclast fromosteosclerotic mice was not able to transport V-ATPases tothe ruffled membrane that suggested that an interactionwith the cytoskeleton might be vital for that specializedtargeting In 1999 a direct interaction between microfila-ments was detected in osteoclasts and reconstituted usingisolated kidney V-ATPases [216] In addition to showing thatthis binding interaction was present evidence was presentedthat the percentage of V-ATPases bound to microfilamentschanged with the activation state of the cells Resorbingosteoclasts displayed little binding between V-ATPase andmicrofilaments and little colocalization between the twoUnactivated osteoclasts in contrast displayed a high percent-age of V-ATPase bound to microfilaments and high levels of

colocalization It was then shown that the B-subunit of V-ATPases contained a high affinity microfilament binding site[62] Both the B2 isoform which is found in ubiquitouslyand at high levels in osteoclasts and B1 which is restrictedto certain kidney epithelial cells and a few other epithelialcells bound microfilaments with similar affinities and intactV-ATPases could be competed fromfilaments by competitionwith recombinant B2- or B1-subunits [62]

The precise aminoacids required for binding to microfil-aments were identified using fusion proteins mutagenesisand peptide analysis and conservative substitutions thateliminated microfilament binding were identified [63] B-subunits that lacked microfilament-binding activity weretested in osteoclasts and in yeast In osteoclasts the mutantB-subunit assembled with other V-ATPase components butwere not targeted to the ruffled plasma membrane [217]Thissuggested that microfilament-binding activity was requiredfor the specialized transport of V-ATPases required for osteo-clast activity In yeast actin binding activity was not requiredfor growth on standard laboratory media but was requiredfor growth in the presence of sublethal concentrations ofcertain pharmacological toxins [217]

Studies from Dictyostelium provide a potential under-lying mechanism for the V-ATPase-microfilament bindinginteraction V-ATPase recruited the Wiscott-Aldrich andScar Homolog (WASH) complex to acidic vesicles TheWASH complex then triggered local actin polymerizationinto microfilaments and V-ATPase bound the newly formeddynamicmicrofilaments and this was required for the sortingof V-ATPases into a daughter vesicle [218] (Figure 9) TheWASH complex is evolutionarily conserved and it will be ofinterest to determine whether theWASH complex is involvedin sorting of V-ATPases required for osteoclast formation andruffled membrane formation

93 Aldolase and Glycolytic Enzymes In 2001 Lu and col-leagues reported that V-ATPase binds directly to the gly-colytic enzyme aldolase and presented evidence that thatinteraction occurred in osteoclasts and proximal tubules ofthe kidney [219] This suggested the possibility that the gly-colyticmetabolon consisting of binding interactions betweenvarious glycolytic enzymes that increases the efficiency ofglycolysis [220ndash222] may at least in some cells includeV-ATPases [80] This idea is attractive because it wouldprovide a ready source of both ATP and protons for theV-ATPase as both are produced by glycolysis Evidence hasbeen provided that this interaction between V-ATPase andglycolytic enzymes occurs in diverse species ranging fromhumans to yeast [7 75 76]

94 Arf6 and ARNO Both ARF6 a small regulatory GTPasewhich is a member of the Ras superfamily and its activatorARNO (cytohesin2) bind V-ATPases [6] Arf6 bind thetransmembrane c-subunit and ARNO binds the a2-subunit[6] When this occurs ARNO can then binds ARF6 whichenhances the rate of nucleotide of change a process that acti-vates [223 224] ARF6 ARF6 regulates membrane traffickingand cytoskeletal organization in various cells [223 224]


A B C D E

V-ATPaseWashMicrofilament

Figure 9 Proposedmechanism bywhich binding ofmicrofilamentsby V-ATPase can be linked to actin polymerization stimulatedby the WASH complex [218] (A) V-ATPases in a vesicle (orany membrane) may recruit the (B) WASH complex which thenstimulates actin polymerization (C) Actin filaments elongate byaddition of monomers at the boundary of the membrane (C) and(D) V-ATPases bind to an actin in the polymerizing filament and aredrawn away from the original membrane as the filament elongates(D) At some point the V-ATPase-rich membrane separates fromthe mother vesicle to form a new vesicle (E) Actin filamentsdepolymerize and the WASH complex is released

Importantly the ability of the a2-subunit to bind ARNOwas dependent on the luminal pH of the associated vesicleallowingV-ATPase to recruit ARF6 and activate it in responseto pH [6]

95 Mammalian Target of Rapamycin Complex 1 (mTORC1)The mTORC1complex protein kinase has a vital role asa nutrientenergyredox sensor and controller of proteinsynthesis [10 225] It is composed of mTor regulatoryndashassociated protein of mTor (raptor) mammalian lethal withSEC13 protein (MLST8) proline-rich AKT1 substrate 40(PRAS40) and DEP domain-containing mTOR-interactingprotein (DEPTOR) Controllers of mTORC1 include insulingrowth factors aminoacids and oxidative stress [9 225 226]

V-ATPase is required for aminoacids present in the lumenof the lysosome to activate mTORC1 on the outside of thelysosome [8ndash11 227] This is achieved through aminoacidsensitive interaction between the V-ATPase and Ragulatora scaffolding complex that links the Rag GTPase to thelysosome The Rag GTPase then recruits mTorc1 to thelysosomal surface where it is activated and phosphorylatesdownstream targets like the translation inhibitor 4E-BP1 andthe kinase S6K to promote protein synthesis [225]

96 V-ATPase and Exosomes Exosomes are a class ofsecreted vesicles [228ndash230]The lumen of these vesicles oftencontains cytosolic proteins like the cytoskeletal proteins actinand myosin glycolytic enzymes and mRNAs and microR-NAs The membranes are oriented so that the cytosolicdomain of plasmamembrane proteins (when they are presentin exosomes) faces the lumen and the extracellular domainis exposed to the external milieu Communication medi-ated by exosomes includes interaction with cell surfaces and

the delivery of proteins mRNA and microRNAs to targetcells V-ATPase subunits have been identified in exosomesbutmost importantlymolecular genetic analysis in C elegansindentified an a-subunit of V-ATPase as being vital for therelease of certain types of exosomes [231]

97 V-ATPases and Cancer Specialized V-ATPases have beenshown to be involved in cancer growth and metastasisCancer cells were shown to express high levels of V-ATPasein their plasma membranes [232 233] The a3- and a4-isoforms were shown to be expressed at high levels inbreast cancer cells [110] and knockdown of the a3-subunitby RNA interference was shown to disrupt the capacity ofcancer cells to metastasize in a mouse model [111] V-ATPaseinhibitors have been the subject of interest as potential toolsfor treating cancer both for direct effects and to preventmultidrug resistance [9 81 177 178 234ndash241] Despite theclear involvement of V-ATPases in cancer to date therapeuticuse of V-ATPase-targeted agents has not reached the clinicThe key may be in identifying regulatory processes (eithertargeting or regulation of enzymatic activity) that are vital forcancer growth and metastasis but not for most cells Becauseof similarities in the mechanisms involved in osteoclastbone resorption and cancer invasion efforts to take rationalapproaches to blocking V-ATPase activity in osteoclasts mayprove useful in identifying new agents for treating cancer[41 114 242]

10 Summary

V-ATPases play a central role in the physiology of eukaryoticcells It is increasingly apparent that their ability to pumpprotons is integrated with many cell processes often in sur-prising ways potentially regulating vesicular trafficking andcytoskeletal rearrangements via interaction with Arf6 andARNO regulating vesicular trafficking through associationswithmicrofilaments coupling glycolysis to acidifying vesiclesand extracellular spaces sensing and responding to nutrientlevels and directly linking to the RAS and WNT signalingIn hindsight it is perhaps not surprising that these roles andothers would be taken up by V-ATPases they are amongthe oldest most complex and powerful of enzymes It islogical that evolution would find ways to fit the cellrsquos needsto the structure and functions of this enzyme We expectthat as more information emerges we will learn that V-ATPases are tied to more surprising processes and that theseties once understood will open doors to new understandingof physiology that will result in new approaches to thetreatment and prevention of pathologies It can be expectedthat soon atomic level structures of V-ATPases will becomeavailable that will provide great insight and that these willbe indispensable tools for the next generation of V-ATPaseresearchers At the same time in order to make use ofdetailed structural knowledge greater understanding of thecell biology of V-ATPases and their place in the networks ofregulation within cells will be vital Because of the immensecomplexities this will likely be the work not of decades but ofgenerations


Conflict of Interests

The author declares that he has no conflict of interestsregarding the publication of this paper

References

[1] A Hinton S Bond and M Forgac ldquoV-ATPase functions innormal and disease processesrdquo Pflugers Archiv European Journalof Physiology vol 457 no 3 pp 589ndash598 2009

[2] R Roberts ldquoLysosomal cysteine proteases structure functionand inhibition of cathepsinsrdquo Drug News and Perspectives vol18 no 10 pp 605ndash614 2005

[3] J Ljusberg B Ek-Rylander and G Andersson ldquoTartrate-resistant purple acid phosphatase is synthesized as a latentproenzyme and activated by cysteine proteinasesrdquo BiochemicalJournal vol 343 no 1 pp 63ndash69 1999

[4] Y Moriyama M Maeda and M Futai ldquoThe role of V-ATPasein neuronal and endocrine systemsrdquo Journal of ExperimentalBiology vol 172 pp 171ndash178 1992

[5] D Brown S Breton D A Ausiello and V MarshanskyldquoSensing signaling and sorting events in kidney epithelial cellphysiologyrdquo Traffic vol 10 no 3 pp 275ndash284 2009

[6] A Hurtado-Lorenzo M Skinner J El Annan et al ldquoV-ATPase interacts with ARNO and Arf6 in early endosomes andregulates the protein degradative pathwayrdquo Nature Cell Biologyvol 8 no 2 pp 124ndash136 2006

[7] M Merkulova A Hurtado-Lorenzo H Hosokawa et alldquoAldolase directly interacts with ARNO and modulates cellmorphology and acidic vesicle distributionrdquo American Journalof PhysiologymdashCell Physiology vol 300 no 6 pp C1442ndashC14552011

