Prenylation of S. cerevisiae Chs4p affects chitin synthase III activity and

chitin chain length

Kariona A. GrabiMska*, Paula Magnelli, and Phillips W. Robbins

Department of Molecular and Cell Biology, School of Dental Medicine,

Boston University, 715 Albany Street, Evans 408 Boston, MA 02118

Running title: Chitin synthesis in S. cerevisiae

Key Words: Saccharomyces cerevisiae, chitin synthase, cell wall, prenylation, farnesylation

*Corresponding author: Kariona A. GrabiMska, Department of Molecular and Cell Biology,

School of Dental Medicine, Boston University, 715 Albany Street, Evans 4 Boston, MA 02118

Telephone # (617) 414-1058. Fax #(617) 414-1041.E-mail: karionag@bu.edu

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ABSTRACT

Chs4p (Cal2/Csd4/Skt5) was identified as a protein factor physically interacting

with Chs3p, the catalytic subunit of chitin synthase III (CSIII), and is indispensable for its

enzymatic activity in vivo. Chs4p contains a putative farnesyl attachment site at the C-

terminal end (CVIM-motif) conserved in Chs4p of S. cerevisiae and other fungi. Several

previous reports questioned the role of Chs4p prenylation in chitin biosynthesis. In this

study we reinvestigated the function of Chs4p prenylation. We provide evidence that

Chs4p is farnesylated by showing that purified Chs4p is recognized by anti-farnesyl

antibody, is a substrate for farnesyl transferase (FTase) in vitro, and that inactivation of

FTase increases the amount of unmodified Chs4p in the yeast cells. We demonstrate that

abolishing Chs4p prenylation causes ~60 % decrease in CSIII activity which is correlated

with a ~30 % decrease in the chitin content, and with increased resistance to the chitin

binding compound, Calcofluor white. Furthermore, we show that lack of Chs4p

prenylation decreases the average chain length of the chitin polymer. Prenylation of

Chs4p, however, is not a factor which mediates plasma membrane association of the

protein. Our results provide evidence that the prenyl moiety attached to the Chs4p is a

factor modulating the activity of CSIII both in vivo and in vitro.

INTRODUCTION

Chitin, a linear N-acetylglucosamine (GlcNAc) polymer, is a minor but essential

structural component of the yeast cell wall - the organelle responsible for the maintaining

of cell shape and osmotic stability (5, 23). The majority (90 %) of chitin in the cell wall

including chitin in the bud scars, in the lateral wall, and the polymer converted to the

chitosan in spore walls is synthesized by chitin synthase III (CSIII) encoded by CHS3

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gene (35). A number of proteins have been identified that are necessary for the proper

activity of Chs3p. Chs7p is required for export of Chs3p from ER, whereas Chs5p and

Chs6p are involved in the proper delivery of Chs3p to the plasma membrane (35).

Chs4p (Cal2/Csd4/Skt5) is a regulatory subunit of the CSIII complex,

indispensable for its enzymatic activity in vivo in vegetative cells (5, 32, 35, 37, 40). It

has been demonstrated that Chs4p interacts directly with Chs3p and is responsible for the

localization of Chs3p to the septin ring thorough the interaction with the scaffolding

protein Bni4p (11). Chs4p contains a possible farnesyl attachment site at the C-terminal

end (CaaX-motif) which is conserved between S. cerevisiae Chs4p and other fungi

including human pathogens Candida albicans (39) and Cryptococcus neoformans (1).

The possibility that Chs4p is prenylated is enhanced by the fact that the CVIM motif is

preceded by a lysine rich amino acid stretch (29). Thus, Chs4p is predicted to be

prenylated by the Prenylation Prediction Suite (http://mendel.imp.ac.at/sat/PrePS/).

However, the role of prenylation of Chs4p in chitin biosynthesis or even the occurrence

of this modification was questioned in several reports (5, 11, 32, 39, 40).

In yeast, 35 proteins, including many important for cell growth, differentiation,

morphology and stress response, require post-translational modification by covalent

attachment of an isoprenoid lipid (prenylation) for proper function (Proteome

Bioknowledge Library: www.incyte.com). Prenylated proteins are posttranslationally

modified by formation of cysteine thioethers with the isoprenoid lipids farnesol (C-15) or

geranylgeraniol (C-20) at or near to the carboxyl terminus. Prenylation is specified by the

amino acid sequence motifs CaaX, CC and CaC at the carboxyl end of the protein where

a is an aliphatic amino acid and X is any amino acid. The CaaX sequence is substrate for

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farnesyl transferase (FTase) (the known biological substrates have X= S, M, A or Q)

unless X=L, which results in a substrate for geranylgeranyl transferase I (GGTase I). The

CC and CaC motifs, present in the Rab family of low molecular mass G-proteins, are

digeranylgeranylated. Typically, prenylation by CaaX protein prenyltransferases is

accompanied by further posttranslational processing, most often involving cleavage of

the carboxy-terminal tripeptide (-aaX) followed by carboxymethylation of the carboxy

terminus (9, 38).

Like other lipid modifications, prenylation has been viewed as a mechanism for

post-translational attachment of proteins to membranes. However, now it appears that

lipid modification by protein prenyltransferases has a more complex role: for example,

the farnesyl and geranylgeranyl moieties are directly involved in protein-protein

interactions as well as in protein-membrane interaction (28, 38).

Since clinical studies in progress are exploring the antitumor activity of FTase

inhibitors as potential therapeutic agents (2), prenylation attracts the attention of many

laboratories. In order to decrease the costs associated with de novo drug design and

accelerate the development of new chemotherapeutics, FTase inhibitors are currently

investigated as agents for protozoan pathogens (14). Since deletion of FTase catalytic

subunit (RAM1) is lethal in the pathogenic fungus Cryptococcus neoformans, in contrast

to the case in Saccharomyces cerevisiae (44), FT inhibitors may be suitable as antifungal

drugs.

In this study we have reinvestigated the function of Chs4p farnesylation and

shown that prenylation of Chs4p does not affect membrane anchoring of Chs4p; however

it does affect the catalytic properties of CSIII.

