Autophagic vacuoles rapidly fuse with pre-existing lysosomes in cultured
hepatocytes
B. PAIGE LAWRENCE and WILLIAM J. BROWN*
Department of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, NY 14853, USA
*Author for correspondence
Summary
Autophagic vacuoles (AVs) arise when membranes of theER sequester parts of the cytoplasm, forming a new,double-membraned vacuole, to which lysosomal en-zymes are then delivered. To investigate the mechanismof lysosomal enzyme delivery to nascent AVs, amino acid(AA) starvation and glucagon treatment were used toinduce autophagy in a cultured cell system using rathepatocytes (Fu5C8 cells). The induction of autophagywas assayed using biochemical, morphometric andimmunocytochemical techniques. In these cells, AAstarvation resulted in a fivefold increase in total cellularproteolysis, and sixfold and 4.5-fold increases in thevolume and surface densities of AVs, respectively. Usingan antibody against the mannose 6-phosphate receptor(MPR) and two sizes of colloidal gold to label separatelyand track the endosomal and lysosomal compartments,the time course of endosomal and lysosomal fusion with
AVs was analyzed in detail. On the basis of theseexperiments, we found that AVs rapidly fuse with pre-existing lysosomes, but seldom with a prelysosomalcompartment (PLC). Using immunoperoxidase, stainingfor the MPR was infrequently observed in associationwith any AVs. However, at early times following theinduction of autophagy (<2 h), many autophagicvacuoles stained positively for the lysosomal enzymecathepsin D. Consistent with these results, treatment ofcells with tunicamycin had no effect on autophagy-induced proteolysis. We conclude that lysosomal enzymedelivery to nascent AVs occurs primarily by the fusion ofpre-existing mature lysosomes, with a much smallercontribution by MPRs or the PLC.
Classical autophagy is the fascinating process by whichcells sequester and degrade parts of their own cyto-plasm, including organelles (for reviews, see Amentaand Brocher, 1981; Glaumann et al., 1981; Holtzman,1989; Marzella and Glaumann, 1987). Through thisprocess a unique type of degradative organelle isformed, called an autophagic vacuole. Autophagy isbelieved to occur in all eukaryotic cells at a low, basallevel, where it is presumably involved in the degra-dation of cellular proteins, and the normal turnover ofunnecessary or dysfunctional organelles. It also plays arole in embryonic programmed cell death and musclecell necrosis during certain types of muscular dystrophy(Glaumann et al., 1981; Kalimo et al., 1988). Auto-phagy can be stimulated above this basal level by avariety of conditions, including: an increase in theconcentration of glucagon, insulin withdrawal, aminoacid starvation, removal of barbiturates like phenobar-bitol, and treatment with microtubule-depolymerizingdrugs (Arstila et al., 1974; Ericsson, 1969; Marzella and
Glaumann, 1980b; Masaki et al., 1987; Mortimore andSchworer, 1977; Pfeifer et al., 1978).
Autophagic vacuoles (AVs) form through a peculiartwo-stage process. First, parts of the cell are seques-tered by membranes of the endoplasmic reticulum(ER). Second, lysosomal enzymes are somehow de-livered to this novel compartment. The organelleformed by the first step is called a nascent AV, orautophagosome (AVj), and is unlike any other cellularcompartment. By definition AViS contain intact andrecognizable parts of the cell: mitochondria, smallvesicles and tubules, and masses of cytoplasm, includ-ing ribosomes and sometimes even cytoskeletal struc-tures (Amenta and Brocher, 1981; Glaumann et al.,1981; Holtzman, 1989). As a consequence of thedouble-membrane ER wrapping around portions ofcytoplasm during the engulfment process, AV;S aresurrounded by more than one membrane, quite often asmany as four. After the formation of nascent AVs,lysosomal enzymes are rapidly delivered, forming adegradative AV, or autophagolysosome (AVd). Unliketheir predecessors, AVds have only a single limiting
516 B. P. Lawrence and W. J. Brown
membrane, so somehow the extra membranes arereadily hydrolyzed. The half-life of AVs has beenestimated to be about 9 min, indicating that theircontents are quickly degraded (Glaumann et al., 1981;Pfeiferet al., 1978).
Using immunocytochemical methods and a variety ofantibodies, several investigators have convincinglydemonstrated that AV membranes are derived from ER(Dunn, 1990a; Furuno et al., 1990; Ueno et al., 1991).In contrast, the precise intracellular sources from whichlysosomal enzymes are delivered to AVs has not beenclearly determined. There are several conceivableroutes through which lysosomal enzyme delivery mightoccur. They could be targeted to AVs by mannose 6-phosphate residues and mannose 6-phosphate receptors(MPR), just as they are during the formation ofheterophagic lysosomes (Brown et al., 1986; Kornfeldand Mellman, 1989). Alternatively, newly synthesizedlysosomal enzymes could be delivered by some un-known receptor/pathway. Two other possible means ofenzyme delivery involve the direct fusion of AVs withcomponents of the endosomal system, principally theMPR-enriched prelysosomal compartment (PLC)(Brown et al., 1986; Griffiths et al., 1988), or with pre-existing mature lysosomes. There have been numerousattempts to resolve this issue, with no definitive answer.Evidence from work done by Ericsson (1969) and Lee etal..(1989) indicates that AVjS fuse readily with pre-existing lysosomes. However, Tooze et al. (1990) andGordon and Seglen (1988) have reported that AV,smerge with a prelysosomal (endocytic) compartment.These differing results have led to uncertainty as towhich is the principal source of lysosomal enzymes.One potential shortcoming of these studies is that eachwas concerned independently with the delivery oflysosomal and endosomal tracers to AVs. Thus, while itis possible to find markers from both endocytic andlysosomal compartments associated with AVs, therelative contributions of both compartments to theconversion of an AVj into an AVd was not simul-taneously determined. .