[8] L Bar-Peled L D Schweitzer R Zoncu and D M SabatinildquoRagulator Is a GEF for the Rag GTPases that signal amino acidlevels to mTORC1rdquo Cell vol 150 pp 1196ndash1208 2012

[9] A Efeyan R Zoncu and D M Sabatini ldquoAmino acids andmTORC1 from lysosomes to diseaserdquo Trends in MolecularMedicine vol 18 pp 524ndash533 2012

[10] S Pena-Llopis S Vega-Rubin-de-Celis J C Schwartz et alldquoRegulation of TFEB and V-ATPases by mTORC1rdquo The EMBOJournal vol 30 no 16 pp 3242ndash3258 2011

[11] R Zoncu L Bar-Peled A Efeyan SWang Y Sancak andDMSabatini ldquomTORC1 senses lysosomal amino acids through aninside-out mechanism that requires the vacuolar H+-ATPaserdquoScience vol 334 no 6056 pp 678ndash683 2011

[12] J A T Dow S A Davies Y Guo S GrahamM E Finbow andK Kaiser ldquoMolecular genetic analysis of V-ATPase function inDrosophila melanogasterrdquo Journal of Experimental Biology vol200 no 2 pp 237ndash245 1997

[13] G-H Sun-Wada Y Murata A Yamamoto H Kanazawa YWada and M Futai ldquoAcidic endomembrane organelles arerequired for mouse postimplantation developmentrdquo Develop-mental Biology vol 228 no 2 pp 315ndash325 2000

[14] T Oka and M Futai ldquoRequirement of V-ATPase for ovulationand embryogenesis in Caenorhabditis elegansrdquo The Journal ofBiological Chemistry vol 275 no 38 pp 29556ndash29561 2000

[15] A L Munn and H Riezman ldquoEndocytosis is required for thegrowth of vacuolar H+-ATPase-defective yeast identification ofsix new END genesrdquo Journal of Cell Biology vol 127 no 2 pp373ndash386 1994

[16] P M Kane ldquoThe long physiological reach of the yeast vacuolarH+-ATPaserdquo Journal of Bioenergetics and Biomembranes vol 39pp 415ndash421 2007

[17] M Toei R Saum and M Forgac ldquoRegulation and isoformfunction of the V-ATPasesrdquo Biochemistry vol 49 no 23 pp4715ndash4723 2010

[18] S P Muench J Trinick and M A Harrison ldquoStructural diver-gence of the rotary ATPasesrdquo Quarterly Reviews of Biophysicsvol 44 no 3 pp 1ndash46 2011

[19] A N Smith R C Lovering M Futai J Takeda D Brown andF E Karet ldquoRevised nomenclature for mammalian vacuolar-type H+-ATPase subunit genesrdquo Molecular Cell vol 12 no 4pp 801ndash803 2003

[20] Y-P Li W Chen Y Liang E Li and P Stashenko ldquoAtp6i-deficient mice exhibit severe osteopetrosis due to loss ofosteoclast-mediated extracellular acidificationrdquo Nature Genet-ics vol 23 no 4 pp 447ndash451 1999

[21] E M Serrano R D Ricofort J Zuo N Ochotny M FManolson and L S Holliday ldquoRegulation of vacuolar H+-ATPase in microglia by RANKLrdquo Biochemical and BiophysicalResearch Communications vol 389 no 1 pp 193ndash197 2009

[22] F Barvencik I Kurth T Koehne et al ldquoCLCN7 and TCIRG1mutations differentially affect bone matrix mineralizationin osteopetrotic individualsrdquo Journal of Bone and MineralResearch 2013

[23] G-H Sun-wada T Toyomura Y Murata A Yamamoto MFutai and Y Wada ldquoThe a3 isoform of V-ATPase regulatesinsulin secretion from pancreatic 120573-cellsrdquo Journal of Cell Sci-ence vol 119 no 21 pp 4531ndash4540 2006

[24] T Toyomura Y Murata A Yamamoto et al ldquoFrom lysosomesto the plasma membrane Localization of vacuolar type H+-ATPase with the a3 isoform during osteoclast differentiationrdquoThe Journal of Biological Chemistry vol 278 no 24 pp 22023ndash22030 2003

[25] J-C Scimeca A Franchi C Trojani et al ldquoThe gene encodingthe mouse homologue of the human osteoclast-specific 116-kDaV-ATPase subunit bears a deletion in osteosclerotic (ococ)mutantsrdquo Bone vol 26 no 3 pp 207ndash213 2000

[26] J-C Scimeca D Quincey H Parrinello et al ldquoNovel mutationsin the TCIRG1 gene encoding the a3 subunit of the vacuo-lar proton pump in patients affected by infantile malignantosteopetrosisrdquoHumanMutation vol 21 no 2 pp 151ndash157 2003

[27] A Frattini P J Orchard C Sobacchi et al ldquoDefects in TCIRG1subunit of the vacuolar proton pump are responsible for a subsetof human autosomal recessive osteopetrosisrdquo Nature Geneticsvol 25 no 3 pp 343ndash346 2000

[28] PA StehbergerN Schulz K E Finberg et al ldquoLocalization andregulation of the ATP6V0A4 (a4) vacuolar H+-ATPase subunitdefective in an inherited form of distal renal tubular acidosisrdquoJournal of the American Society of Nephrology vol 14 no 12 pp3027ndash3038 2003

[29] E E Norgett Z J Golder B Lorente-Canovas N Ingham KP Steel and F E Karet Frankl ldquoAtp6v0a4 knockout mouse is amodel of distal renal tubular acidosis with hearing loss withadditional extrarenal phenotyperdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 109 pp13775ndash13780 2012

[30] N Kawamura H Tabata G-H Sun-Wada and YWada ldquoOpticnerve compression and retinal degeneration in Tcirg1 mutantmice lacking the vacuolar-type H+-ATPase a3 subunitrdquo PLoSONE vol 5 no 8 Article ID e12086 2010


[31] A N Smith J Skaug K A Choate et al ldquoMutations inATP6N1B encoding a new kidney vacuolar proton pump 116-kD subunit cause recessive distal renal tubular acidosis withpreserved hearingrdquo Nature Genetics vol 26 no 1 pp 71ndash752000

[32] P M Kane ldquoTargeting reversible disassembly as a mechanismof controlling V-ATPase activityrdquo Current Protein amp PeptideScience vol 13 pp 117ndash123 2012

[33] M Diepholz D Venzke S Prinz et al ldquoA different confor-mation for EGC stator subcomplex in solution and in theassembled yeast V-ATPase possible implications for regulatorydisassemblyrdquo Structure vol 16 no 12 pp 1789ndash1798 2008

[34] M Lu S Vergara L Zhang L Shannon Holliday J Aris andS L Gluck ldquoThe amino-terminal domain of the E subunit ofvacuolar H+-ATPase (V-ATPase) interacts with the H subunitand is required for V-ATPase functionrdquoThe Journal of BiologicalChemistry vol 277 no 41 pp 38409ndash38415 2002

[35] R A Oot and S Wilkens ldquoDomain characterization andinteraction of the yeast vacuolar ATPase subunit C with theperipheral stator stalk subunits E and Grdquo The Journal ofBiological Chemistry vol 285 no 32 pp 24654ndash24664 2010

[36] R A Oot L S Huang E A Berry and S Wilkens ldquoCrystalstructure of the yeast vacuolar ATPase heterotrimeric EGC

ℎ119890119886119889

peripheral stalk complexrdquo Structure vol 20 no 11 pp 1881ndash1892 2012

[37] R A Oot and S Wilkens ldquoSubunit interactions at the V1-Vointerface in yeast vacuolar ATPaserdquo The Journal of BiologicalChemistry vol 287 no 16 pp 13396ndash13406 2012

[38] T Inoue and M Forgac ldquoCysteine-mediated cross-linkingindicates that subunit C of the V-ATPase is in close proximityto subunits E and G of the V1 domain and subunit a of the V0domainrdquo The Journal of Biological Chemistry vol 280 no 30pp 27896ndash27903 2005

[39] Y Arata J D Baleja and M Forgac ldquoCysteine-directed cross-linking to subunit B suggests that subunit E forms part of theperipheral stalk of the vacuolar H+-ATPaserdquo The Journal ofBiological Chemistry vol 277 no 5 pp 3357ndash3363 2002

[40] N Kartner Y Yao K Li G J Crasto A Datti andM F Manol-son ldquoInhibition of osteoclast bone resorption by disruptingvacuolar H+-ATPase a3-B2 subunit interactionrdquo The Journal ofBiological Chemistry vol 285 no 48 pp 37476ndash37490 2010

[41] N Kartner and M F Manolson ldquoV-ATPase subunit interac-tions the long road to therapeutic targetingrdquo Current Proteinamp Peptide Science vol 13 pp 164ndash179 2012

[42] T Hirata A Iwamoto-Kihara G-H Sun-Wada T Okajima YWada and M Futai ldquoSubunit rotation of vacuolar-type protonpumpingATPase Relative rotation of the G and c subunitsrdquoTheJournal of Biological Chemistry vol 278 no 26 pp 23714ndash237192003

[43] J A Mindell ldquoLysosomal acidification mechanismsrdquo AnnualReview of Physiology vol 74 pp 69ndash86 2012

[44] D Kasper R Planells-Cases J C Fuhrmann et al ldquoLoss of thechloride channel CIC-7 leads to lysosomal storage disease andneurodegenerationrdquo EMBO Journal vol 24 no 5 pp 1079ndash1091 2005

[45] A R Graves P K Curran C L Smith and J A Mindell ldquoTheClminusH+ antiporter ClC-7 is the primary chloride permeationpathway in lysosomesrdquo Nature vol 453 no 7196 pp 788ndash7922008

[46] Q Liu P M Kane P R Newman andM Forgac ldquoSite-directedmutagenesis of the yeast V-ATPase B subunit (Vma2p)rdquo

The Journal of Biological Chemistry vol 271 no 4 pp 2018ndash2022 1996

[47] Q Liu X-H Leng P R Newman E Vasilyeva P M Kane andM Forgac ldquoSite-directed mutagenesis of the yeast V-ATPase asubunitrdquoThe Journal of Biological Chemistry vol 272 no 18 pp11750ndash11756 1997