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MATERIALS AND METHODS

Strains, plasmids, and growth conditions. Strains and plasmids used in this

study are listed in Table 1 and oligonucleotide primers in Table 2.

To generate the mutation leading to the abolition of Chs4p farnesylation (Cysteine

693 Serine substitution) directly at the chromosomal locus, we adapted the loxP/Cre

based disruption system (16). The homologous recombination cassette was amplified by

PCR using oligonucleotides comprising the 3’ region complementary to the sequence in

the template (pUG27 marker plasmid) flanking the disruption cassette and 50 5’

nucleotides that anneal to the STOP codon and the region upstream (CHS4CtoS primer)

and the region downstream of the STOP codon (CHS4DR primer) of the CHS4 gene. In

the forward primer the TGT codon for cysteine was replaced with AGT codon for serine.

After transformation of the linear cassette into yeast cells, selected transformants were

checked by PCR for correct integration of the cassette using CH4-1920 and KanB pairs

of primers. Introduction of mutation was verified by sequencing of the PCR product.

Finally, the marker gene was removed by expression of recombinase Cre leaving behind

a single LoxP site. As a control strain we used a yeast strain with LoxP introduced site at

the chromosomal locus after the STOP codon of CHS4 gene. To obtain this strain, yeast

cells were transformed with the PCR cassette generated using CHS4wt and CHS4DR

primers and the pUG27 plasmid as a template. In order to change farnesylation of the

CVIM motif to the geranylgeranylation motif CVLL, we used a mutagenesis cassette

generated with primers: CHS4DR and CHS4LL. The CHS4LL forward primer last

codons ATT for isoleucine and ATG for methionine were replaced with TTG and TTA,

both coding for leucine. Comparison of growth rate, sensitivity to Calcofluor white

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(CFW) and chitin content of parental BY4741 and KG103B yeast strain containing LoxP

sequence in terminator region of CHS4 gene did not reveal any difference.

In order to construct the trp1 auxotrophic KG101B yeast strain, we decided to

delete only the first 312 nucleotides of TRP1 ORF to leave intact the putative YDR008C

ORF located on the complementary strand of DNA. Deletion was accomplished by the

method described by Gueldner et al., (16). The deletion cassette was amplified with DF-

TRP1 and DR-TRP1 primers from the pUG27 plasmid used as a template, then

transformed into BY4741 yeast cells. Transformants able to grow on medium lacking

histidine and requiring tryptophan were isolated, and correct insertion of cassette was

verified by PCR with TRP1UP and KanB primers pair. Finally, the marker gene was

removed by expression of recombinase Cre.

In order to construct a yeast strain expressing TAP-Chs4p (33, 34), KG101B cells

were transformed with PCR cassette amplified with pBS1761 plasmid as a template and

TAP1-CHS4 and TAP2-CHS4 primers pair. Transformants able to grow on medium

lacking tryptophan were isolated, then correct insertion of cassette was verified by PCR

with primers: CHS4 –290R: and F1-CBP. Marker TRP1 gene was removed by expression

of recombinase Cre.

TAPCHS4B5 and TAPCHS4B6 were obtained by mating TAPCHS4B3 or

TAPCHS4B2 with BY4741ram1F0"Sporulation of the diploid cells and tetrad dissection

was done by standard yeast genetics methods. Yeast cells were cultured in 2% (w/v)

bactopeptone, 1% (w/v) yeast extract supplemented with 2% glucose (w/v) (YPD).

Synthetic minimal media (SD) were made of 0.67% (w/v) yeast nitrogen base 2% (w/v)

glucose, supplemented with auxotrophic requirements. For solid media, agar (Difco) was

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added to YPD or SD at 2% (w/v) final concentration. Bacterial cells carrying pET30a

plasmid were grown in LB medium (1% [w/v] bactotryptone, 0.5% [w/v] yeast extract,

1% NaCl [w/v]) supplemented with kanamycin (25 mg/l) and chloramphenicol (34mg/l).

Heterologues expression and purification of Chs4p from Eschericha coli. WT

and mutated at prenylation box (C693S) CHS4 alleles were amplified by PCR using yeast

genomic DNA as a template and CHS4BamHF as a forward primer, and Chs4-R or Chs4-

C693S-R as a reverse primer. PCR products were cloned into pGEM Easy T vector

(Promega) and BamHI/NotI insert was subcloned into the pET30a vector (Novagen) in

such a way that a poly-His sequence and S-tag were added to the N-terminus of Chs4p.

pET30a-CHS4 and pET30a-chs4(C693S) were each transformed into The Rosetta™ 2

strain of E. coli. E coli cells in logarithmic growth phase were induced to express Chs4p

by incubation with 1mM IPTG (Isopropyl-ß-D-thiogalactopyranosid) for 4 h at 30 °C.

The harvested cells were lysed by sonication. WT and mutated Chs4p were purified by

means of N-terminal poly-His using a nickel-column according to the Invitrogen protocol

and checked on SDS-PAGE and Western Blot using monoclonal mouse IgG raised

against S-tag (Novagen).

Miscellaneous methods. Protein level was determined on microtiter plates by the

Bradford method (Bio-Rad Laboratories). Protein samples were resolved by SDS

NuPAGE-NOVEX Bis-Tris 4-12% gel or 8 % Precise SDS-PAGE (Pierce) under

reducing conditions and transferred to polyvinylidene difluoride (PVDF) membranes,

blocked in 5% milk and probed with antibodies. Chs3p was recognized with polyclonal

rabbit antibodies (25), plasma membrane marker Gas1p with polyclonal rabbit IgG (kind

gift of L. Popolo, Università degli Studi di Milano), cytoplasm marker Pgk1p with

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monoclonal mouse antibodies 22C5 (Molecular Probes), ER membrane marker Dpm1p

with monoclonal 5C5 mouse antibodies (Molecular Probes), and TAPtag fused proteins

with Peroxidase-Anti-Peroxidase Soluble Complex (PAP) rabbit IgG coupled with HRP

(SIGMA) or anti-Chs3p IgG. Farnesylated purified CBP-Chs4p was detected with rabbit

anti-farnesyl antiserum (SIGMA). As secondary antibodies Horseradish peroxidase-

conjugated anti-mouse antibodies or anti-rabbit antibodies (Promega) were used. Binding

was visualized with the Western Lightning Chemiluminescence Reagent (PerkinElmer)

according to the manufacturer's instructions.