In this study, we have measured the relativecontributions of both the endosomal and lysosomalcompartments to the delivery of material to newlyformed AVs. For this purpose, we developed a systemfor studying autophagy in a non-transformed, culturedrat hepatocyte cell line, Fu5C8, using either amino acid(AA) starvation alone or in combination with thehormone glucagon to stimulate autophagy. The associ-ation of endosomal and lysosomal compartments withAVs was analysed both qualitatively and quantitativelyusing compartments that were separately labeled withdifferent sizes of colloidal gold, and the possibleinvolvement of the MPR in the delivery of enzymes toAVs was investigated using immunocytochemical andbiochemical methods. Our results are consistent withthe rapid and direct association of pre-existing lyso-somes with AVs, rather than a requirement for newenzymes and subsequent targeting, or the significantinvolvement, of the PLC.
Materials and methods
MaterialsL-[2,3,4,5-3H]leucine at —110 Ci/mmol and Ecolume scintil-lation counting solution were purchased from ICN Radio-chemicals, Inc. (Costa Mesa, CA). Fluorescent antibodyreagents were from Organon Teknika-Cappel (Malvern, PA),and Fab fragments of sheep anti-rabbit IgG conjugated withhorseradish peroxidase (HRP) were from BioSys S.A.(Compiegne, France). Tetrachloroauric (III) acid, diamino-benzidine hydrochloride (Type II), saponin, glucagon and cellculture media were from Sigma Chemical Co. (St. Louis,MO). Leupeptin (hydrogen sulfate) was from BoehringerMannheim (Indianapolis, IN). Fetal bovine serum and MEMvitamin supplement were from Gibco Laboratories (GrandIsland, NY). Spurr's resin and other reagents for electronmicroscopy were obtained from Electron Microscopy Sci-ences (Fort Washington, PA).
Cell cultureFu5C8 cells were grown in minimal essential medium (MEM)supplemented with 5% fetal bovine serum (FBS), at 37°C inan atmosphere of 95% air and 5% CO2. All incubations wereconducted under these conditions. To induce autophagy byA A starvation, the cells were washed three times with Earle'sbalanced salts solution, containing vitamins (EBSS), and thenincubated in EBSS for various lengths of time. For otherexperiments, autophagy was induced with EBSS containing86 nM glucagon (Dunn, 1990a).
Electron microscopyFollowing various experimental conditions, cultured hepato-cytes were fixed with 1% paraformaldehyde, 3% glutaralde-hyde, 0.1 M sodium cacodylate, pH 7.4, 2 [M CaCl2, for 1.5 hat 25°C. After washing with 0.1 M sodium cacodylate, pH 7.4,the cells were post-fixed with 1% OsO4 in 0.1 M sodiumcacodylate, pH 7.4, for 2 h, at 4°C. Osmicated samples wererinsed with 0.1 M sodium maleate buffer, pH 5.15, and thenen bloc stained with 1% uranyl acetate, in 0.1 M sodiummaleate buffer, pH 6.0, for 1 h at 25°C. After rinsing with 0.1M sodium maleate buffer, pH 5.15, the samples weredehydrated in graded ethanol solutions, removed from plasticculture dishes with propylene oxide, and embeded in Spurr'sresin. The samples were thin sectioned, stained with leadcitrate and uranyl acetate, and examined using a Philips 301electron microscope.
Immunoperoxidase cytochemistry was carried out as de-scribed by Brown and Farquhar (1984), except that 0.01%saponin was used in all solutions containing detergent.Polyclonal antibodies against the cation-independent MPRand the lysosomal enzyme cathepsin D were used at 1:100dilutions, and have been previously described (Brown andFarquhar, 1987; Brown et al., 1986; Park et al., 1991a). Thehorseradish peroxidase-conjugated sheep anti-rabbit Fabantibody was used at a 1:500 dilution.
Colloidal gold of two different sizes (8 and 25 nm) wasprepared separately by methods outlined by Handley (1989),and stabilized both with BSA in 0.005 M NaCl, pH 7.4 (withK2CO3), and with 0.2% PEG (8000). Prior to each exper-iment, the uniformity and size of each preparation weredetermined by electron microscopy.
Morphometric analysis of autophagy in culturedFu5C8 hepatocytesA morphometric analysis was conducted using techniquesdescribed by Weibel (1979). To induce autophagy, the cells
Autophagic vacuoles rapidly fuse with lysosomes 517
were AA starved from 0 to 72 h prior to fixation andprocessing for electron microscopy, as described above. Foreach time point, 40 micrographs were randomly taken atxl,800 and xl8,000 magnifications. The negatives weremagnified three times and projected onto a double lattice testsystem (C100) for point counting. The volume density (Vv)and surface density (5V) of the following organelles wereevaluated: autophagosomes (AV|), lysosomes, endosomesand multivesicular bodies, mitochondria, Golgi complex andsmooth and rough endoplasmic reticulum (ER). The Vv andSv of the cytoplasm and nuclei were also determined. Strictmorphological criteria were used to define each compartment.Specifically, only organelles bounded by more than onelimiting membrane, and which contained recognizable partsof the cell, were included in the AV| category. The lysosomalcompartment included all single membrane-bounded degra-dative organelles, including autophagolysosomes (AVd). Theendosomal category included all non-coated vesicles, tubularelements, multilamellar bodies and multivesicular bodies.Owing to the test system used, only the surface density for theER was determined. Originally, the smooth and the rough ERwere evaluated separately, but were combined because it wasoften difficult to distinguish between the two on a projectedmicrograph. All of the volume and surface densities wereexpressed as a percentage of the total cytoplasmic volume orsurface density. Comparisons were made between the averagevalues from the micrographs taken of each different sample.The standard deviation and standard error were calculated bythe method of Cochran (1953), and the differences betweenunstarved and starved cells were evaluated by Student's f-test(PssO.05).