[48] K J MacLeod E Vasilyeva J D Baleja and M ForgacldquoMutational analysis of the nucleotide binding sites of the yeastvacuolar proton-translocatingATPaserdquoThe Journal of BiologicalChemistry vol 273 no 1 pp 150ndash156 1998

[49] N Hernando P David M Tarsio et al ldquoThe presence of thealternatively spliced A2 cassette in the vacuolar H+-ATPasesubunit a prevents assembly of the V1 catalytic domainrdquoEuropean Journal of Biochemistry vol 266 no 1 pp 293ndash3011999

[50] N HernandoM Bartkiewicz P Collin-Osdoby P Osdoby andR Baron ldquoAlternative splicing generates a second isoform of thecatalytic A subunit of the vacuolar H+-ATPaserdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 92 no 13 pp 6087ndash6091 1995

[51] R Alzamora R FThali F Gong et al ldquoPKA regulates vacuolarH+-ATPase localization and activity via direct phosphorylationof the A subunit in kidney cellsrdquo The Journal of BiologicalChemistry vol 285 no 32 pp 24676ndash24685 2010

[52] R Alzamora M M Al-Bataineh W Liu et al ldquoAMP-activatedprotein kinase regulates the vacuolar H+-ATPase via directphosphorylation of the A subunit (ATP6V1A) in the kidneyrdquoRenal PhysiologymdashAmerican Journal of Physiology vol 305 ppF943ndashF956 2013

[53] K R Hallows R Alzamora H Li et al ldquoAMP-activated proteinkinase inhibits alkaline pH- and PKA-induced apical vacuolarH+-ATPase accumulation in epididymal clear cellsrdquo AmericanJournal of PhysiologymdashCell Physiology vol 296 no 4 pp C672ndashC681 2009

[54] T Rieg and J A Dominguez Rieg ldquoConnecting type A inter-calated cell metabolic state to V-ATPase function phospho-rylation does matterrdquo American Journal of PhysiologymdashRenalPhysiology vol 305 pp F1105ndashF1106 2013

[55] B vanHille H Richener P Schmid I Puettner J R Green andG Bilbe ldquoHeterogeneity of vacuolar H+-ATPase differentialexpression of two human subunit B isoformsrdquo BiochemicalJournal vol 303 no 1 pp 191ndash198 1994

[56] P Bernasconi T Rausch I Struve L Morgan and L Taiz ldquoAnmRNA from human brain encodes an isoform of the B subunitof the vacuolarH+-ATPaserdquoThe Journal of Biological Chemistryvol 265 no 29 pp 17428ndash17431 1990

[57] B S Lee L S Holliday B Ojikutu I Krits and S L GluckldquoOsteoclasts express the B2 isoform of vacuolar H+-ATPaseintracellularly and on their plasma membranesrdquo AmericanJournal of PhysiologymdashCell Physiology vol 270 no 1 pp C382ndashC388 1996

[58] T C Sudhof V A Fried D K Stone P A Johnston and X-SXie ldquoHuman endomembrane H+ pump strongly resembles theATP-synthetase of Archaebacteriardquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 86 no16 pp 6067ndash6071 1989

[59] F E Karet K E Finberg R D Nelson et al ldquoMutations inthe gene encoding B1 subunit of H+-ATPase cause renal tubularacidosis with sensorineural deafnessrdquo Nature Genetics vol 21no 1 pp 84ndash90 1999

[60] R L Miller P Zhang M Smith et al ldquoV-ATPase B1-subunitpromoter drives expression of EGFP in intercalated cells of


kidney clear cells of epididymis and airway cells of lung intransgenic micerdquo American Journal of PhysiologymdashCell Physi-ology vol 288 no 5 pp C1134ndashC1144 2005

[61] S Breton T Wiederhold V Marshansky N N Nsumu VRamesh and D Brown ldquoThe B1 subunit of the H+ATPase is aPDZ domain-binding protein colocalization with NHE-RF inrenal B-intercalated cellsrdquo The Journal of Biological Chemistryvol 275 no 24 pp 18219ndash18224 2000

[62] L S Holliday M Lu B S Lee et al ldquoThe amino-terminaldomain of the B subunit of vacuolar H+-ATPase containsa filamentous actin binding siterdquo The Journal of BiologicalChemistry vol 275 no 41 pp 32331ndash32337 2000

[63] S-H Chen M R Bubb E G Yarmola et al ldquoVacuolar H+-ATPase binding to microfilaments regulation in response tophosphatidylinositol 3-kinase activity and detailed character-ization of the actin-binding site in subunit Brdquo The Journal ofBiological Chemistry vol 279 no 9 pp 7988ndash7998 2004

[64] J Zuo S Vergara S Kohno and L S Holliday ldquoBiochemicaland functional characterization of the actin-binding activityof the B subunit of yeast vacuolar H+-ATPaserdquo Journal ofExperimental Biology vol 211 no 7 pp 1102ndash1108 2008

[65] A N Smith K J Borthwick and F E Karet ldquoMolecular cloningand characterization of novel tissue-specific isoforms of thehuman vacuolar H+-ATPase C G and d subunits and theirevaluation in autosomal recessive distal renal tubular acidosisrdquoGene vol 297 no 1-2 pp 169ndash177 2002

[66] O Drory F Frolow and N Nelson ldquoCrystal structure of yeastV-ATPase subunit C reveals its stator functionrdquo EMBO Reportsvol 5 no 12 pp 1148ndash1152 2004

[67] J Lebownz R Ott and M Teale ldquoBand cbntrifugation for thecharacterization of proteins and protein complexesrdquoTheFASEBJournal vol 12 no 8 p A1394 1998

[68] K J Parra and P M Kane ldquoReversible association betweenthe V1 and V0 domains of yeast vacuolar H+-ATPase is anunconventional glucose-induced effectrdquoMolecular and CellularBiology vol 18 no 12 pp 7064ndash7074 1998

[69] O Vitavska H Wieczorek and H Merzendorfer ldquoA novel rolefor subunit C in mediating binding of the H+-V-ATPase to theactin cytoskeletonrdquoThe Journal of Biological Chemistry vol 278no 20 pp 18499ndash18505 2003

[70] O Vitavska H Merzendorfer and H Wieczorek ldquoThe V-ATPase subunit C binds to polymeric F-actin as well as tomonomeric G-actin and induces cross-linking of actin fila-mentsrdquo The Journal of Biological Chemistry vol 280 no 2 pp1070ndash1076 2005

[71] H Wieczorek K W Beyenbach M Huss and O VitavskaldquoVacuolar-type proton pumps in insect epitheliardquo Journal ofExperimental Biology vol 212 no 11 pp 1611ndash1619 2009

[72] G-H Sun-Wada YMurataMNamba A Yamamoto YWadaandM Futai ldquoMouse proton pump ATPase C subunit isoforms(C2-a and C2-b) specifically expressed in kidney and lungrdquoTheJournal of Biological Chemistry vol 278 no 45 pp 44843ndash44851 2003

[73] S P Muench M Huss C F Song et al ldquoCryo-electronmicroscopy of the vacuolar ATPase motor reveals its mechani-cal and regulatory complexityrdquo Journal ofMolecular Biology vol386 no 4 pp 989ndash999 2009


[75] M Lu Y Y Sautin L SHolliday and S LGluck ldquoTheglycolyticenzyme aldolase mediates assembly expression and activity of

vacuolar H+-ATPaserdquo The Journal of Biological Chemistry vol279 no 10 pp 8732ndash8739 2004

[76] M Lu D Ammar H Ives F Albrecht and S L GluckldquoPhysical interaction between aldolase and vacuolarH+-ATPaseis essential for the assembly and activity of the proton pumprdquoThe Journal of Biological Chemistry vol 282 no 34 pp 24495ndash24503 2007

[77] S Avnet P G Di S Lemma et al ldquoV-ATPase is a candidatetherapeutic target for Ewing sarcomardquo Biochimica et BiophysicaActa vol 1832 pp 1105ndash1116 2013

[78] S I Galkina G F Sudrsquoina and T Klein ldquoMetabolic regulationof neutrophil spreading membrane tubulovesicular extensions(cytonemes) formation and intracellular pH upon adhesion tofibronectinrdquo Experimental Cell Research vol 312 no 13 pp2568ndash2579 2006

[79] S R Sennoune and R Martinez-Zaguilan ldquoVacuolar H+-ATPase signaling pathway in cancerrdquo Current Protein amp PeptideScience vol 13 pp 152ndash163 2012

[80] Y Su A Zhou R S Al-Lamki and F E Karet ldquoThe a-subunitof the V-type H+-ATPase interacts with phosphofructokinase-1in humansrdquoThe Journal of Biological Chemistry vol 278 no 22pp 20013ndash20018 2003

[81] S Lebreton J Jaunbergs M G Roth D A Ferguson and JK De Brabander ldquoEvaluating the potential of vacuolar ATPaseinhibitors as anticancer agents and multigram synthesis of thepotent salicylihalamide analog saliphenylhalamiderdquo Bioorganicand Medicinal Chemistry Letters vol 18 no 22 pp 5879ndash58832008

[82] SNakamura ldquoGlucose activatesH+-ATPase in kidney epithelialcellsrdquo American Journal of PhysiologymdashCell Physiology vol 287pp 97ndash105 2004

[83] R Graf A LepierW R Harvey andHWieczorek ldquoA novel 14-kDa V-ATPase subunit in the tobacco hornworm midgutrdquo TheJournal of Biological Chemistry vol 269 no 5 pp 3767ndash37741994

[84] T Fujiwara A Kawai F Shimizu et al ldquoCloning sequencingand expression of a novel cDNA encoding human vacuolarATPase (14-kDa subunit)rdquo DNA Research vol 2 no 3 pp 107ndash111 1995

[85] S-B Peng B P Crider S J Tsai X-S Xie and D K StoneldquoIdentification of a 14-kDa subunit associated with the catalyticsector of clathrin-coated vesicle H+-ATPaserdquo The Journal ofBiological Chemistry vol 271 no 6 pp 3324ndash3327 1996

[86] H Imamura C Ikeda M Yoshida and K Yokoyama ldquoThe Fsubunit of thermus thermophilus V1-ATPase promotes ATPaseactivity but is not necessary for rotationrdquo The Journal ofBiological Chemistry vol 279 no 17 pp 18085ndash18090 2004