Prenylation of Chs4p in vivo. In order to purify Chs4p, a modified TAPtag

purification method was applied (33). Yeast cells expressing TAP-Chs4p (TAPCHS43B)

or TAP-Chs4pC693S

(TAPCHS4B) were grown overnight in YPD medium to late

logarithmic phase (2-3 OD600 units/ml). Cells were collected by centrifugation, washed

with water, and then disrupted in IPP1000 buffer (10 mM Tris HCl pH 8, 1M NaCl, 0.5

% Nonylphenyl-polyethylene glycol [NP 40, SIGMA], 2 mM phenylmethylsulfonyl

fluoride [PMSF], Protease Inhibitor Cocktail [SIGMA]) by Vortexing with glass beads.

Lysates were clarified by 15 min. centrifugation at 1500 g and centrifuged at 100,000 g

for 90 min at 4°C.

The supernatant was incubated overnight at 4flC on a rotating platform with 100 伊

ol of IgG Sepharose 6 FAST Flow (Amersham Biosciences). The IgG Sepharose beads

were collected by centrifugation, washed extensively with IPP buffer, equilibrated with

TEV cleavage buffer (10 mM Tris HCL pH 8, 150 mM NaCl, 0.1% NP40, 0.5 EDTA,

1mM DTT), and suspended in 50 ol of TEV cleavage buffer containing 3ol (30 units) of

TEV protease (Invitrogen). The mixture was incubated during 3 h at 16flC on a rotating

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platform in order to cleave the CBP-Chs4p fusion protein from ProtA tag. The mixture

was centrifuged (20,000 g) in order to separate the upper phase eluate containing CBP-

Chs4 fusion protein from IgG sepharose beads. Protein samples were separated by

protein electorophoresis and analyzed by immunoblotting.

Farnesyl transferase activity. 1.5 og of Chs4p (wt or mutated at the

farnesylation box) purified from bacterial cells was incubated with 4 oM farnesyl

pyrophosphate-(1-3H (N) (15Ci/mmol, ARC) in 30 ol reaction mixture containing 10

mM TrisHCl pH7.5, 10 mM MgCl2, 5 oM ZnCl2, 0.1mM PMSF and 0.2 mg of

recombinant S. cerevisiae farnesyltransferase (SIGMA). The reaction mixture was

incubated at 30ºC for 60 min, stopped by addition 1ml of TEV buffer, concentrated using

Amicon ultra filter device (Millipore) and proteins were separated by SDS PAGE gel and

blotted onto PVDF membrane. Farnesylated protein was visualized by autoradiography.

In order to asses the level of prenylation in wt background and in the cells lacking

the catalytic subunit of farnesyltransferase (Ram1p), yeast strains expressing TAP

versions of wt and mutated form of Chs4p in the RAM1 knockout genetic background

were constructed. Cells were disrupted in IPP1000 buffer and the protein extract (100 000

x g supernatant) was incubated for 3 hr with at 4flC on a rotating platform with IgG

Sepharose 6 FAST Flow (10ol of Sepharose was incubated with the extract made from

0.25 g of cells to ensure saturation of the resin with the protein). The IgG Sepharose

beads were collected by centrifugation, washed extensively with IPP buffer, equilibrated

with FTase buffer lacking farnesyl pyrophosphate and FTase and, 10 ol of resin with

immobilized protein was used for FTase assay in 60 ol reaction mixture as described

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previously. After reaction, the resin was washed 5 times with 1ml of TEV buffer and the

radioactivity was measured by scintillation counting.

Subcellular fractionations. The analysis of organelles by differential

centrifugation was performed according to the methods described (21, 42) with

modifications.

To study membrane association of Chs4p, 0.6 gram of yeast cells in mid-log

phase (0.5-0.8 OD600) were harvested, washed with ice-cold 10 mM NaN3, 10 mM KF, 50

mM Tris-HCl pH 7.5 buffer and then with 50 mM Tris-HCl pH 7.5, 1 mM EDTA buffer.

The cells were suspended in 1.2 ml of hypoosmotic lysis buffer (HOB: 200 mM sorbitol,

1 mM EDTA, 50 mM Tris HCL pH 7.5, 2 mM PMSF, protease inhibitor cocktail

[SIGMA]) and broken by agitation with glass beads. Lysates were cleared by 5 min

centrifugation at 500 g. The cleared cell lysate was mixed with an equal volume of 200

mM sodium carbonate or HOB containing 1 M NaCl, 2 % Triton X100 or 2% SDS. After

incubation for 1 hour on ice, samples were centrifugated at 200,000 g for 1 hour to

separate soluble and particulate fraction. The pellet was then resuspended in HOB in the

same volume as the corresponding soluble fraction. 5 ol of each fraction was subjected to

protein electorophoresis and immunoblotting.

Chitin content measurement. Chitin content was measured by an assay adapted

for microtiter plates as described previously (6) with minor modifications. Chitin polymer

was digested with chitinase c (Interspex) in McIlvaine's Buffer, pH 6.0 during 3 h.

Chitin synthase III activity. Chitin synthase III activity was measured by the

colorimetric assay adapted for microtiter plates as described (25). The enzyme source

was prepared as follows: cells were harvested by centrifugation, washed once with water

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and resuspended in 20 mM Tris HCl pH 8.0 buffer containing 2 mM PMSF and protease

inhibitor cocktail (SIGMA), and broken by Vortexing with 425-600 micron glass beads.

The cell free extract was clarified by 15 minutes centrifugation (1500 g) and then

centrifugated 1h for 100,000 g. The membrane fraction was dissolved in 20 mM Tris HCl

pH 8.0 buffer supplemented with protease inhibitors. Trypsin pretreatment of enzyme

source was done according to the method described for measurement of chitin synthase II

activity (25).