Biochemical assay for autophagic protein degradationIt has been previously established that differences in intra-cellular proteolysis can be measured by monitoring changes inradiolabeled amino acids and proteins released into theculture medium (Ballard, 1987; Berger and Dice, 1987).Trichloroacetic acid (TCA) precipitates proteins but not freeamino acids, so it is a convenient tool for separating labeledproteins from amino acids, providing a simple, reproducible,biochemical assay for changes in intracellular protein degra-dation. To measure the total cellular proteolysis, cells werelabeled to isotopic steady-state (>16 h) with 0.5 jiCi/ml L-[2,3,4,5-3H]leucine in MEM, containing 5% FBS. Followingextensive washing with EBSS, containing a 10-fold excess ofunlabeled leucine (10 x Leu), samples were incubated at 37°Cfor various lengths of time in either MEM, containing 5%FBS and 10 x Leu, or in EBSS, containing 10 x Leu. Theculture medium was collected and clarified by centrifugation,to remove any dead cells or debris. An equal volume of ice-cold 10% TCA was added to half of the medium. After a 5=1 hincubation at 4°C, the samples were microcentrifuged for 10min to pellet all of the TCA-insoluble material. Followingremoval of the supernatant, the pellets were dissolved in 20 ,ulof 1 M NaOH. Both the TCA-soluble and TCA-insolubleradioactivity were determined using a scintillation counter.These data were expressed as the average TCA-soluble/TCA-insoluble radioactivity for each time point.
Results
The induction of autophagy in cultured Fu5C8hepatocytesAA starvation alone or in combination with glucagonstimulated the formation of autophagic vacuoles (AV)
Fig. 1. Autophagic vacuoles in AA-starved cultured Fu5C8hepatocytes. This electron micrograph illustrates the typicalmorphology of AVs before (AVi; nascent AV) and after(AVd, or degradative AV) the delivery of lysosomalenzymes. Autophagy was stimulated by switching the cellsfrom MEM, with 5% FBS to EBSS, followed byincubation at 37°C for 12 h. Bar, 0.50 }xra.
in Fu5C8 cells, as monitored both biochemically andmorphologically. The electron micrograph in Fig. 1shows two representative AVs from an AA-starvedFu5C8 cell, which clearly illustrates the distinct mor-phological differences between an AVj and an AVd.The AVj is surrounded by multiple membranes andcontains clearly discernable cytoplasmic contents,whereas the AVd is surrounded by only one membraneand contains partially disrupted organelles. Stimulationof intracellular proteolysis during autophagy was deter-mined by labeling cells to isotopic steady-state with[3H]leucine and measuring the release of TCA-solubleradioactivity into the medium. Upon the induction ofautophagy, either by AA starvation alone or incombination with glucagon, there was a fivefoldincrease in protein degradation (Fig. 2). Morphologicaland biochemical findings support the conclusion thatthis increased proteolysis is due to autophagy, not someother mechanism (Ballard, 1987; Berger and Dice,1986; Marzella and Glaumann, 1980a). Our obser-vations also showed that when autophagy was inducedby A A starvation plus glucagon, many AVs were seenas early as 15 min, whereas a similar number of AVs wasnot observed until after at least 2 h of AA starvationalone. Differences in cell viability between fed andstarved cells were negligible, as measured by trypanblue exclusion, the release of the soluble cytoplasmicprotein lactate dehydrogenase, and Lowry proteinassays (Lowry et al., 1951), indicating that changes inthe release of amino acids were not due to cell death(data not shown).
Morphometric analysis of the formation of AVs incultured hepatocytesA more specific and quantitative assay for the induction
518 B. P. Lawrence and W. J. Brown
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Fig. 2. The stimulation of autophagy resulted in a fivefoldincrease in total cellular proteolysis. Cells were labeled toisotopic steady-state with [3H]leucine prior to the inductionof autophagy. Changes in the amount of degradation weredetermined by measuring the release of TCA-solubleradioactivity into the culture media. To correct for thefinding that fed cells continued their normal secretion,while starved cells did not, the data were plotted as theratio of TCA-soluble/TCA-insoluble radioactivity.Autophagy was induced at time=0 h. (A) Proteolysismeasured over a 48 h period in AA-starved and fed cells.(B) Proteolysis measured over a 12 h period in AA-starvedand fed cells in the presence or absence of 86 nMglucagon. The values plotted are averages of triplicatesamples. Fed cells ( • • ) ; AA-starved cells (O O);fed cells plus 86 nM glucagon ( • #) ; and, AA-starvedcells plus 86 nM glucagon (A A).
of autophagy was conducted, using morphometricmethods, to measure changes in the volume and surfacedensities occupied by AVs over a 72 h period (Fig. 3).Following 6 h of AA starvation, a fivefold increase inthe volume density (Vv) and surface density (Sv) of theAV, compartment was observed. This appeared to be
20 40 60 80
Time (h)
B
0 20 40 60Time (h)
Fig. 3. AA starvation resulted in a fivefold increase innascent AVs. A morphometric analysis of AA-starvedcultured Fu5C8 cells was conducted. Autophagy wasinduced at time=0 h. The data for the nascent AVs areexpressed as a percentage of the total cytoplasmic volumedensity or surface density; 40 micrographs were countedfor each sample. The error bars indicate the standarddeviation of each sample, compared to the control(unstarved) cells. (A) Volume density (Vv); (B) surfacedensity (5V).
the peak of AV formation, and these values slowlydecreased to about threefold higher than the control by24 h. By 48 h the volume density was back to the basallevel, but the surface density was still about 50%higher. The morphometric data correspond remarkablywell with the biochemical data, in terms of the peakappearance of AVs and the peak in proteolysis(compare Figs 2A and 3).