[87] K J Parra K L Keenan and P M Kane ldquoThe H subunit(VMA13p) of the yeast V-ATPase inhibits the ATPase activityof cytosolic V1 complexesrdquoThe Journal of Biological Chemistryvol 275 no 28 pp 21761ndash21767 2000

[88] Z Zhou S-B Peng B P Crider P Andersen X-S Xie and DK Stone ldquoRecombinant SFD isoforms activate vacuolar protonpumpsrdquoThe Journal of Biological Chemistry vol 274 no 22 pp15913ndash15919 1999

[89] M Geyer H Yu R Mandic et al ldquoSubunit H of the V-ATPase binds to the medium chain of adaptor protein complex2 and connects nef to the endocytic machineryrdquoThe Journal ofBiological Chemistry vol 277 no 32 pp 28521ndash28529 2002

[90] X Lu H Yu S-H Liu F M Brodsky and B M PeterlinldquoInteractions between HIV1 Nef and vacuolar ATPase facilitate


the internalization of CD4rdquo Immunity vol 8 no 5 pp 647ndash6561998

[91] RMandic O T Fackler M Geyer T Linnemann Y-H Zhengand B M Peterlin ldquoNegative factor from SIV binds to thecatalytic subunit of the V-ATPase to internalize CD4 and toincrease viral infectivityrdquo Molecular Biology of the Cell vol 12no 2 pp 463ndash473 2001

[92] T Oka Y Murata M Namba et al ldquoA4 a unique kidney-specific isoform of mouse vacuolar H+-ATPase subunit ardquo TheJournal of Biological Chemistry vol 276 no 43 pp 40050ndash40054 2001

[93] T Oka T Toyomura K Honjo Y Wada and M FutaildquoFour subunit a isoforms of Caenorhabditis elegans vacuolarH+-ATPase cell-specific expression during developmentrdquo TheJournal of Biological Chemistry vol 276 no 35 pp 33079ndash33085 2001

[94] M F Manolson B Wu D Proteau et al ldquoSTV1 gene encodesfunctional homologue of 95-kDa yeast vacuolar H+-ATPasesubunit Vph1prdquoThe Journal of Biological Chemistry vol 269 no19 pp 14064ndash14074 1994

[95] F Peri and C Nusslein-Volhard ldquoLive imaging of neuronaldegradation by microglia reveals a role for v0-ATPase a1 inphagosomal fusion in vivordquo Cell vol 133 no 5 pp 916ndash9272008

[96] B Fischer A Dimopoulou J Egerer et al ldquoFurther character-ization of ATP6V0A2-related autosomal recessive cutis laxardquoHuman Genetics vol 131 pp 1761ndash1773 2012

[97] V Hucthagowder E Morava U Kornak et al ldquoLoss-of-function mutations in ATP6V0A2 impair vesicular traffickingtropoelastin secretion and cell survivalrdquo Human MolecularGenetics vol 18 no 12 pp 2149ndash2165 2009

[98] U Kornak E Reynders A Dimopoulou et al ldquoImpairedglycosylation and cutis laxa caused bymutations in the vesicularH+-ATPase subunit ATP6V0A2rdquoNature Genetics vol 40 no 1pp 32ndash34 2008

[99] E Morava R A Wevers M A Willemsen and D LefeberldquoCobbleston-like brain dysgenesis and altered glycosylation incongenital cutis laxa debri typerdquo Neurology vol 73 no 14 pp1164ndash1165 2009

[100] E Morava M Guillard D J Lefeber and R A WeversldquoAutosomal recessive cutis laxa syndrome revisitedrdquo EuropeanJournal of Human Genetics vol 17 no 9 pp 1099ndash1110 2009

[101] C Noordam S Funke N V Knoers et al ldquoDecreased bonedensity and treatment in patients with autosomal recessive cutislaxardquo Acta Paediatrica vol 98 no 3 pp 490ndash494 2009

[102] M Guillard A Dimopoulou B Fischer et al ldquoVacuolarH+-ATPase meets glycosylation in patients with cutis laxardquoBiochimica et BiophysicaActa vol 1792 no 9 pp 903ndash914 2009

[103] C Pietrement G-H Sun-Wada N Da Silva et al ldquoDistinctexpression patterns of different subunit isoforms of the V-ATPase in the rat epididymisrdquo Biology of Reproduction vol 74no 1 pp 185ndash194 2006

[104] U Kornak A Schulz W Friedrich et al ldquoMutations in the a3subunit of the vacuolar H+-ATPase cause infantile malignantosteopetrosisrdquo Human Molecular Genetics vol 9 no 13 pp2059ndash2063 2000

[105] T Nishi and M Forgac ldquoMolecular cloning and expressionof three isoforms of the 100-kDa a subunit of the mousevacuolar proton-translocatingATPaserdquoThe Journal of BiologicalChemistry vol 275 no 10 pp 6824ndash6830 2000

[106] T Toyomura T Oka C Yamaguchi Y Wada and M FutaildquoThree subunit a isoforms of mouse vacuolar H+-ATPasePreferential expression of the 1205723 isoform during osteoclastdifferentiationrdquoThe Journal of Biological Chemistry vol 275 no12 pp 8760ndash8765 2000

[107] J P Mattsson X Li S-B Peng et al ldquoProperties of threeisoforms of the 116-kDa subunit of vacuolar H+-ATPase from asingle vertebrate species cloning gene expression and proteincharacterization of functionally distinct isoforms in Gallusgallusrdquo European Journal of Biochemistry vol 267 no 13 pp4115ndash4126 2000

[108] G-H Sun-Wada H Tabata N Kawamura M Aoyama and YWada ldquoDirect recruitment of H+-ATPase from lysosomes forphagosomal acidificationrdquo Journal of Cell Science vol 122 no14 pp 2504ndash2513 2009

[109] T Schinke A F Schilling A Baranowsky et al ldquoImpairedgastric acidification negatively affects calcium homeostasis andbone massrdquo Nature Medicine vol 15 no 6 pp 674ndash681 2009

[110] A Hinton S R Sennoune S Bond et al ldquoFunction of asubunit isoforms of the V-ATPase in pH homeostasis and invitro invasion of MDA-MB231 human breast cancer cellsrdquo TheJournal of Biological Chemistry vol 284 no 24 pp 16400ndash16408 2009

[111] T Nishisho K Hata M Nakanishi et al ldquoThe a3 isoformvacuolar type H+-ATPase promotes distant metastasis in themouse B16 melanoma cellsrdquo Molecular Cancer Research vol 9no 7 pp 845ndash855 2011

[112] E J Toro J Zuo D A Ostrov et al ldquoEnoxacin directly inhibitsosteoclastogenesis without inducing apoptosisrdquo The Journal ofBiological Chemistry vol 287 pp 17894ndash17904 2012

[113] J Zuo J Jiang S-H Chen et al ldquoActin binding activity ofsubunit B of vacuolar H+-ATPase is involved in its targeting toruffled membranes of osteoclastsrdquo Journal of Bone and MineralResearch vol 21 no 5 pp 714ndash721 2006

[114] E J Toro D A Ostrov T J Wronski and L S HollidayldquoRational identification of enoxacin as a novel V-ATPase-directed osteoclast inhibitorrdquoCurrent Proteinamp Peptide Sciencevol 13 pp 180ndash191 2012

[115] A N Smith K E Finberg C A Wagner et al ldquoMolecularcloning and characterization of Atp6n1b A novel fourthmurinevacuolar H+-ATPase a-subunit generdquo The Journal of BiologicalChemistry vol 276 no 45 pp 42382ndash42388 2001

[116] H Nishigori S Yamada H Tomura et al ldquoIdentification andcharacterization of the gene encoding a second proteolipidsubunit of human vacuolar H+-ATPase (ATP6F)rdquo Genomicsvol 50 no 2 pp 222ndash228 1998

[117] B Powell L A Graham and T H Stevens ldquoMolecular charac-terization of the yeast vacuolar H+-ATPase proton porerdquo TheJournal of Biological Chemistry vol 275 no 31 pp 23654ndash23660 2000

[118] A R Flannery L A Graham and T H Stevens ldquoTopologicalcharacterization of the c c1015840 and c

10158401015840

subunits of the vacuolarATPase from the yeast Saccharomyces cerevisiaerdquo The Journalof Biological Chemistry vol 279 no 38 pp 39856ndash39862 2004

[119] S-H Lee J Rho D Jeong et al ldquoV-ATPase V0 subunit d2-deficient mice exhibit impaired osteoclast fusion and increasedbone formationrdquoNatureMedicine vol 12 no 12 pp 1403ndash14092006

[120] T Kim H Ha N Kim et al ldquoATP6v0d2 deficiency increasesbone mass but does not influence ovariectomy-induced bonelossrdquo Biochemical and Biophysical Research Communicationsvol 403 pp 73ndash78 2010


[121] M Sambade and P M Kane ldquoThe yeast vacuolar proton-translocating ATPase contains a subunit homologous to themanduca sexta and bovine e subunits that is essential forfunctionrdquo The Journal of Biological Chemistry vol 279 no 17pp 17361ndash17365 2004

[122] H Merzendorfer M Huss R Schmid W R Harvey andH Wieczorek ldquoA novel insect V-ATPase subunit M97 isglycosylated extensivelyrdquo The Journal of Biological Chemistryvol 274 no 24 pp 17372ndash17378 1999

[123] J Ludwig S Kerscher U Brandt et al ldquoIdentification and char-acterization of a novel 92-kDa membrane sector- associatedprotein of vacuolar proton-ATPase from chromaffin granulesrdquoThe Journal of Biological Chemistry vol 273 no 18 pp 10939ndash10947 1998

[124] F Supek L Supekova SMandiyan Y-C E PanHNelson andN Nelson ldquoA novel accessory subunit for vacuolar H+-ATPasefrom chromaffin granulesrdquoThe Journal of Biological Chemistryvol 269 no 39 pp 24102ndash24106 1994

[125] F Getlawi A Laslop H Schagger J Ludwig J Haywoodand D Apps ldquoChromaffin granule membrane glycoprotein TVis identical with Ac45 a membrane-integral subunit of thegranulersquos H+-ATPaserdquo Neuroscience Letters vol 219 no 1 pp13ndash16 1996