Degree of polymerization of chitin. Chain length was estimated according to

Kang et al. (22). To obtain pure chitin, d-1,3-glucan and mannan were removed by

digesting isolated cell walls with 2 mg/ml of Zymolyase X100 (Seikagaku). In order to

remove the remaining d-1,6-glucan attached to chitin chains, the washed pellet was later

digested with 4 units/ml of d-1,6 endoglucanase (27). The chitin pellet was reduced in

100 ol of 0.1M NaOH, with 500oCi of NaB3H4 (NEN, 100mCi/mmol) for 6h at 25ºC.

The reaction was stopped with 200ol 0.5M acetic acid. After extensive washing, the

reduced chitin was digested in 0.2 ml of 0.1M KPO4 pH 6.0 with 0.015 units of Serratia

chitinase (Sigma) for 16h at 37ºC. This crude endochitinase preparation, containing

hexosaminidase activity, digests chitin to free N-acetylglucosamine (GlcNAc) (27). After

digestion the total GlcNAc was estimated using the Morgan Elson assay, as described

above. To determine the amount of terminal residues, the supernatants were

chromatographed in a P4 Biogel column (1 x 120 cm). Three major species were present:

free N-acetylglucosamintol, and two oligosaccharides with an apparent mass of GlcNAc-

glucitol and GlcNAc-glucose-glucitol (residual glucan stubs attached to the chitin

reducing end). The molarity of these tritiated species was calculated according to the

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radioactivity of 2.25 omol of GlcNAcOL isolated by P4 chromatography after reducing

GlcNAc under the same conditions described above. The degree of polymerization (DP)

was calculated as the omol of GlcNAc/omol of total alditols.

RESULTS

Chs4p is prenylated. Although Chs4p contains a possible farnesyl attachment

site at the C-terminal end (CVIM motif) which is conserved between Chs4p of S.

cerevisiae and a number of other fungi, the role of farnesylation of Chs4p in chitin

biosynthesis was questioned by several reports (5, 11, 32, 39, 40). Since previously wild

type or mutated Chs4p was expressed from a plasmid, we suspected that the effect of the

mutation might be masked by variation in the protein expression level; therefore in this

study we used only yeast strains expressing Chs4p from the genome. The lack of the

phenotype in previous studies may be also due to the fact that in some cases chitin level

was measured by CFW or Wheat Germ Agglutinin coupled with FITC binding assay (11,

40). These methods may be not accurate enough to observe small differences in chitin

level.

We confirmed that when purified from bacterial source, Chs4p is substrate for

yeast farnesyltransferase in vitro (1A). To reinvestigate whether Chs4p is prenylated in

vivo, we constructed yeast strains expressing Chs4p with N-terminal TAP (ProtA -

protease TEV cleavage site - CBP) -tag mutated at C-terminal CVIM farnesylation motif

(C693S substitution) (33). The affinity purification on IgG-Sepharose from yeast extracts

gave TAP-Chs4p and TAP-Chs4pC693S

with the same efficiency (Fig.1Bi). CBP-tagged

forms of the proteins were then released into solution from the ProtA bound to the IgG-

Sepharose by treatment with protease TEV. Antibodies raised against N-acetyl-S-

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farnesyl-L-cysteine which recognizes farnesyl or with less specificity geranylgeranyl-

modified proteins (3, 24) were able to recognize wild type Chs4p but not the mutated

protein (Fig. 1Bii). The presence of wt and mutated CBP-Chs4p in the eluate was

confirmed by mass spectrometry analysis of the purified proteins (data not shown). This

result confirmed that Chs4p is prenylated in vivo.

Since prenylation is an irreversible process, we assume that if Chs4p is

preferentially farnesylated, inactivation of endogenous FTase will increase the amount of

unmodified Chs4p available as a substrate for reaction in vitro, even if cross-specificity

between FTase and GGTaseI are observed (29, 31, 41). To prove the involvement of

FTase in modification of Chs4p, we immobilized TAP-Chs4p expressed in wt yeast cells

or cells lacking the catalytic subunit of FTase (Ram1p) on IgG-Sepharose beads and used

the resin-Chs4p as substrates for FTase assays in vitro. As a negative control we used the

resin carrying Chs4p C693S mutant. Our results (1C) show that Chs4p isolated from wt

yeast cells is a poor substrate for FTase. On the other hand, inactivation of FTase

increases the amount of Chs4p in the cell available for reaction in vitro which indicates

that Chs4p under normal conditions is farnesylated, not geranylgeranylated.

Prenylation of Chs4p is not essential for its plasma membrane localization or

membrane association. All known prenylated proteins are found, at least to some extent,

bound to cellular membranes, and prenylation has often been viewed as a mechanism for

post-translational attachment of proteins to membranes (38). Chs4p is also known to be a

membrane protein, so we determined whether farnesylation of Chs4p influenced its

membrane association or plasma membrane localization.

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To test membrane association of Chs4p, cell free protein extracts from cells

expressing TAP-Chs4p and TAP-Chs4pC693S

were fractionated into supernatant (soluble)

and pellet (membrane-associated) portions by centrifugation. TAP-Chs4p and TAP-

Chs4pC693S

were found in the pellet fraction (Fig.2A). To test whether lack of prenylation

changes the membrane association of Chs4p, protein extracts were treated with sodium

chloride and sodium carbonate to disrupt peripheral or protein–protein associations,

or

with Triton X-100 or Sodium Dodecyl Sulfate (SDS) to disrupt integral membrane

association. TAP-Chs4p as well as TAP-Chs4pC693S

were solubilized to similar extents by

treatment with sodium chloride, sodium carbonate or SDS, but Triton X-100 (a widely

used non-ionic surfactant for recovery of membrane components under mild non-

denaturing condition) had no effect in either case, indicating that membrane association

of Chs4p is independent of farnesylation.

In order to determine whether prenylation affects Chs4p subcellular distribution,

the cells were converted to spheroplasts, lysed by osmotic shock, and

membranes were

separated by differential centrifugation (13,000 g for 10 min) to obtain a

Golgi/endosome-rich fraction (S) and a plasma membrane/ endoplasmic reticulum-rich

fraction (PM/ER) fraction (P). As can be seen, prenylation did not change distribution of

Chs4p between the S and P fractions. Chs4p populated both fractions. However, in

contrast to the catalytic subunit of CSIII (Chs3p) it is present to the greater extent in the

Golgi/endosome-rich fraction (Fig 2B).