Compared to the large increase in AVs, the changesin the volume and surface densities of other organelleswere less dramatic (Table 1). Upon stimulation ofautophagy, the Sv of the ER decreased slightly, to about90% of the control surface density. Although there wasno change in either the Vv or Sv of lysosomes, theendosomal Vv and Sv decreased to about half of the
Autophagic vacuoles rapidly fuse with lysosomes 519
Table 1. Summary of morphometric analysis of AA-starved cultured Fu5C8 hepatocytes
% control
Table 2. Intracellular distribution of Aus
% of organelles with Aug
AVj
GC
END
LYS
MITO
ER
Svvvsvv.SvVvSv
SvSv
6h
5105101631155766110*100*80102*94
12 h
310270127124455496*859410997
24 h
320240123114394594:
819511598
Values are expressed as a percentage of the level in unstarved,control cells. Forty micrographs were counted for each sample.
AV|, nascent AV; GC, Golgi complex; END, endosomalcompartments; LYS, lysosomes, including AVds; MITO,mitochondria; and ER, smooth and rough ER.
*Variation was not statistically significant, compared to thecontrol.
basal level, the mitochondrial Vv decreased to about90%, and their Sv rose to 115% of the level in unstarvedcells. Both the Vv and Sv of the Golgi complex increasedslightly, to about 120% of the level in unstarved cells.The Vv and Sv of the cells and nuclei were unaffected byautophagy (data not shown).
Pre-existing lysosomes rapidly associate with nascentAVsTo investigate the role of lysosomes and the PLC duringthe formation of AVs, we examined the delivery ofendosomal and lysosomal markers to AVs at the onsetof autophagy. Based upon the work of many labs, theconditions necessary to specifically label temporallydiscrete compartments of the endosome/lysosome path-way have been established (Brown et al., 1986; Griffithset al., 1988; Kornfeld and Mellman, 1989; Park et al.,1991a). By endocytic uptake, lysosomes were labeledwith 8 nm colloidal gold (Au8) and endosomes,including the PLC, were labeled with 25 nm colloidalgold (Au25). Prior to using this method for studyingautophagy, it was necessary to verify that the Au8particles were located primarily in pre-existing lyso-somes. Previous work has shown that mature lysosomesare devoid of MPRs (Brown et al., 1986; Griffiths et al.,1988; Kornfeld and Mellman, 1989; Sahagian andNeufeld, 1983), so the distribution of endocytosed Au8in MPR-positive (MPR+) and MPR-negative (MPR")compartments was determined after various pulse-chase conditions. Cells were incubated for variouslengths of time with Au8, rinsed extensively, andincubated without Au8 to chase the internalized goldparticles into lysosomes. The cells were fixed andprocessed for indirect immunoperoxidase cyto-chemistry using an antibody against the MPR. The Au8particles were observed within morphologically identifi-able lysosomes and prelysosomes, and were very rarelyfound in small vesicular structures or on the cell surface
Length ofpulse (h)
No. ofmicrographs
countedMPR"
(lysosomes)MPR+
(PLC)
0.512
16
32323264
91(48)84(49)85 (50)95 (229)
9(5)16(9)15(9)5(12)
To label lysosomes with Au8, Fu5C8 cells were incubated at37°C in MEM, containing 5% FBS and Au8 for 0.5 to 16 h. Aftergold uptake, the cells were rinsed extensively and incubated inmedium without Aus for 2 h prior to fixation. Micrographs weretaken at a primary magnification of x22,000, and the numbers oforganelles that contained Au8 or MPR staining were counted.Organelles that contained Au8 but lacked MPR were defined aslysosomes, and those that contained both Au8 and MPR weredefined as PLC. The values in parenthesis are the number oforganelles counted. MPR immunoperoxidase reaction product inthe ER, Golgi complex, coated vesicles and coated pits was notcounted.
(see Fig. 4). Quantitation of these observations (Table2) indicates that after both long and short incubations inAu8, 85-95% of the gold particles were in lysosomes(i.e. Au8
+/MPR~), and the other 5-15% of the goldparticles were in the PLC (i.e. Au8
+/MPR+).Since the 0.5 and 16 h pulse times led to the highest
percentage of Au8 being delivered to mature lysosomesin the control experiments (i.e. > 90% found in MPR~organelles), both of these conditions were used to labellysosomes before autophagy was stimulated. Auto-phagy was induced simultaneously with continuousexposure to Au2s, in the presence of 0.5 mM leupeptin,which was added to prolong the half-life of AVds(Amenta and Brocher, 1981; Kovacs et al., 1982;Marzella and Glaumann, 1987). The cells were fixedand processed for immunoperoxidase cytochemistryusing an antibody against the MPR. AVs that containedthe lysosomal marker (Au8
+) were considered to havefused with a pre-existing lysosome, and AVs thatcontained either Au25 or MPR staining were deemed tohave fused with endosomes or the PLC. After 5 and 15min of autophagy induction, many AVds were found tocontain Au8, the lysosomal marker; however, Au25 andMPR reaction product were rarely observed withinAViS or AVds (Fig. 4A-D). At later times (1-2 h), mostof the AVds were still only labeled with the lysosomallabel (Fig. 4E and F). Some Au25 was observed inAVds, as expected when an endocytic tracer progressesthrough the endosomal and lysosomal systems (Fig. 41);however, even after 1-2 h of autophagy, MPR stainingof AVds was infrequent. Occasionally, the Au25 andMPR labeling were present inside AVjS becauseendosomes were autophagocytosed (Fig. 4G and H).Au25 particles were found in endocytic vesicles through-out the cytoplasm, and abundant MPR labeling wasfound in the PLC, Golgi cisternae and Golgi-associatedvesicles, the TGN, coated pits and vesicles and ERmembranes, similar to its distribution in other cells(Brown and Farquhar, 1984, 1987; Brown et al., 1986;Griffiths et al., 1988; Kornfeld and Mellman, 1989).