[126] E J R Jansen J C M Holthuis C McGrouther J P HBurbach and G J M Martens ldquoIntracellular trafficking of thevacuolar H+-ATPase accessory subunit Ac45rdquo Journal of CellScience vol 111 no 20 pp 2999ndash3006 1998

[127] V T G Schoonderwoert E J R Jansen and G J M MartensldquoThe fate of newly synthesized V-ATPase accessory subunitAc45 in the secretory pathwayrdquo European Journal of Biochem-istry vol 269 no 7 pp 1844ndash1853 2002

[128] D Q Yang S Feng W Chen H Zhao C Paulson and YP Li ldquoV-ATPase subunit ATP6AP1 (Ac45) regulates osteoclastdifferentiation extracellular acidification lysosomal traffickingand protease exocytosis in osteoclast-mediated bone resorp-tionrdquo Journal of Bone and Mineral Research vol 27 no 8 pp1695ndash1707 2012

[129] E J Jansen and G J Martens ldquoNovel insights into V-ATPase functioning distinct roles for its accessory subunitsATP6AP1Ac45 and ATP6AP2(pro) renin receptor rdquo CurrentProtein amp Peptide Science vol 13 pp 124ndash133 2012

[130] H Feng T Cheng N J Pavlos et al ldquoCytoplasmic terminusof vacuolar type proton pump accessory subunit Ac45 isrequired for proper interaction with V0 domain subunits andefficient osteoclastic bone resorptionrdquoThe Journal of BiologicalChemistry vol 283 no 19 pp 13194ndash13204 2008

[131] GNguyen F Delarue C Burckle L Bouzhir T Giller and J-DSraer ldquoPivotal role of the reninprorenin receptor in angiotensinII production and cellular responses to reninrdquo The Journal ofClinical Investigation vol 109 no 11 pp 1417ndash1427 2002

[132] M Augsten C Hubner M Nguyen W Kunkel A Hartland R Eck ldquoDefective hyphal induction of a Candida albicansphosphatidylinositol 3-phosphate 5-kinase null mutant on solidmedia does not lead to decreased virulencerdquo Infection andImmunity vol 70 no 8 pp 4462ndash4470 2002

[133] G Nguyen ldquoRenin and prorenin receptor in hypertensionwhatrsquos newrdquo Current Hypertension Reports vol 13 pp 79ndash852011

[134] G Nguyen ldquoRenin (pro)renin and receptor an updaterdquo Clini-cal Science vol 120 pp 169ndash178 2011

[135] G Nguyen and D N Muller ldquoThe biology of the (Pro)Reninreceptorrdquo Journal of the American Society of Nephrology vol 21pp 18ndash23 2010

[136] C-M Cruciat B Ohkawara S P Acebron et al ldquoRequirementof prorenin receptor and vacuolar H+-ATPase-mediated acidi-fication for Wnt signalingrdquo Science vol 327 no 5964 pp 459ndash463 2010

[137] C Cousin D Bracquart A Contrepas P Corvol L Mullerand G Nguyen ldquoSoluble form of the (pro)renin receptorgenerated by intracellular cleavage by furin is secreted inplasmardquo Hypertension vol 53 no 6 pp 1077ndash1082 2009

[138] K Kinouchi A Ichihara M Sano et al ldquoThe (Pro)renin recep-torATP6AP2 is essential for vacuolar H+-ATPase assembly inmurine cardiomyocytesrdquoCirculation Research vol 107 no 1 pp30ndash34 2010

[139] K Kinouchi A Ichihara and H Itoh ldquoFunctional character-ization of (pro)renin receptor in association with V-ATPaserdquoFrontiers in Bioscience vol 16 no 8 pp 3216ndash3223 2011

[140] A E Cuadra Z Shan C Sumners and M K Raizada ldquoA cur-rent view of brain renin-angiotensin system Is the (pro)reninreceptor themissing linkrdquo Pharmacology andTherapeutics vol125 no 1 pp 27ndash38 2010

[141] M Azizi and G Wuerzner ldquoRationale for combining blockersof the renin-angiotensin systemrdquo Seminars in Nephrology vol27 no 5 pp 544ndash554 2007

[142] R V Durvasula and S J Shankland ldquoActivation of a local reninangiotensin system in podocytes by glucoserdquo American Journalof PhysiologymdashRenal Physiology vol 294 no 4 pp F830ndashF8392008

[143] P Garcia S Schwenzer J Slotta et al ldquoInhibition ofangiotensin-converting enzyme stimulates fracture healing andperiosteal callus formationmdashrole of a local renin-angiotensinsystemrdquo British Journal of Pharmacology vol 159 no 8 pp1672ndash1680 2010

[144] S Satofuka A Ichihara N Nagai et al ldquoSuppression of ocularinflammation in endotoxin-induced uveitis by inhibiting non-proteolytic activation of proreninrdquo Investigative Ophthalmologyamp Visual Science vol 47 no 6 pp 2686ndash2692 2006

[145] M Tahmasebi S Barker J R Puddefoot and G P VinsonldquoLocalisation of renin-angiotensin system (RAS) componentsin breastrdquo British Journal of Cancer vol 95 no 1 pp 67ndash742006

[146] Y Asaba M Ito T Fumoto et al ldquoActivation of renin-angiotensin system induces osteoporosis independently ofhypertensionrdquo Journal of Bone andMineral Research vol 24 no2 pp 241ndash250 2009

[147] Y Izu F Mizoguchi A Kawamata et al ldquoAngiotensin IItype 2 receptor blockade increases bone massrdquo The Journal ofBiological Chemistry vol 284 no 8 pp 4857ndash4864 2009

[148] H Shimizu H Nakagami M K Osako et al ldquoPreventionof osteoporosis by angiotensin-converting enzyme inhibitor inspontaneous hypertensive ratsrdquo Hypertension Research vol 32no 9 pp 786ndash790 2009

[149] S M Bernhard K Seidel J Schmitz et al ldquoThe (pro)reninreceptor ((P)RR) can act as a repressor of Wnt signallingrdquoBiochemical Pharmacology vol 84 pp 1643ndash1650 2012

[150] B S Lee ldquoRegulation of V-ATPase expression in mammaliancellsrdquo Current Protein amp Peptide Science vol 13 pp 107ndash1162012

[151] T K Kundu and M R S Rao ldquoCpG islands in chromatinorganization and gene expressionrdquo Journal of Biochemistry vol125 no 2 pp 217ndash222 1999


[152] A Razin and R Shemer ldquoDNA methylation in early develop-mentrdquo Human Molecular Genetics vol 4 pp 1751ndash1755 1995

[153] P Caiafa and M Zampieri ldquoDNA methylation and chromatinstructure the puzzling CpG islandsrdquo Journal of Cellular Bio-chemistry vol 94 no 2 pp 257ndash265 2005

[154] H Cedar and Y Bergman ldquoProgramming of DNAmethylationpatternsrdquo Annual Review of Biochemistry vol 81 pp 97ndash1172012

[155] R Chatterjee and C Vinson ldquoCpG methylation recruitssequence specific transcription factors essential for tissue spe-cific gene expressionrdquo Biochimica et Biophysica Acta vol 1819pp 763ndash770 2012

[156] MMonk ldquoEpigenetic programming of differential gene expres-sion in development and evolutionrdquo Developmental Geneticsvol 17 no 3 pp 188ndash197 1995

[157] F Jouret C Auzanneau H Debaix et al ldquoUbiquitous andkidney-specific subunits of vacuolar H+-ATPase are differen-tially expressed during nephrogenesisrdquo Journal of the AmericanSociety of Nephrology vol 16 no 11 pp 3235ndash3246 2005

[158] M Katoh and M Katoh ldquoHuman FOX gene family (review)rdquoInternational Journal of Oncology vol 25 no 5 pp 1495ndash15002004

[159] D L Lacey E Timms H-L Tan et al ldquoOsteoprotegerinligand is a cytokine that regulates osteoclast differentiation andactivationrdquo Cell vol 93 no 2 pp 165ndash176 1998


[161] G E Beranger D Momier J-M Guigonis M Samson G FCarle and J-C Scimeca ldquoDifferential binding of poly(ADP-ribose) polymerase-1 and JunDFra2 accounts for RANKL-induced Tcirg1 gene expression during osteoclastogenesisrdquoJournal of Bone andMineral Research vol 22 no 7 pp 975ndash9832007

[162] G E Beranger D Momier N Rochet et al ldquoRANKL treat-ment releases the negative regulation of the poly(ADP-ribose)polymerase-1 on Tcirg1 gene expression during osteoclastogen-esisrdquo Journal of Bone and Mineral Research vol 21 no 11 pp1757ndash1769 2006

[163] S Smith ldquoThe world according to PARPrdquo Trends in BiochemicalSciences vol 26 no 3 pp 174ndash179 2001

[164] P Caiafa T Guastafierro and M Zampieri ldquoEpigeneticspoly(ADP-ribosyl)ation of PARP-1 regulates genomic methyla-tion patternsrdquo The FASEB Journal vol 23 no 3 pp 672ndash6782009

[165] D Quenet R R El V Schreiber and F Dantzer ldquoThe role ofpoly(ADP-ribosyl)ation in epigenetic eventsrdquoThe InternationalJournal of Biochemistry amp Cell Biology vol 41 pp 60ndash65 2009

[166] M Chevanne C Calia M Zampieri et al ldquoOxidative DNAdamage repair and parp 1 and parp 2 expression in Epstein-Barrvirus-immortalized B lymphocyte cells from young subjectsold subjects and centenariansrdquo Rejuvenation Research vol 10no 2 pp 191ndash204 2007

[167] T Guastafierro B Cecchinelli M Zampieri et al ldquoCCCTC-binding factor activates PARP-1 affecting DNA methylationmachineryrdquoThe Journal of Biological Chemistry vol 283 no 32pp 21873ndash21880 2008

[168] S-P Wang I Krits S Bai and B S Lee ldquoRegulation ofenhanced vacuolarH+-ATPase expression inmacrophagesrdquoTheJournal of Biological Chemistry vol 277 no 11 pp 8827ndash88342002