We also did not observe a difference in sedimentation of wt and mutated Chs4p

on a step sucrose/EDTA density gradient (data not shown).These results confirm that

abolishing Chs4p prenylation does not prevent trafficking to the plasma membrane.

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Mutation of the farnesylation site confers resistance to Calcofluor white.

Since Calcofluor white (CFW) is a fluorescent dye that intercalates with nascent chitin

chains, preventing microfibril assembly and inhibiting growth of yeast strains, the

sensitivity to CFW is often an indicator of changes in cellular chitin level (13, 36).

Expecting that prenylation of Chs4p could influence chitin biosynthesis, we compared the

growth rate of chs4 mutants and the corresponding wild type yeast strain on medium

supplemented with CFW. The results in Fig.3 show that chs4-C693S yeast cells harboring

the nonprenylated version of Chs4p are more resistant to CFW than wild type cells and

less resistant than chs4F yeast cells. To confirm the role of prenylation of Chs4p, we also

constructed yeast strain chs4-I695L,M696L expressing Chs4p with the C-terminal CVLL

motif (the CVLL motif present in yeast Rho1p was proven experimentally to be a

substrate for geranylgeranyl transferase Type I [31]). Geranylgeranylation of Chs4p only

partially restores the sensitivity to CFW.

Farnesylation of Chs4p affects chitin synthase III activity in vitro and chitin

content. Next, we examined the effect of mutagenesis of the Chs4p farnesylation site on

the cellular chitin content and CSIII activity in different growth conditions. As shown in

Table 3, abolishing farnesylation causes approximately a 30 - 40% decrease in chitin

content under various conditions of growth (CFW or glucosamine supplementation) or in

various genetic backgrounds, including those that induce the cell wall stress response

(deletion of FKS1 or GAS1 gene [15, 43]). However, the rate of increase in chitin levels

produced by CFW addition in wild type and mutated yeast cells was very similar. In both

cases maximal levels of chitin were achieved after 4-6 hours of exposure to CFW and

half of this value after ~2 hours (data not shown). Since the kinetics of response to CFW

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treatment is similar in yeast strains bearing wild type or mutated CHS4 gene, we can

assume that prenylation of Chs4p does not contribute directly to the mechanism of the

cell wall stress response.

Reported here changes in the cellular chitin content are well correlated with the

observed 60% decrease in CSIII specific activity (Fig.4A). Restoring prenylation by

introducing the CVLL geranylgeranylation motif is able only to partially restore the wild

type phenotype. Treatment of the enzyme source with trypsin is able to increase CSIII in

chs4-C603S and chs4F mutants to the level measured in the parental strain (Fig.3B),

which indicates that proteolytic treatment activates CSIII in a way that is independent of

the Chs4p function.

Chs4p prenylation affects chitin chain length. We have noticed that cells

carrying nonprenylated Chs4p are much more resistant to CFW (Fig 3) than expected

from a 30 % reduction in chitin content (Table 3). CFW binds to the insoluble chitin

microfibrills in the cell wall. If Chs4p somehow determines chitin chain length, its

partial loss of function will affect not only total chitin content but the structure of the

microfribrills as well. To confirm our supposition, we measured average chitin chain

length. For chitin isolated from wild type cells the degree of polymerization was 60 units,

while from chs4-C693S it was 45 (Table 4). Mutations (fks1F and gas1F) inducing cell

wall stress response lead to a two to four-fold increase in length. Nevertheless, the effect

of chs4-C693S on chitin structure is observed in these genetic backgrounds as well. We

also observed that a CFW induced cell wall stress response leads to four-fold increase in

chitin length. However, treatment with glucosamine, which activates chitin synthesis

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without induction of the cell wall integrity pathway (7) has only a minor effect on the

length of the polymer.

DISCUSSION

Previous studies suggested two roles for Chs4p in chitin synthesis. One of its

proposed functions is activation of Chs3p catalytic activity (5, 11, 32, 40). Two-hybrid

analysis indicates that this process depends on direct interaction between the catalytic

subunit, Chs3p, and Chs4p (5, 11, 32). DeMarini and coworkers (11) revealed the second

role of Chs4p, anchoring Chs3p to the septins via Bin4p, which confers septum

localization to CSIII. Two facts indicate that the two roles are separable: a) delocalized

chitin is present in bni4 but not in the double bni4, chs4 mutant, and b) it is known that a

truncated version of Chs4p allows chitin synthesis but does not localize Chs3p to the

septum (11). If Chs4p has other function than stimulation of CSIII activity at the lateral

wall (for example recruiting Chs3p to the specific scaffolding proteins) remains to

establish.

In previous reports no phenotype related to chitin synthesis was attributed to loss

of the potential prenylation site (CVIM) in Chs4p (5, 11, 32, 39, 40). Two reasons led us

to reinvestigate the role of Chs4p farnesylation. First, a prenylation motif is present in a

number of Chs4p homologues, and second, we also realized that C-terminal tagging of

CHS4 gene in the genomic locus affects chitin content (data not shown). For the first time

we demonstrate here that the intact prenylation motif of Chs4p is indispensable for full

activity of CSIII. Lack of the farnesylation consensus sequence causes approximately a

60% decrease in CSIII activity which leads to a substantial lowering in chitin content and

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partial resistance to CFW. Reduction in CSIII activity is also correlated with an

approximately 20 % decrease in average chitin chain length.

We used several approaches to prove that Chs4p is prenylated. We have shown

that unfarnesylated recombinant Chs4p is a substrate for FTase in vitro. The protein

isolated from yeast cells reacts with serum raised against N-acetyl-S-farnesyl-L-cysteine

which recognizes farnesyl or with less specificity geranylgeranyl-modified proteins. This

confirms the supposition that Chs4p is prenylated in vivo. The occurrence of

farnesylation but not geranylgeranylation is strongly supported by two facts. First,

deletion of the gene encoding the catalytic subunit of FTase increases the amount of

Chs4p which upon purification is the substrate for the enzyme in vitro. Second, changing

the farnesylation CVIM site to the known gernylgeranylation CVLL motif (31) corrects

only partially the phenotype caused by the mutation abolishing Chs4p prenylation. At this

point, however, we can not tell whether the phenotype induced by the CVIM to CVLL

motif substitution is due to the difference in structure of the attached prenyl group or

lower efficiency of prenylation by geranylgeranyl transferase I.