520 B. P. Lawrence and W. J. Brown
Fig. 4. A lysosomal marker, Au8, is rapidly delivered toAVs. To label lysosomes, cells were incubated for 30 minin MEM, containing 5% FBS and Au8, followed by a 2 hincubation in the same medium, except without Au8. Toinduce autophagy, cells were incubated in EBSS,containing 86 nM glucagon and 0.5 mM leupeptin and Au25
for 0 to 60 min The cells were fixed and processed forimmunoperoxidase cytochemistry, using a polyclonalantibody against the MPR. (A) 0 min, (B-D) 5 min, (G)15 min and (E,F,I) 60 min after the stimulation ofautophagy, in the continuous presence of Au25.(A) Distribution of the lysosomal marker Au8 (arrowheads)and MPR staining in fed cells (control). Note that most ofthe Au8 is in organelles that are MPR", although MPRreaction product is visible in Golgi cisternae (GC) andvesicles throughout the cell. As early as 5 min afterstimulating autophagy, both AVjS and AVds were observed,and many contained lysosomal marker Au8 (B-D). After 60min of uptake and autophagy most of the AVs containedonly Au8 (E,F). The endosomal marker, Au25 (arrow), andMPR staining were infrequently observed in AVs, evenafter 60 min (I). Occasionally, Au25- or MPR-labeledendosomes were observed within AV|S (G,H). LYS,lysosome; PLC, prelysosomal compartment. Bars: A-F,0.50 fim; G-I, 0.25 ,um.
To understand better the interaction of nascent AVswith lysosomes and endosomes, the results from theexperiments described above were quantitated. Ran-dom electron micrographs were taken and the numberof AVjS, AVds, lysosomes and endosomes that con-tained Au8, Au25, or both sizes of colloidal gold, werecounted and categorized. Strict morphological criteriawere adhered to: only organelles that were surroundedby 3=2 membranes and contained recognizable parts of
Fig. 5. Lysosomes fuse with AVs earlier and morefrequently than with endosomes. Fu5C8 cells wereincubated at 37°C in MEM, containing 5% FBS and Au8
for 30 min (A,B) or 16 h (C), and then chased for 2 h inMEM containing 5% FBS to label lysosomes. Then,autophagy was induced in the continuous presence of Au25
to label endosomes. Cells were fixed and processed forimmunocytochemistry using an antibody against the MPR.In order to determine the relative contribution ofendosomal and lysosomal contents to AVs, the numbers ofAV|S, AVds, endosomes and lysosomes with Au8, Au25 andMPR reaction product were counted and categorized asfollows: Au8~/MPR~, Au8
+/MPR" Au8+/MPR+ or
Au8~/MPR+ (A,B), Au8+/Au2 5
+, Au8+/Au2 5" and
Aug~/Au25+ (C). These data were plotted several different
ways: (A) the percentage of the total number of organellesthat contained Au8; (B) the distribution of Au8 and MPRsin AVds; (C) and the distribution of the lysosomal andendosomal markers, Au8 and Au25, in AVds. As seen inA, after only 5 min of stimulating autophagy, there was anincrease in the percentage of Au8
+ AVs, and by 60 min~40% of all Au8
+ organelles were AVs. As shown in Band C, while about half of the AVs counted containedAu8, there was very little increase in the percentage ofMPR+ or Au2s+ AVs. Throughout these experiments, thenumber of AVds and lysosomes with Au8 was about 50%of all the AVs and lysosomes observed; 50-100 photographsof each sample were taken at an average primarymagnification of x22,000.