[169] S Jeyaraj D Dakhlallah S R Mill and B S Lee ldquoHuRstabilizes vacuolar H+-translocating ATPase mRNA duringcellular energy depletionrdquo The Journal of Biological Chemistryvol 280 no 45 pp 37957ndash37964 2005

[170] S C Jeyaraj D Dakhlallah S R Hill and B S Lee ldquoExpressionand distribution of HuR during ATP depletion and recovery inproximal tubule cellsrdquo American Journal of PhysiologymdashRenalPhysiology vol 291 no 6 pp F1255ndashF1263 2006

[171] C F Lopez S O Nielsen P B Moore and M L KleinldquoUnderstanding naturersquos design for a nanosyringerdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 101 pp 2987ndash2992 2004

[172] A P Levy ldquoHypoxic regulation of VEGF mRNA stability byRNA-binding proteinsrdquo Trends in Cardiovascular Medicine vol8 pp 246ndash250 1998

[173] W Wang H Furneaux H Cheng et al ldquoHuR regulatesp21 mRNA stabilization by UV lightrdquo Molecular and CellularBiology vol 20 no 3 pp 760ndash769 2000

[174] I E Gallouzi C M Brennan M G Stenberg et al ldquoHuRbinding to cytoplasmic mRNA is perturbed by heat shockrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 97 pp 3073ndash3078 2000

[175] O Hobert ldquoGene regulation by transcription factors andMicroRNAsrdquo Science vol 319 no 5871 pp 1785ndash1786 2008

[176] D T OrsquoConnor G Zhu F Rao et al ldquoHeritability and genome-wide linkage in US and australian twins identify novel genomicregions controlling chromogranin a implications for secretionand blood pressurerdquo Circulation vol 118 no 3 pp 247ndash2572008

[177] A Hernandez G Serrano-Bueno J R Perez-Castineira and ASerrano ldquoIntracellular proton pumps as targets in chemother-apy V-ATPases and cancerrdquo Current Pharmaceutical Designvol 18 pp 1383ndash1394 2012

[178] M Huss and H Wieczorek ldquoInhibitors of V-ATPases old andnew playersrdquo Journal of Experimental Biology vol 212 no 3 pp341ndash346 2009

[179] A Qin T S Cheng N J Pavlos Z Lin K R Dai andM H Zheng ldquoV-ATPases in osteoclasts structure functionand potential inhibitors of bone resorptionrdquo The InternationalJournal of Biochemistry amp Cell Biology vol 44 pp 1422ndash14352012

[180] G Werner H Hagenmaier H Drautz A Baumgartner andH Zahner ldquoBafilomycins a new group of macrolide antibioticsproduction isolation chemical structure and biological activ-ityrdquoThe Journal of Antibiotics vol 37 pp 110ndash117 1984

[181] E J Bowman A Siebers and K Altendorf ldquoBafilomycins aclass of inhibitors of membrane ATPases frommicroorganismsanimal cells and plant cellsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 85 no21 pp 7972ndash7976 1988

[182] H Kinashi K Sakaguchi T Higashijima and T MiyazawaldquoStructures of concanamycins B and Crdquo The Journal of Antibi-otics vol 35 no 11 pp 1618ndash1620 1982

[183] H Kinashi K Someno and K Sakaguchi ldquoIsolation andcharacterization of concanamycins A B and Crdquo The Journal ofAntibiotics vol 37 no 11 pp 1333ndash1343 1984

[184] B J Bowman and E J Bowman ldquoMutations in subunit c of thevacuolar ATPase confer resistance to bafilomycin and identifya conserved antibiotic binding siterdquo The Journal of BiologicalChemistry vol 277 no 6 pp 3965ndash3972 2002


[185] E J Bowman L A Graham T H Stevens and B J BowmanldquoThe bafilomycinconcanamycin binding site in subunit c ofthe V-ATPases from Neurospora crassa and SaccharomycescerevisiaerdquoThe Journal of Biological Chemistry vol 279 no 32pp 33131ndash33138 2004

[186] M Huss G Ingenhorst S Konig et al ldquoConcanamycin A thespecific inhibitor of V-ATPases binds to the VO subunit crdquoThe Journal of Biological Chemistry vol 277 no 43 pp 40544ndash40548 2002

[187] F Sasse H Steinmetz G Hofle and H Reichenbach ldquoArcha-zolids new cytotoxic macrolactones fromArchangium gephyra(Myxobacteria) Production isolation physico-chemical andbiological propertiesrdquo The Journal of Antibiotics vol 56 no 6pp 520ndash525 2003

[188] M Huss F Sasse B Kunze et al ldquoArchazolid and apicularennovel specific V-ATPase inhibitorsrdquo BMC Biochemistry vol 6article 13 2005

[189] D Menche J Hassfeld F Sasse M Huss and H WieczorekldquoDesign synthesis and biological evaluation of novel analoguesof archazolid a highly potent simplified V-ATPase inhibitorrdquoBioorganic and Medicinal Chemistry Letters vol 17 no 6 pp1732ndash1735 2007

[190] S Bockelmann D Menche S Rudolph et al ldquoArchazolid Abinds to the equatorial region of the c-ring of the vacuolar H+-ATPaserdquoThe Journal of Biological Chemistry vol 285 no 49 pp38304ndash38314 2010

[191] M R Boyd C Farina P Belfiore et al ldquoDiscovery of anovel antitumor benzolactone enamide class that selectivelyinhibits mammalian vacuolar-type (H+)-ATPasesrdquo Journal ofPharmacology and ExperimentalTherapeutics vol 297 no 1 pp114ndash120 2001

[192] C Chouabe V Eyraud P Da Silva et al ldquoNew mode of actionfor a knottin protein bioinsecticide Pea Albumin 1 subunit b(PA1b) is the first peptidic inhibitor of V-ATPaserdquo The Journalof Biological Chemistry vol 286 no 42 pp 36291ndash36296 2011

[193] G J Crasto N Kartner Y Yao et al ldquoLuteolin inhibitionof V-ATPase a3ndashd2 interaction decreases osteoclast resorptiveactivityrdquo Journal of Cellular Biochemistry vol 114 no 4 pp 929ndash941 2013

[194] M C Yu J H Chen C Y Lai C Y Han and W CKo ldquoLuteolin a non-selective competitive inhibitor of phos-phodiesterases 1ndash5 displaced [3H]-rolipram from high-affinityrolipram binding sites and reversed xylazineketamine-inducedanesthesiardquo European Journal of Pharmacology vol 627 pp269ndash275 2010

[195] D K Shin M H Kim S H Lee T H Kim and S YKim ldquoInhibitory effects of luteolin on titaniumparticle-inducedosteolysis in a mouse modelrdquo Acta Biomaterialia vol 8 pp3524ndash3531 2012

[196] G B Sun X Sun M Wang et al ldquoOxidative stress suppressionby luteolin-induced heme oxygenase-1 expressionrdquo Toxicologyand Applied Pharmacology vol 265 pp 229ndash240 2012

[197] D A Ostrov A T Magis T J Wronski et al ldquoIdentificationof enoxacin as an inhibitor of osteoclast formation and boneresorption by structure-based virtual screeningrdquo Journal ofMedicinal Chemistry vol 52 no 16 pp 5144ndash5151 2009

[198] E J Toro J Zuo A Guiterrez et al ldquoBis-enoxacin inhibits boneresorption and orthodontic toothmovementrdquo Journal of DentalResearch vol 92 no 10 pp 925ndash931 2013

[199] P M Kane ldquoDisassembly and reassembly of the yeast vacuolarH+-ATPase in vivordquo The Journal of Biological Chemistry vol270 no 28 pp 17025ndash17032 1995

[200] J-P Sumner J A T Dow F G P Earley U Klein D Jagerand HWieczorek ldquoRegulation of plasmamembrane V-ATPaseactivity by dissociation of peripheral subunitsrdquo The Journal ofBiological Chemistry vol 270 no 10 pp 5649ndash5653 1995

[201] E S Trombetta M Ebersold W Garrett M Pypaert and IMellman ldquoActivation of lysosomal function during dendriticcell maturationrdquo Science vol 299 no 5611 pp 1400ndash1403 2003

[202] Y Y Sautin M Lu A Gaugler L Zhang and S L GluckldquoPhosphatidylinositol 3-kinase-mediated effects of glucose onvacuolar H+-ATPase assembly translocation and acidificationof intracellular compartments in renal epithelial cellsrdquoMolecu-lar and Cellular Biology vol 25 no 2 pp 575ndash589 2005

[203] R Graf W R Harvey and H Wieczorek ldquoPurification andproperties of a cytosolic V1-ATPaserdquo The Journal of BiologicalChemistry vol 271 no 34 pp 20908ndash20913 1996

[204] AM SmardonM Tarsio and PM Kane ldquoTheRAVE complexis essential for stable assembly of the yeast V-ATpaserdquo TheJournal of Biological Chemistry vol 277 no 16 pp 13831ndash138392002

[205] J S Cohen P M Kane and M F Manolson ldquoPossible roleof phosphorylation in V1-V0 dissociation yeast V-AtpaserdquoMolecular Biology of the Cell vol 6 p 333 1995

[206] O Oehlke C Schlosshardt M Feuerstein and E RoussaldquoAcidosis-induced V-ATPase trafficking in salivary ducts is ini-tiated by cAMPPKACREB pathway via regulation of Rab11bexpressionrdquo The International Journal of Biochemistry amp CellBiology vol 44 pp 1254ndash1265 2012

[207] L A Graham A R Flannery and T H Stevens ldquoStructure andassembly of the yeast V-ATPaserdquo Journal of Bioenergetics andBiomembranes vol 35 pp 301ndash312 2003

[208] S Birman F-MMeunier B Lesbats J-P LeCaer J Rossier andM Israel ldquoA 15 kDa proteolipid found in mediatophore prepa-rations from Torpedo electric organ presents high sequencehomology with the bovine chromaffin granule protonophorerdquoFEBS Letters vol 261 no 2 pp 303ndash306 1990

[209] M J Bayer C Reese S Buhler C Peters and A MayerldquoVacuole membrane fusion V0 functions after trans-SNAREpairing and is coupled to the Ca2+-releasing channelrdquo Journalof Cell Biology vol 162 no 2 pp 211ndash222 2003