In almost all instances, prenylated proteins are membrane associated and protein

prenylation is often viewed as a modification which serves to increase protein

hydrophobicity, producing membrane association for proteins that otherwise lack

membrane affinity. Since Chs4p lacks any predicted transmembrane domain, one might

expect that membrane association of Chs4p would be at least to a certain extent

prenylation dependent. This prediction was not borne out, since wild type and

nonprenylated versions of Chs4p behave similarly in membrane association experiments,

and both are partially solubilized by 100 mM sodium carbonate or 0.5 NaCl as is typical

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for peripheral membrane proteins. Further, similar to the catalytic subunit of CSIII,

Chs3p, Chs4p is not sensitive to Triton X100 treatment (Fig.2A). However, it is

completely solubilized by the anionic detergent SDS. This result is compatible with the

results of DeMarini and co-workers (11), which show that localization of the Chs4p to

the septum depends on interaction with Bni4p and Chs3p in a manner independent of the

presence of CaaX box. It also suggests that localization of Chs4p to the lateral wall

depends on the interaction with Chs3p, and perhaps also other proteins. Separation of

membranes by differential centrifugation (Fig.2B) or on sucrose density gradients 9data

not shown) indicates that prenylation of Chs4p does not affect its endomembrane

trafficking. Furthermore, staining of wild type and chs4-C693S yeast strains with the

chitin binding dye CFW did not reveal chitin delocalization in the mutant cells (not

shown). However, we could not exclude more subtle changes in Chs4p localization that

might be difficult to detect by standard molecular biology methods. For example,

prenylation may be involved in efficient loading of Chs4p to the plasma membrane.

Unfortunately, in contrast to Chs3p trafficking which has been subject of numerous

studies (35, 42, 43), little is known about Chs4p trafficking apart from the fact that both

Chs3p and Chs4p relocalize in response to stress conditions which leads to chitin

deposition in the lateral wall (8, 15). It will be of interest to study the route of Chs4p

trafficking in greater detail. Such information may be crucial in a final understanding the

role of Chs4p prenylation.

Although protein prenylation may facilitate anchoring of proteins to lipid

membrane, data suggesting its role in protein interaction and activation are accumulating

(12, 17, 19, 28, 30, 38). Our data support the proposition of Magee and Seabra (36) which

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stresses the role of prenyl groups in protein-protein interaction in addition to its role in

membrane binding. Since prenylation does not affect membrane association of Chs4p but

clearly affects CSIII enzymatic activity and alters chitin chain length, is possible that the

farnesyl group attached to Chs4p interacts with a hydrophobic pocket in Chs3p,

modifying the structure of the CSIII complex, and in turn influencing the disengagement

of the nascent polymer from the enzymatic complex. This hypothesis is in good

agreement with the proposition that Chs4p is a direct activator of Chs3p (5, 11, 32, 40).

Other possible explanations of the described phenotype should be also taken in account.

The farnesyl group may be necessary, for example, for interactions with other protein

factors than Chs3p, or with the membrane bilayer during the assembly of the CSIII

complex. Here, one of the obvious candidates is Bni4p. However, we do not observe that

inactivation of BNI4 enhances or suppress CFW resistance of the chs4-C697S mutant in

comparison to the wild type background (data not shown).

There does not seem to be a specific role of farnesylation of Chs4p in the cell wall

stress response. As mentioned before, a defect in prenylation affects chitin synthesis in a

manner independent of the localization of chitin synthesis and the induction of the cell

wall integrity pathway. Also Chs3p requires Shc1p during sporulation as an alternative to

the Chs4p activating subunit (37). However, Shc1p does not posses a prenylation motif.

In spore cell walls, chitin is a substrate for chitin deacetylase, which forms chitosan (10).

Thus, the different structural requirement for the final product of Chs3p under different

conditions may not require prenylation of Shc1p.

Cell wall composition changes during growth, budding, mating, sporulation, and

stress response, and these dynamic processes require synthesis of new sets of proteins as

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well as remodeling of the crosslinking of ß-1,3- and ß-1,6-glucans

(23). The CSIII

synthesized polymer is attached to different acceptors (d-1,3-glucan at the bud neck and

d-1,6- glucan in the lateral wall) depending on the deposition site. However, both types

have the same polydisperse size profile (7). In this study we observe a new characteristic

feature of cell wall remodeling during stress response, an increase in chitin content

coupled with an increase in average chitin chain length (Table 4). This increase in chain

length is clearly associated with an increase in the rate of chitin synthesis. Our suggestion

that changes in chitin chain length is one of the features of the cell wall stress response is

confirmed by the fact that treatment with glucosamine, which stimulates chitin synthesis

in the lateral wall without induction of the cell wall integrity pathway (6) has only a

minor effect on the length of the polymer. At this point we can only speculate on the

mechanism which leads to the changes in chitin chain length. One possibility involves

changes in the kinetic properties of CSIII and/or chain length termination mechanism, i.e.

the rate of the synthesis may increase without a change in the kinetics of chain

termination under some circumstances.