Autophagic vacuoles rapidly fuse with lysosomes 521
the cell were counted as AVjs; organelles that containedrecognizable parts of the cell but had only one limitingmembrane were deemed AVds. Lysosomes were de-fined morphologically (see Dean and Barrett, 1976;Holtzman, 1989, for reviews) and by the presence ofAu8; similarly, endosomes were defined morphologi-cally and by the presence of Au2s and/or staining for theMPR. Using the labeling conditions described above,>90% of the Au8 was in lysosomes (MPR~) prior to thestimulation of autophagy; likewise, only about 10% ofthe MPR+ organelles contained Au8. Fig. 5 shows therapid appearance of the Au8 lysosomal label in AVds.To illustrate this more clearly, we have plotted thisseveral different ways. First, when plotted as a functionof the percentage of the total Au8
+ organelles, therewas a steady increase in the percentage of MPR~ AVds,
with a reciprocal decrease in the percentage of Au8found in heterophagic lysosomes (Fig. 5A). After 60min of stimulating autophagy, 50% of the Au8-containing organelles were MPR~ AVds. When thedistribution of these markers was plotted as thepercentage of the total number of AVds observed,similar results were obtained (Fig. 5B,C). At 5 minafter the induction of autophagy, 40-50% of all theAVds contained only Au8, while <10% contained MPRstaining. Over the course of an hour, more AVs formedbut the relative distribution of Au8 and MPR stainingwithin them remained unchanged (Fig. 5B). WhenAu25, delivered at the onset of autophagy, was used tolabel endosomes a similar observation was made. After15 min of autophagy, 70% of the AVds contained onlyAu8, whereas 2% contained Au8 and AU25. Even 2 hafter the induction of autophagy, in the continuouspresence of Au25, less than 5% of the AVds werelabeled with this endosomal marker (Fig. 5C). Exceptfor its rare appearance in an autophagocytosed vesicle,AU25 was never found in AVJS; hence the absence of thiscategory from the graphs. Although not shown in thegraphs, the intracellular distribution of MPRs was notsignificantly altered by the stimulation of autophagy. Atall times, most of the MPR staining was in organellesthat lacked Au8 and had the morphological character-istics described for the PLC. The endosomal label Au2smoved through the endosomal compartment with thekinetics expected of an endocytic tracer. After 5 min ofuptake it was found inside small vesicles near theplasma membrane. By 15-30 min, Au25 was observed invesicles and tubules typical of the PLC, and after 2 h itappeared along with Au8 in lysosomes (data notshown).
Only a small population of AVs are labeled byantibodies against the MPRAt any one time, <10% of all the AV;S and AVdsobserved were labeled by our anti-MPR antibody(quantitative data not shown for AVJS; see Fig. 5B forAVds). To investigate this more fully, cultured Fu5C8cells were AA starved for various lengths of time beforefixation and immunoperoxidase staining. Consistently,both AVjS and AVds were found throughout thecytoplasm, but only occasionally were any AVs stainedwith the MPR antibody (Fig. 6A,B). If labeled, theimmunoperoxidase reaction product was typically inone or two patches on the multiple membranessurrounding the AVj. Even after long periods of A Astarvation, the distribution of the MPR appeared to bethe same as it was in unstarved cells. A parallelexperiment was conducted using a polyclonal antibodyagainst the lysosomal enzyme cathepsin D. Even afterjust 2 h of AA starvation (without glucagon), thecathepsin D antibody heavily stained both lysosomesand AVs (Fig. 6C,D), providing direct evidence thatlysosomal enzymes have been delivered to AVs.
Newly synthesized, glycosylated lysosomal enzymesare not necessary for autophagic degradationTo gain a clearer understanding of the potential role of
the MPR in the delivery of lysosomal enzymes to AVs,the effect of tunicamycin treatment on autophagicsequestration and degradation was investigated usingboth morphological and biochemical methods. Tunica-mycin is a potent inhibitor of N-linked glycosylation,and therefore inhibits the proper targeting of lysosomalenzymes via MPRs (Rosenfeld et al., 1982; Von Figuraet al., 1979). Cells were labeled to isotopic steady-statewith [3H]leucine, then incubated in the presence orabsence of 2 jug/ml tunicamycin for 3 h, to abrogate fullythe proper targeting of new enzymes. After thisincubation, autophagy was induced by A A starvation,in either the absence or presence of tunicamycin. Theresults showed that tunicamycin treatment did notsignificantly alter the observed increase in degradationinduced by stimulating autophagy, nor did it have ameasurable effect on proteolysis in unstarved cells (Fig.7). The finding that tunicamycin does not inhibitautophagy was further demonstrated when a parallelexperiment was evaluated at the electron microscopylevel. Although not quantified, numerous AVs wereobserved in the tunicamycin-treated, AA-starved cells(data not shown). Using immunoprecipitations, weverified that 2 ,ug/ml tunicamycin completely inhibitedlysosomal enzyme glycosylation and intracellular tar-geting in Fu5C8 cells (data not shown). These resultsindicate that glycosylated, newly synthesized lysosomalenzymes are not necessary for the autophagy-inducedincrease in proteolysis, nor are they needed for theconversion of an AVj into a degradative organelle.
Discussion
We have found that newly formed AVs have rapidaccess to lysosomal contents. Combining gold labelingand an antibody against the MPR to distinguishbetween lysosomes and the PLC, we have conductedexperiments to determine which organelle initially fuseswith AVs. Both our work and that of Dunn (1990a,b)have shown that the simultaneous induction of auto-phagy with AA starvation and glucagon treatmentcauses AVs to form very rapidly. In fact, they formwithin the same time frame that is used to define variousendosomal compartments (Brown et al., 1986; Park etal., 1991b; Schmid et al., 1988), permitting thedetermination of which marker arrives in AVs first: Au8from lysosomes or Au25 from endosomes. Within 5 minafter the stimulation of autophagy, numerous AVdscontained the lysosomal marker Au8, whereas nonecontained the endosomal marker Au25, and <10% wereMPR+. After longer periods of time more AVs wereobserved; however, AVds were still primarily labeledwith Au8, not Au25 or MPR staining, and AVjS werepredominantly unlabeled. In addition to determiningwhich compartment merges with AVs first, quantitationof these experiments provided information regardingthe relative distributions of lysosomal and endosomalcontents in AVs. We conclude from these experimentsthat direct fusion with pre-existing lysosomes accountsfor the bulk of the lysosomal enzymes delivered to AVs.