[210] C Peters M J Bayer S Buhler J S Andersen M Mann andAMayer ldquoTrans-complex formation by proteolipid channels inthe terminal phase of membrane fusionrdquo Nature vol 409 no6820 pp 581ndash588 2001

[211] P R Hiesinger A Fayyazuddin S Q Mehta et al ldquoThe v-ATPase V0 subunit a1 is required for a late step in synapticvesicle exocytosis in Drosophilardquo Cell vol 121 no 4 pp 607ndash620 2005

[212] T Galli P S McPherson and P De Camilli ldquoThe V0 sector ofthe V-ATPase synaptobrevin and synaptophysin are associatedon synaptic vesicles in a Triton X-100-resistant freeze-thawingsensitive complexrdquoThe Journal of Biological Chemistry vol 271no 4 pp 2193ndash2198 1996

[213] J Di Giovanni S Boudkkazi S Mochida et al ldquoV-ATPasemembrane sector associates with synaptobrevin to modulateneurotransmitter releaserdquo Neuron vol 67 no 2 pp 268ndash2792010

[214] N Morel J-C Dedieu and J-M Philippe ldquoSpecific sortingof the a1 isoform of the V-H+ATPase a subunit to nerveterminals where it associates with both synaptic vesicles and thepresynaptic plasma membranerdquo Journal of Cell Science vol 116no 23 pp 4751ndash4762 2003


[215] I Nakamura N Takahashi N Udagawa et al ldquoLack of vacuolarproton ATPase association with the cytoskeleton in osteoclastsof osteosclerotic (ococ) micerdquo FEBS Letters vol 401 no 2-3pp 207ndash212 1997

[216] B S Lee S L Gluck and L S Holliday ldquoInteraction betweenvacuolar H+-ATPase andmicrofilaments during osteoclast acti-vationrdquoThe Journal of Biological Chemistry vol 274 no 41 pp29164ndash19171 1999


[218] M Carnell T Zech S D Calaminus et al ldquoActin polymer-ization driven by WASH causes V-ATPase retrieval and vesicleneutralization before exocytosisrdquo Journal of Cell Biology vol193 no 5 pp 831ndash839 2011

[219] M Lu L S Holliday L Zhang W A Dunn Jr and SL Gluck ldquoInteraction between aldolase and vacuolar H+-ATPase Evidence for direct coupling of glycolysis to the ATP-hydrolyzing proton pumprdquoThe Journal of Biological Chemistryvol 276 no 32 pp 30407ndash30413 2001

[220] M E CampanellaH ChuN JWandersee et al ldquoCharacteriza-tion of glycolytic enzyme interactions with murine erythrocytemembranes in wild-type and membrane protein knockoutmicerdquo Blood vol 112 no 9 pp 3900ndash3906 2008

[221] M E Campanella H Chu and P S Low ldquoAssembly andregulation of a glycolytic enzyme complex on the humanerythrocyte membranerdquo Proceedings of the National Academy ofSciences of the United States of America vol 102 pp 2402ndash24072005

[222] R E Weber W Voelter A Fago H Echner E Campanellaand P S Low ldquoModulation of red cell glycolysis interactionsbetween vertebrate hemoglobins and cytoplasmic domains ofband 3 red cell membrane proteinsrdquo American Journal ofPhysiologymdashRegulatory Integrative and Comparative Physiologyvol 287 no 2 pp R454ndashR464 2004

[223] J G Donaldson ldquoMultiple roles for Arf6 sorting structuringand signaling at the plasmamembranerdquoThe Journal of BiologicalChemistry vol 278 no 43 pp 41573ndash41576 2003

[224] J K Schweitzer A E Sedgwick and C DrsquoSouza-SchoreyldquoARF6-mediated endocytic recycling impacts cell movementcell division and lipid homeostasisrdquo Seminars in Cell andDevelopmental Biology vol 22 no 1 pp 39ndash47 2011

[225] M Laplante andDM Sabatini ldquoRegulation ofmTORC1 and itsimpact on gene expression at a glancerdquo Journal of Cell Sciencevol 126 pp 1713ndash1719 2013

[226] Y Sancak and D M Sabatini ldquoRag proteins regulate amino-acid-induced mTORC1 signallingrdquo Biochemical Society Trans-actions vol 37 no 1 pp 289ndash290 2009

[227] Y Xu A Parmar E Roux et al ldquoEpidermal growth factor-induced vacuolar (H+)-ATPase assembly a role in signaling viaMtorc1 activationrdquoThe Journal of Biological Chemistry vol 287pp 26409ndash26422 2012

[228] A Bobrie M Colombo G Raposo and C Thery ldquoExo-some secretion molecular mechanisms and roles in immuneresponsesrdquo Traffic vol 12 no 12 pp 1659ndash1668 2011

[229] A J OrsquoLoughlin C A Woffindale and M J Wood ldquoExosomesand the emerging field of exosome-based gene therapyrdquoCurrentGene Therapy vol 12 pp 262ndash274 2012

[230] S Pant H Hilton and M E Burczynski ldquoThe multifacetedexosome biogenesis role in normal and aberrant cellular

function and frontiers for pharmacological and biomarkeropportunitiesrdquo Biochemical Pharmacology vol 83 no 11 pp1484ndash1494 2012

[231] I Kolotuev A Apaydin and M Labouesse ldquoSecretion ofhedgehog-related peptides and WNT during Caenorhabditiselegans developmentrdquo Traffic vol 10 no 7 pp 803ndash810 2009

[232] S R Sennoune D Luo and R Martinez-Zaguilan ldquoPlas-malemmal vacuolar-type H+-ATPase in cancer biologyrdquo CellBiochemistry and Biophysics vol 40 pp 185ndash206 2004

[233] S R Sennoune K Bakunts GMMartınez et al ldquoVacuolarH+-ATPase in human breast cancer cells with distinct metastaticpotential distribution and functional activityrdquo American Jour-nal of Physiologymdash Cell Physiology vol 286 no 6 pp C1443ndashC1452 2004

[234] E J Bowman and B J Bowman ldquoV-ATPases as drug targetsrdquoJournal of Bioenergetics and Biomembranes vol 37 pp 431ndash4352005

[235] W Castillo-Avila M Abal S Robine and R Perez-TomasldquoNon-apoptotic concentrations of prodigiosin (H+Clminus sym-porter) inhibit the acidification of lysosomes and induce cellcycle blockage in colon cancer cellsrdquo Life Sciences vol 78 pp121ndash127 2005

[236] A DeMilito E Iessi M Logozzi et al ldquoProton pump inhibitorsinduce apoptosis of human B-cell tumors through a caspase-independent mechanism involving reactive oxygen speciesrdquoCancer Research vol 67 no 11 pp 5408ndash5417 2007

[237] J H Lim J W Park M S Kim S K Park R S Johnson and YS Chun ldquoBafilomycin induces the p21-mediated growth inhi-bition of cancer cells under hypoxic conditions by expressinghypoxia-inducible factor-1120572rdquo Molecular Pharmacology vol 70pp 1856ndash1865 2006

[238] K Niikura ldquoEffect of a V-ATPase inhibitor FR202126 in syn-geneic mouse model of experimental bone metastasisrdquo CancerChemotherapy and Pharmacology vol 60 pp 555ndash562 2007

[239] M Perez-Sayans J M Somoza-Martın F Barros-AngueiraP G Diz J M G Rey and A Garcıa-Garcıa ldquoMultidrugresistance in oral squamous cell carcinoma the role of vacuolarATPasesrdquo Cancer Letters vol 295 no 2 pp 135ndash143 2010

[240] W Shen X Zou M Chen et al ldquoEffects of diphyllin as a novelV-ATPase inhibitor on gastric adenocarcinomardquoEuropean Jour-nal of Pharmacology vol 667 no 1ndash3 pp 330ndash338 2011

[241] H You J Jin H Shu et al ldquoSmall interfering RNA targetingthe subunit ATP6L of proton pump V-ATPase overcomeschemoresistance of breast cancer cellsrdquo Cancer Letters vol 280no 1 pp 110ndash119 2009

[242] L S Holliday ldquoEditorial [hot topic vacuolar H+-ATPasetargeting a ldquoHousekeepingrdquo enzyme for drug development(Guest Editor L ShannonHolliday)rdquoCurrent Proteinamp PeptideScience vol 13 pp 105ndash106 2012

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


ATPADP

VGLUT

Glutamate release

Clathrin-mediatedendocytosis

Secretoryvesicles

Endocytosis

Coated vesicle

Sorting endosome

Recycling vesicle

Recyclingendosome

MultivesicularbodyLysosome

Late endosome

rec return to GolgiMannose-6-phospahate

Mannose-6-phospahate recwith bound cathepsinto late endosome

ATPADP

ATP

ADP

ATPADP

ATPADP

ATPADP

Role of Vsecretory

-ATPase in vesicle fusion

GlutamateH+

H+

Figure 1 Roles of V-ATPases in cell physiology V-ATPases have vital roles in theGolgi apparatusMutations in the a2-subunit which is foundin the Golgi result in a form of cutis laxa due to impaired glycosylation In secretory pathways energy from a proton gradient generated byV-ATPases may be used to load molecules like glutamate V-ATPases in endosomal compartments are involved in disassociating complexeslike the mannose-6 phosphate receptor from cathepsins Acid-dependent enzymes may also become active due to V-ATPase acidification tomodify proteins in the lumen of the vesiclesThere is also increasing evidence that the V-ATPase may be able to recruit regulators of vesiculartrafficking like ARF6 V-ATPases in the lysosome provide an acidic environment for the degradation of proteins by acidic proteases

by mTORC1 requires ATPase activity by the V-ATPase [8ndash11] This central nutrient sensing apparatus is a vital nodein physiological signaling but also represents a potentialldquoAchilles heelrdquo of cancer cells which may be made vulnerableas a more sophisticated understanding of the role of theV-ATPase in the process emerges [9]