Several aspects of the present study are relevant to the regulation of Chs3p

activity in vivo. In the first place, it appears that while farnesylation of the enzyme may

not influence the extent of binding of Chs4p to Chs3p or to membranes, it has a major

effect on the catalytic activity of the holoenzyme. This can easily be envisioned as an

effect on the conformation of the catalytic domain of the protein. Prenylation of Chs4p

does not change the Km value with respect to UDP-N-acetyl-D-glucosamine

(approximately 1 mM), but it increases Vmax of the intact protein. Indeed, this may be

its only function. If this conclusion is true, how can one explain the observations that

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increases in the level of Chs4p (but not Chs3p) increase chitin synthase activity (5, 40),

and that overproduction of the nonfarnesylated form of the enzyme is able to restore

chitin synthesis to wild type levels (5, 32)? The obvious answer would seem to be that

under normal circumstances Chs3p is not saturated with Chs4p, and that cellular levels of

Chs4p represent one of the factors that regulate the availability of active enzyme. Other

factors that may produce increased chitin synthesis include moderately increased

transcription of CHS4 and CHS3 (6, 18, 20, 40) and alteration in localization of the

enzyme (8, 15, 42). However, this attractive hypothesis needs to be proven. Our data

strongly encourage reconstruction in vitro of the active CSIII complex to finally

understand the role of Chs4p and explain the function of its prenylation.

ACKNOWLEDMENTS This work was supported by a grant from the National Institutes of Health GM31318 to PWR.

The authors are grateful to Dr. Laura Popolo form Università degli Studi di Milano for providing

anti Gas1p antibodies and to Dr. Martin Steffen from Boston University Medical School for

Mass Spectrometry analysis.

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FIGURE LEGENDS

FIG. 1. Chs4p is prenylated. (A) Chs4p is a substrate for farnesyl transferase in vitro.

Wt and mutated at CaaX prenylation box Chs4p purified from E. coli cells were used as

an acceptor for 3H labeled farnesyl group in an in vitro reaction with farnesyl transferase.

The proteins, after reaction, were separated by NuPAGE BisTris/SDS, blotted onto

PVDF membrane, and the position of prenylated protein was visualized by

autoradiography.

(B) Chs4p is prenylated in vivo. Wild type (left lane) or mutated at the farnesylation site

(right lane) Chs4p tagged with TAP (ProtA-TEV-CBP) epitope on the N-terminus was

purified from yeast cells and immobilized on the IgG sepharose beads. Then, CBP-Chs4p

(~82 kDa) was removed from TAP-Chs4p (~97 kDa) by cleavage with protease TEV.

IgG sepharose beads suspension samples (before [i] and after TEV cleavage) and a final

eluate containing purified wt or mutated CBP-Chs4p were analyzed by NuPAGE

BisTris/SDS and immunoblotting. TAP tagged proteins were recognized with PAP

antibodies (i) and prenylated CBP-Chs4p with anti-farnesyl antibodies (ii). (C)

Inactivation of FTase increases the level of unmodified Chs4p in cells. TAP-Chs4p

expressed in wt yeast cells or cells lacking the catalytic subunit of FTase (Ram1p) was

immobilized on IgG-Sepharose beads and used as a substrate for the FTase assay in vitro.

To measure background, the resin carrying TAP-Chs4pC693S mutant was used.

FIG.2. Prenylation does not affect subcellular distribution of Chs4p.

(A) Extraction of TAP-Chs4p. The total homogenate prepared in hypo-osmotic buffer

(HOB) was treated with 100 mM Na2CO3, 0.5 NaCl or 1% Triton X-100 (TX100) and

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the soluble and insoluble fractions were separated by centrifugation at 200,000 g for 1 h.

Treatment with 4% Triton X 100 also does not solubilize Chs3p or Chs4p (not shown).

(B) Chs4p and Chs3p fractionation by differential centrifugation of membranes from wt

and mutated (Chs4pC693S

) cells. Total membranes from Con A coated spheroplasts were

separated by differential centrifugation (13,000 g for 10 min) into a PM/ER fraction (P)

and a Golgi/endosome-rich fraction (S). The protein composition of fractions obtained

from differential centrifugations and sucrose gradients were analyzed by SDS/nuPAGE

(PM marker: Gas1p, Chs3p; ER membrane marker Dpm1p, Soluble fraction marker:

Pgk1p)

FIG. 3. Mutation in the CVIM motif of Chs4p leads to resistance to Calcofluor

white. Substitution C693S blocks farnesylation of Chs4p. Substitution M696L and I695L

changes the farnesylation CVIM sequence to the gernylgeranylation CVLL (present in

Rho1p) motif. Cells grown overnight in liquid YPD medium were diluted to a final

concentration of 0.8 OD units per 1 ml in water, then 3 ol of each suspension and three

subsequent 10-fold serial dilutions were each spotted onto YPD, YPD +50 og/ml or 200

og/ml Calcofluor white plates. Cells were incubated at 30ºC for 3 days.

FIG. 4. Mutation of the CVIM motif of Chs4p affects chitin synthse III activity.

Substitution C693S blocks farnesylation of Chs4p. Substitution M696L and I695L

changes farnesylation CVIM to gernylgeranylation CVLL motif.

(A) Chitin synthase III activity was measured by the nonradioacive method as described

(25). As an enzyme source, the total membrane fractions from yeast cells grown 6 h at 30

ºC in YPD liquid medium, YPD supplemented with glucosamine (15 mmol) or with

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Calcofluor white (25 og/ml) were used. Cultures for chitin synthase III assay were

inoculated with a saturated overnight culture and grown to midlog phase.

(B) Influence of trypsinzation on CSIII activity. As an enzyme source the total

membrane fractions from yeast cells grown 6 h at 30 ºC in YPD liquid medium, and

pretreated in the presence (+Trypsin) or absence of (-Trypsin) of protease were used.

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Table 1. Strains and Plasmids

Strain/plasmid Genotype/Description

Yeast strains:

Source or

ref.