Autophagic vacuoles rapidly fuse with lysosomes 523
Fig. 6. Immunoperoxidase localization of the MPR and cathepsin D in AA-starved hepatocytes. To induce autophagy, cellswere incubated in EBSS for 0.5 to 24 h prior to fixation and processing for immunoperoxidase cytochemistry. (A,B)Staining for the MPR labeled the Golgi complex (GC), prelysosomal compartment (PLC) and small vesicles and tubulesthroughout the cytoplasm. Although numerous AVs were observed, staining of AVjS and AVds was rare. When it wasobserved, MPR reaction product was in small patches on the multilayer AV: membrane (B). (C,D) Numerous cathepsin D-labeled lysosomes and AVds were observed early on in AV formation. AV|S were not labeled with this antibody. Note themultiple membranes (arrow) in the upper half of the AVj in C. It seems likely that this AV has just received hydrolyticenzymes, and has not fully degraded the extra membranes characteristic of an AV;. The AVd in D has a lysosome fusedwith it. Bars: A-C, 0.50 /im; D, 0.25 jun.
To study autophagy, we established a model systemusing the cultured rat hepatocyte cell line Fu5C8.Because cultured Fu5C8 hepatocytes had not been usedfor studying autophagy, morphometric and biochemicalanalyses were conducted prior to the actual executionof experiments. This quantitative analysis provided uswith definitive information regarding the kinetics and
extent of AV formation and total cellular proteolysis,and demonstrated that autophagy induced in Fu5C8cells is morphologically and biochemically identical tothat observed in liver cells treated in situ (Deter et al.,1967; Dunn, 1990a,b; Ericsson, 1969; Lardeux andMortimore, 1987; Marzella and Glaumann, 1980b;Mortimore and Schworer, 1977). When autophagy was
524 B. P. Lawrence and W. J. Brown
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5 10Time (h)
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Fig. 7. Treatment with tunicamycin does not inhibitautophagic degradation. [3H]leucine-labeled cells weretreated for 3 h with 2 ,ug/ml tunicamycin prior to theinduction of autophagy. After this incubation, the cellswere either refed or switched to EBSS for AA starvation,in the continuous presence of the drug. Degradation wasmonitored by the release of TCA-soluble radioactivity intothe medium. Identical control samples were not treatedwith tunicamycin, but were otherwise the same. The valuesshown are averages of triplicate samples. Fed cells(• • ) ; A A starved cells (O O); fed cells plus 2,ug/ml tunicamycin ( • • ) ; AA-starved cells plus 2iUg/ml tunicamycin (A A).
stimulated by AA starvation alone, the maximumvolume and surface densities of AVjS were reached afterabout six hours, and then the cells appeared to recovergradually. We found no significant change in thevolume and surface densities of lysosomes, which aresimilar to results reported by Lee et al. (1989) from amorphometric analysis of autophagy in murine terato-carcinoma cells. They reported a 60% decrease in thevolume density of dense bodies, and proposed that thiswas due to the convergence of lysosomes, to provide thenecessary hydrolases for autophagic degradation. Mar-zella and Glaumann (1987) explain this finding bysuggesting that as the volume of the degradative AVsexpands there is a concomitant loss of morphologicallyidentifiable heterophagic lysosomes. Our data areconsistent with this explanation, since the lysosomalcompartment (autophagic plus heterophagic lyso-somes) does not expand following the stimulation ofautophagy and, as we have shown, lysosomes rapidlyfuse with AVs.
Our evidence indicates that lysosomes, not endo-somes or the PLC, are the principal source of lysosomalenzymes for AVs. The portion of the AVs that wereMPR+ or Au25
+ (<10% of all the AVs counted) couldbe a result of the steady-state exchange of contentsbetween the PLC and mature lysosomes, or the fusionof the PLC with a small population of AVs, and it would
be difficult to distinguish between these two possi-bilities. The lack of AU25 within most AVds was not dueto an inability of Fu5C8 cells to endocytose these goldparticles. Similarly, abundant immunoperoxidase reac-tion product was observed in the rough ER, Golgicomplex, the PLC and coated pits on the surface of thecells. AV staining with anti-MPR antibodies was lessabundant than the labeling of coated pits, which containonly 10% of the total cellular pool of MPRs (Jin et al.,1989; Sahagian and Neufeld, 1983), suggesting that theinfrequent staining of AVs with our MPR antibody wasnot due to detection limitations of this method. We didobserve significant immunocytochemical staining ofAVjS with antibodies against cathepsin D, consistentwith the theory that lysosomes fuse directly with AVs.
The possibility that much of the Au8 was retained in aprelysosomal endosome, rather than progressing intomature lysosomes was excluded. Using well-establishedpulse-chase times, we defined mature lysosomes asorganelles that contained Au8 and lacked MPRs(Brown et al., 1986; Griffiths et al., 1988; Kornfeld andMellman, 1989; Sahagian and Neufeld, 1983) and foundthat after long and short periods of uptake, followed bya 2 h chase, 90-95% of the Au8 labeled lysosomes (i.e.Au8
+/MPR~organelles), while the other 5-10% was inthe PLC (i.e. Au8
+/MPR+ compartments). Therefore,90-95% of the Au8 delivered to AVs must have comefrom mature lysosomes. In a similar experiment, using<*2-macroglobulin-conjugated to gold (o^M-gold), Grif-fiths et al. (1988) reported that after a 2 h pulse and a 2 hchase, 85% of the o^M-gold remained in MPR+
compartments, but after a 24 h chase, 70% of the a2M-gold was in lysosomes (MPR~), while 30% was in thePLC (MPR+). Their results differ from those reportedhere in that the endocytic marker (a^M-gold) chasedinto lysosomes much more slowly, and more of itrecycled to an MPR+ compartment. These dissimilari-ties may be inherent in the different markers used, orthey could be cell-type-specific variations; in either casethese data illustrate the importance of determining thedistribution of a fluid-phase marker in lysosomes andprelysosomes, for the purposes of accurately accountingfor the contribution of each of these compartments.