Given the importance of acidification it is not surprisingthat most eukaryotic cells cannot survive if V-ATPase activityis lost either due to the administration of a selective inhibitoror molecular genetic loss of a ubiquitous subunit [12ndash14]An important exception is the yeast Saccharomyces cerevisiaewhich is able to take up protons from acidic media tocompensate for lack of V-ATPase activity V-ATPase-deficientyeast thrive in media at pH 5 but fail to grow in alkalinemedia This allows knockouts and replacement knockouts tobe performed to analyze functions of specific subdomains ofV-ATPase subunits [15] Yeast has become one of the mostpowerful model systems for studying the V-ATPase [16] Thefact that the V-ATPase is highly conserved also makes theyeast V-ATPase a semiuniversal model for understanding thebasic mechanisms of the pump

2 Structure and EnzymaticFunction of V-ATPases

The mammalian V-ATPase is composed of 13 subunits thatcan be divided into 8 peripheral proteins which is also called

the V1 and 5 membrane intrinsic proteins are called V0[17 18] (Figure 2) The V-ATPase is referred to formally asATP6 [19] V1 subunits are ATP6V1A (A-subunit) ATP6V1B(B-subunit) ATP6V1C (C-subunit) ATP6V1D (D-subunit)ATP6V1E (E-subunit) ATP6V1F (F-subunit) ATP6V1G (G-subunit) and ATP6V1H (H-subunit) The V0 domains areATP6V0a (a-subunit) ATP6V0b (crdquo-subunit) ATP6V0c (c-subunit) ATP6V0d (d-subunit) and ATP6V0e (e-subunit)As indicated in Figure 2 many of the subunits have isoformsIn higher organisms there are ubiquitous isoforms andisoforms that are selectively expressed in specific cell typesThese are associated with subsets of V-ATPases that per-form specialized functions However specialized V-ATPasesrepresent a mixture of cell type selective isoforms andubiquitous isoforms Indeed some specialized functions maybe carried out by V-ATPases that are indistinguishable fromthe ubiquitous enzyme

As an example in mammals there are 4 isoforms of the a-subunit ATP6V0a1 (a1) and ATP6V0a2 (a2) are ubiquitouslyexpressed and are associated with subsets of housekeepingV-ATPases ATP6V0a3 (also referred to as TCIRG1 Atp6iTIRC7 and a3) is found at high levels in osteoclasts [20]microglia [21] gastric parietal cells [22] pancreatic beta cells[23] and perhaps a few other cell types and associates with aspecialized subset of V-ATPases that perform nonhousekeep-ing functions Osteoclasts for instance express a1 a2 anda3 [24] The first two are thought to perform housekeeping


d

c

ddd

c

A

D

c

B

F

EG

a

e

ATP

ADP-Pi

ADP

Cytoplasm

H

Lumenextracellular

dd

Vacuolar H+-ATPase

V1

V0c998400998400

H+

H+

C

Isoforms



C1-ubiquitous

C2a-lung

C2b-kidney

E1-acrosome

E2-ubiquitous

G1-ubiquitous


G3-kidney

a1-endomembranes

a2-endomembranes



d1-ubiquitous


















B2

B1

Yeast B

Pyrococcus B




B2

B1







ATP6V1C1

(a)

ATP6V1C2

(b)



















(a)



Actin ring

(b)






































1were


1










c cc

d

A

D

B

C

F

H

G

a

e c998400998400

(a)

c c

d

c

F

D

A B G

E

ae c998400998400

C

H

(b)



50of 12 120583M [40]


50in








c c

d

A

D

BG

E

a

c998400998400c

dda FdC

e

H

(a)

c

d

A

D

BG

F

E

a

c998400998400

dC

e

H

(b)



Signal

c

A

D

B G

E

c998400998400

FC

c

a

c

A

D

B G

HE

c998400998400

FC

e cea

H



















A B C D E









10 Summary





References






































ℎ119890119886119889


























































































10158401015840










































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


d

c

ddd

c

A

D

c

B

F

EG

a

e

ATP

ADP-Pi

ADP

Cytoplasm

H

Lumenextracellular

dd

Vacuolar H+-ATPase

V1

V0c998400998400

H+

H+

C

Isoforms



C1-ubiquitous

C2a-lung

C2b-kidney

E1-acrosome

E2-ubiquitous

G1-ubiquitous


G3-kidney

a1-endomembranes

a2-endomembranes



d1-ubiquitous


















B2

B1

Yeast B

Pyrococcus B




B2

B1







ATP6V1C1

(a)

ATP6V1C2

(b)



















(a)



Actin ring

(b)






































1were


1










c cc

d

A

D

B

C

F

H

G

a

e c998400998400

(a)

c c

d

c

F

D

A B G

E

ae c998400998400

C

H

(b)



50of 12 120583M [40]


50in








c c

d

A

D

BG

E

a

c998400998400c

dda FdC

e

H

(a)

c

d

A

D

BG

F

E

a

c998400998400

dC

e

H

(b)



Signal

c

A

D

B G

E

c998400998400

FC

c

a

c

A

D

B G

HE

c998400998400

FC

e cea

H



















A B C D E









10 Summary





References






































ℎ119890119886119889


























































































10158401015840










































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








B2

B1

Yeast B

Pyrococcus B




B2

B1







ATP6V1C1

(a)

ATP6V1C2

(b)



















(a)



Actin ring

(b)






































1were


1










c cc

d

A

D

B

C

F

H

G

a

e c998400998400

(a)

c c

d

c

F

D

A B G

E

ae c998400998400

C

H

(b)



50of 12 120583M [40]


50in








c c

d

A

D

BG

E

a

c998400998400c

dda FdC

e

H

(a)

c

d

A

D

BG

F

E

a

c998400998400

dC

e

H

(b)



Signal

c

A

D

B G

E

c998400998400

FC

c

a

c

A

D

B G

HE

c998400998400

FC

e cea

H



















A B C D E









10 Summary





References






































ℎ119890119886119889


























































































10158401015840










































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


ATP6V1C1

(a)

ATP6V1C2

(b)



















(a)



Actin ring

(b)






































1were


1










c cc

d

A

D

B

C

F

H

G

a

e c998400998400

(a)

c c

d

c

F

D

A B G

E

ae c998400998400

C

H

(b)



50of 12 120583M [40]


50in








c c

d

A

D

BG

E

a

c998400998400c

dda FdC

e

H

(a)

c

d

A

D

BG

F

E

a

c998400998400

dC

e

H

(b)



Signal

c

A

D

B G

E

c998400998400

FC

c

a

c

A

D

B G

HE

c998400998400

FC

e cea

H



















A B C D E









10 Summary





References






































ℎ119890119886119889


























































































10158401015840










































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









(a)



Actin ring

(b)






































1were


1










c cc

d

A

D

B

C

F

H

G

a

e c998400998400

(a)

c c

d

c

F

D

A B G

E

ae c998400998400

C

H

(b)



50of 12 120583M [40]


50in








c c

d

A

D

BG

E

a

c998400998400c

dda FdC

e

H

(a)

c

d

A

D

BG

F

E

a

c998400998400

dC

e

H

(b)



Signal

c

A

D

B G

E

c998400998400

FC

c

a

c

A

D

B G

HE

c998400998400

FC

e cea

H



















A B C D E









10 Summary





References






































ℎ119890119886119889


























































































10158401015840










































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



































1were


1










c cc

d

A

D

B

C

F

H

G

a

e c998400998400

(a)

c c

d

c

F

D

A B G

E

ae c998400998400

C

H

(b)



50of 12 120583M [40]


50in








c c

d

A

D

BG

E

a

c998400998400c

dda FdC

e

H

(a)

c

d

A

D

BG

F

E

a

c998400998400

dC

e

H

(b)



Signal

c

A

D

B G

E

c998400998400

FC

c

a

c

A

D

B G

HE

c998400998400

FC

e cea

H



















A B C D E









10 Summary





References






































ℎ119890119886119889


























































































10158401015840










































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




















1were


1










c cc

d

A

D

B

C

F

H

G

a

e c998400998400

(a)

c c

d

c

F

D

A B G

E

ae c998400998400

C

H

(b)



50of 12 120583M [40]


50in








c c

d

A

D

BG

E

a

c998400998400c

dda FdC

e

H

(a)

c

d

A

D

BG

F

E

a

c998400998400

dC

e

H

(b)



Signal

c

A

D

B G

E

c998400998400

FC

c

a

c

A

D

B G

HE

c998400998400

FC

e cea

H



















A B C D E









10 Summary





References






































ℎ119890119886119889


























































































10158401015840










































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








1were


1










c cc

d

A

D

B

C

F

H

G

a

e c998400998400

(a)

c c

d

c

F

D

A B G

E

ae c998400998400

C

H

(b)



50of 12 120583M [40]


50in








c c

d

A

D

BG

E

a

c998400998400c

dda FdC

e

H

(a)

c

d

A

D

BG

F

E

a

c998400998400

dC

e

H

(b)



Signal

c

A

D

B G

E

c998400998400

FC

c

a

c

A

D

B G

HE

c998400998400

FC

e cea

H



















A B C D E









10 Summary





References






































ℎ119890119886119889


























































































10158401015840










































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


c cc

d

A

D

B

C

F

H

G

a

e c998400998400

(a)

c c

d

c

F

D

A B G

E

ae c998400998400

C

H

(b)



50of 12 120583M [40]


50in








c c

d

A

D

BG

E

a

c998400998400c

dda FdC

e

H

(a)

c

d

A

D

BG

F

E

a

c998400998400

dC

e

H

(b)



Signal

c

A

D

B G

E

c998400998400

FC

c

a

c

A

D

B G

HE

c998400998400

FC

e cea

H



















A B C D E









10 Summary





References






































ℎ119890119886119889


























































































10158401015840










































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


c c

d

A

D

BG

E

a

c998400998400c

dda FdC

e

H

(a)

c

d

A

D

BG

F

E

a

c998400998400

dC

e

H

(b)



Signal

c

A

D

B G

E

c998400998400

FC

c

a

c

A

D

B G

HE

c998400998400

FC

e cea

H



















A B C D E









10 Summary





References






































ℎ119890119886119889


























































































10158401015840










































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology














A B C D E









10 Summary





References






































ℎ119890119886119889


























































































10158401015840










































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


A B C D E









10 Summary





References






































ℎ119890119886119889


























































































10158401015840










































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




References






































ℎ119890119886119889


























































































10158401015840










































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








ℎ119890119886119889


























































































10158401015840










































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
































































10158401015840










































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology































10158401015840










































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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