BY4741 Mat a his3F1; leu2F1; met15F01; ura3F1; 4

BY4742 Mat７c,his3F1; leu2FＲ= lys2F01; ura3F1; 4

KG101B As for BY4741, trp1F1-312::loxP This study

KG102B As for BY4742, chs4-C693S::loxP This study

KG103B As for BY4742,CHS4::loxP This study

KG109 As for BY4742, chs4-I695L,M696L::loxP This study

TAPCHS4B1 As for KG101B, TAP:: CHS4 This study

TAPCHS4B3 As for KG101B TAP:: CHS4::loxP This study

TAPCHS4B2 As for KG101B, TAP:: chs4-C693S::loxP This study

TAPCHS4B4 As for KG101B, TAP:: chs4-I695L,M696L::loxP This study

TAPCHS4B5 As for KG101B, TRP1 TAP::CHS4::loxP,ram1F::kanMX4 This study

TAPCHS4B6 As for KG101B, TAP:: chs4-C693S::loxP, ram1F::kanMX4 This study

BY741 gas1F As for BY4741, gas1F::kanMX4 45

BY4741 fks1F As for BY4741, fks1F

pUG27 Contains MX6HIS3 disruption cassette 16

pSH47 Expresses recombinase Cre 16

pBS1761 Contains N-terminal Tap tagging cassette 34

pET30a Bacterial Expression vector Novagen

pET30a-CHS4 Expresses Poly-His-Stag-Chs4 This study

pET30a-chs4-C693S Expresses Poly-His-Stag-Chs4-C693S This study

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Table 2. Oligonucleotides Primers

Primer name Sequence

TAP1-CHS4

ACCAGGTTCTCGTCTTTTTTGGTGATAAAGTTAAAATAAAAGGATAAAA

AGAACAAAAGCTGGAGCTCAT

TAP2-CHS4 TGTGATTGCATTAAATGCTTCTTATATGGATGTACCTGCGGTGAACTTGC

CTTATCGTCATCATCAAGTG

CHS4 –290R AGGTACTGACGCAGCTCTAA

F1-CBP ATG GAA AAGAGAAGATGGAAAAAGAA

CHS4DR AGTGTAAACTGTTGCACCTATAAAGAATGAAAACAATCTAGTATGTGTA

CGCATAGGCCACTAGTGGATCTG

CHS4wt ACAAGAAGGATAAACAAGGTAAAAAAAAAAAAGACTGGTAATTATGTA

ACAGCTGAAGCTTCGTCTTCGTACGC

CHS4CtoS ACAAGAAGGATAAACAAGGTAAAAAAAAAAAAGACAGTGTAATTATGT

AACAGCTGAAGCTTCGTCTTCGTACGC

CHS4LL ACAAGAAGGATAAACAAGGTAAAAAAAAAAAAGACTGT GTATTG

TTATAACAGCTGAAGCTTCGTCTTCGTACGC

DF- TRP1 TATTGAGCACGTGAGTATACGTGATTAAGCACACAAAGGCAGCTTGGAG

TCAGCTGAAGCTTCGTCTTCGTACGC

DR-TRP1 CGAGTCTTTTAATAACTGGCAAACCGAGGAACTCTTGGTTTCTTGCCACG

CATAGGCCACTAGTGGATCTG

TRP1UP AGAGGGAGGGCATTGGTGA

CH4-1920 TGCTGAGCAGTCGATGGCAG

KanB GGATGTATGGGCTAAATG

CHS4BamHF

GGATCCATGGCAAGTTCACCGCAGGTAC

Chs4-R

GTACTTACATAATTACACAGTC

Chs4-C693S-R

GTACTTACATAATTACACTGTC

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Table 3. Chitin content

Chitin content [nmol GlcNAc/ mg cells]

Growth conditions/ mutations affecting cell wall structure CHS4 allele

YPD YPD + GlcNH2 YPD+ CFW fks gas1

wt 5.47 (± 0.23) 12.99 (± 0.42) 22.23 (± 3.11) 18.37 (± 1.37) 17.03(± 1.01)

chs4-C693S 3.39 (± 0.28) 6.79 (± 0.40) 12.64 (± 2.14) 11.33 (± 1.31) 13.7 (±1.24)

chs4-I695,M696L 4.19 (± 0.40) 8.9 (± 0.8) 17.47 (± 1.77)

chs4 1.55 (± 0.15)

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Table 4. Degree of Polymerization of Chitin.

Average chitin chain length CHS4 allele

Genetic background

Growth conditions

CHS4 chs4-C693S

Wt 59 (±6.1)* [KG103B] 46 (±1.7)* [KG102B]

Wt + Glucosamine 68 [KG103B]

Wt + CFW 206 [KG103B]

fks1F 114 [KG106B] 86 [KG104B]

gas1F 180 [KG107B] 151 [KG105B]

Chitin chain lengths were estimated as described in Experimental Procedures.

*Data point is an average of 4 independent experiments.

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Figure 1

CA B

150 kDa

75 kDa

50 kDa

37 kDa

wt chs4-C693Swt chs4-C693S

98 kDa

64 kDa

98 kDa

64 kDa

i

ii

0

1000

2000

3000

4000

1 2

[cp

m/r

eact

ion

]wt ram1F

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Figure 2

B

A

S P S P S P S P S P

HOB Na2CO3 NaCl TX100 SDS

wt

S P S P S P S P S P

chs4-C693S

HOB Na2CO3 NaCl TX100 SDS

Chs3p

TAP-Chs4p

Pgk1p

chs4-C693S

S PS P

wt

Chs3p

Gas1pTAP-Chs4pDpm1pAC

CEPT

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Figure 3

0 og/ml 50 og/ml 200 og/ml 200 og/ml

2 days 3 days

chs4Fchs4-C693S

chs4-I695L,M696L

wt

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Figure 4

AB

wt

chs4

C693

S

chs4

I695L

,M69

6L

wt

chs4

C693

S

chs4

I695L

,M69

6L

chs4

F

chit

in s

ynth

ase

III

activ

ity[n

mol

Glc

NA

c/h/

mg

prot

ein]

YPD YPD [GlcNH2] YPD [CFW]

0

5

10

15

20

25

30

35

40

wt

chs4

C693

S

chs4

I695L

,M69

6L

-Trypsin

0

5

10

15

20

25

chit

in s

ynth

ase

III

activ

ity[n

mol

Glc

NA

c/h/

mg

prot

ein]

wt

chs4

C693

Swt

chs4

C693

Sch

s4F

chs3

Fch

s4F

chs3

F

+Trypsin

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Grabinska et al 2006.pdfDISCUSSIONPrevious studies suggested two roles for Chs4p in chitin synthesis. One of its proposed functions is activation of Chs3p catalytic activity (5, 11, 32, 40). Two-hybrid analysis indIn previous reports no phenotype related to chitin synthesisACKNOWLEDMENTSFIGURE LEGENDS