To investigate further a potential role for the MPRduring autophagy, we used tunicamycin to inhibit thetargeting of newly synthesized lysosomal enzymes tothe PLC, and their subsequent delivery to lysosomes.Treatment with tunicamycin had only a small ornegligible effect on autophagic sequestration anddegradation, as determined by morphological andbiochemical methods. The observed fivefold increase indegradation was unaffected by tunicamycin treatment,indicating that for at least the first 24 h of AAstarvation, glycosylated, newly synthesized lysosomalenzymes are not required. Although a quantitativemorphological analysis was not conducted, our obser-vation that AA-starved and tunicamycin-treated cellshad many nascent and degradative AVs supports thebiochemical data. Pooling this evidence together withthe rare MPR and Au2s labeling of AVs, the fact thatthe PLC is not heavily laden with lysosomal enzymes
Autophagic vacuoles rapidly fuse with lysosomes 525
(Sahagian and Neufeld, 1983), and that AVs have ashort half-life (Marzella and Glaumann, 1987; Pfeifer etal., 1978), implies that fusion with the PLC cannotexplain the rapid conversion of an AVj into an AVj.
Indirect evidence that mature lysosomes are theprimary source of lysosomal enzymes was first obtainedby Ericsson (1969) in renal proximal tubule cells andhepatocytes, in which the lysosomes were labeled withan electron-dense marker. Similar to what is reportedhere, Ericsson reported that nascent AVs containedidentifiable cellular components and lacked both thepre-loaded lysosomal marker and lysosomal enzymes;however, shortly after the induction of autophagy, thelysosomal marker was observed in morphologicallyidentifiable degradative AVs. More recently, Lee et al.(1989) conducted an analysis of autophagy in nutrient-deprived transformed cells (F9 12-la teratocarcinomacells), and found that the direct fusion of lysosomeswith sequestered portions of the cytoplasm was also alikely source of degradative enzymes.
Although these studies corroborate our conclusion,they did not separately determine the potential contri-bution from prelysosomal sources, and other studieshave suggested that AVs merge with the endocyticpathway at a prelysosomal point (Gordon and Seglen,1988; Dunn, 1990b; Tooze et al., 1990). For example,Tooze et al. (1990) studied autophagy by inducing theformation of intracisternal granules (ICGs) by injectionof CoCI2 into guinea pig pancreas. ICGs are insolubleaggregates of pancreatic digestive enzymes that form inthe rough ER (Palade, 1956). Using ICGs as a markerfor AVs, and horseradish peroxidase (HRP) as anendocytic tracer, they observed that HRP arrived inAVs about 30 min after uptake, although it was found inendosomes at earlier times. As with other studies, theydid not simultaneously monitor the appearance of alysosomal marker in AVs during earlier times of AVformation; consequently, the relative contributions ofthe endocytic pathway and mature lysosomes could notbe evaluated. Our work suggests that lysosomalenzymes would reach AVs before the endocytosedHRP, because we observed that delivery of endocy-tosed tracers to AVs was rare compared to the deliveryof a lysosomal marker.
Other studies of the role of the MPR duringautophagy have led to mixed conclusions regarding itsinvolvement. Dunn reported that 30% of the AVjS inAA-starved, glucagon-treated perfused rat liver werelabeled by anti-MPR antibodies; however, treatmentwith tunicamycin did not inhibit the delivery oflysosomal enzymes to AVs (Dunn, 1990b), which isconsistent with our results. Using immunogold pro-cedures, Tooze et al. (1990) did not detect any MPRlabeling of initial AVs (Type I); however, later stageAVs (Type II) were heavily labeled. It is not clearwhether or not there are discrepancies between ourwork and that of Dunn and Tooze et al., but theapparent differences could certainly be related todifferences in the types of cells studied, the methodsused to induce autophagy, and the morphologicalcriteria used to categorize the different subclasses of
AVs. Our results suggest that MPR-mediated lysosomalenzyme delivery is not required for autophagy toproceed, and that the intracellular distribution of MPRsis not greatly altered by the stimulation of autophagy.
In this report we have presented evidence oflysosome and AV confluence following the onset ofautophagy in cultured hepatocytes. By separatelylabeling lysosomal and endosomal compartmentswithin the same cell, and quantitating the distributionof markers within these compartments, we were able todetermine the relative contributions of lysosomes andendosomes to the delivery of material to AVs. Ourconclusion that lysosomes rapidly fuse with AVs isconsistent with early studies of autophagic degradationdescribed in perfused organs, such as liver and proximalrenal tubules (Deter et al., 1967; Ericsson, 1969). It isalso consistent with studies demonstrating that AVshave a very short half-life (~9 min) (Pfeifer et al., 1978;Glaumann et al., 1981), implying the need for rapiddelivery of significant amounts of hydrolases, whichcould only come from pre-existing lysosomes Theconclusion that AVs have early access to lysosomescorresponds very well with our other results, whichshowed that induction of autophagic degradation doesnot require the MPR-mediated delivery of lysosomalenzymes, nor does it significantly alter the intracellulardistribution of MPRs. Finally, our data demonstrate aminor contribution by endosomes and the PLC;however, fusion with pre-existing lysosomes appears tobe the principal route of enzyme delivery to AVs.
This work was supported by N1H grant DK37249 to W.J.B.The authors thank Dr. Melvin Rosenfeld (NYU) for the giftof the Fu5C8 cells, and Marian Strang for producing the largenumbers of thin sections needed for this work.
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