THE JOURNALBIOLOGICAL OF CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Val. 266. No. 29, Issue of October 15, pp. 1975&19767,1991 Printed in U.S.A.

Chitinase Is Required for Cell Separation during Growth of Saccharomyces cerevisiae"

(Received for publication, February 28, 1991)

Michael J. KurandaS andPhillips W. Robbins

From the Center for Cancer Research and the Departmentof Biology, Massachusetts Institute of Techrwbgy, Cambridge, Massachusetts 02139

The Saccharomyces cerevisiae chitinase described MATERIALS AND METHODS l by Correaet a. (Correa, J. U., Elango, N., Polacheck, Media and Strain Constructions-S. cereuisiae strains were grown I., andCabib, E. (1982) Biol. Chem. 257, 1392- YPD medium (1% Bacto-yeast extract, 2% Bacto-peptone, 2% J. in 1397)has been cloned and sequenced. Analysis of the glucose) or SD medium (0.67%Bacto-yeast nitrogen base, 2% glucose) derived amino acid sequence suggests that the protein with nutritional supplements appropriate for selections and complecontains four domains: a signal sequence, a catalytic mentation of strain auxotrophies. Escherichia coli was grown Luriain domain,aserine/threonine-richregion,andacarBertani (LB) medium containing 1%Bacto-tryptone, 0.5% Bactoboxyl-terminal domain with high binding affinity for yeast extract, and 0.5% NaCl. LB medium was supplemented when chitin. Most ofthe enzyme produced cells is secreted appropriate with ampicillin (100 pglml). Conditions for crosses and by into the growth medium and is extensively glycosy- sporulation used in strain constructions were standard (3). The strains used in this study are listed in Table I. lated with series of short O-linked mannose oligosaca Cell Wall Suspensions-One milliliter of an (apcharides ranging in from Manzto Mans. Chitinase proximately 2 X 10' cells) was transferred to a overnight culture tube size 1.5-ml Microfuge O-mannosylationwas further examined in the temper- pelleted by centrifugation. The pellet was suspended in 200 pl of and ature-sensitive secretion mutants secl8, sec7, and solution A (0.8% NaC1, 0.02% KCl, 0.12% Na2HP0, 0.02% KHPOJ 8ec6. Oligosaccharidesisolatedfromchitinaseaccucontaining 0.1% Triton X-100. Glass beads were added (0.45-0.5 mm diameter; Braun, Melsungen, Federal Republic of Germany) to a level mulatingin cells atthenonpermissivetemperature revealed ManlandManz associated with the secl8 approximately 2 mm below the meniscus, and the cells were mixed mutant. see6 and see7 accumulatedManz-Mans with a vigorously for 2 min. The broken cell slurry was recovered with a pipette, and the glass beads were washed two times with 0.5 ml of higher proportion of Mans relative to the secreted protein. A significant amount of chitinase is also found solution A with Triton. Washes were pooled with the original slurry, and the cell washed two associatedwith the cell wall through binding COOH- times with 1walls were pelleted. The walls were thensuspended in of ml of solution A and Triton and finally terminal domain to chitin. Disruption of the gene for 200 pl of 0.1 M sodium citrate buffer, pH 3.0. Samples were assayed the enzyme leads to a defectcell separation but does as described below. in not substantially alter the level of cellular chitin. Chitinase Assays-20 pl of culture media, cell wall, or whole cell
suspensions were mixed with 80 pl of 250 pM 4-methylumbelliferyl j3D-N,N',N"-triacetylchitotrioside (Carbohydrates International,Arlo, Sweden) in 0.1 M sodium citrate buffer, pH 3.0, and incubated at 30 ' C for 1 h. The reaction was then diluted into 2.9 ml of0.5 M Chitin,a homopolymer of 81-4-linked N-acetylglucosa- glycine, NaOH buffer, pH 10.4. Debris was removedby centrifugation, mine, is a fibrous cellulose-like material that is an important and the liberated 4-methylumbelliferone was measured with a fluostructural component of fungal cell walls. In Saccharomyces rescence spectrophotometer (excitation at 350 nm, emission at 440 cereuisiae it represents only about 1% of the cell wall, but its nm). Units of activity are defined as nanomoles of 4-methylumbelliper h. specific deposition in the region of the septum is important ferone released Chitinuse from Culture Media by Chitin BidingIsolation of for mechanical stability of this temporary junction between Native chitinase was isolated from saturated yeast cultures grown in mother and daughter cells. The presence of an enzyme that YPD. Strains containingchitinase plasmids were initially grown hydrolyzes chitin, an endochitinase, in Saccharomyces was overnight in 10 ml of SD medium with appropriate nutritional supdescribed in 1982 by Correa et al. (1).We reported cloning of plements at 30 "C. The cultures were then diluted into 500 mlof YPD the gene for this enzyme, CTSl, by a plasmid-based overex- medium and allowed to grow for an additional 16 h, after which the cells was pression in 1987 (2) and have subsequently isolated the en- then were pelleted andathe supernatant collected. The mediumpm). filtered through Millipore-Type HA membrane (0.45 zyme and sequenced the corresponding gene. In the present Chitin used in binding experiments was prepared from purified chitin paper we discuss the structureof this interesting enzyme and (Sigma) which had been boiled in 1%SDS,' 1%j3-mercaptoethanol show that itplays an important role in cell separation during and thenextensively washed with water. For large scale purification of chitinase, 0.5 g of chitin (wet) was growth. added per 500 ml of filtered culture supernatant. The suspension was * This work wassupported by National Institutes of Health Grants swirled overnight on a rotary shaker at 4 "C. The chitin was then CA14051 (to P. Sharp) and GM31318 (to P. W. R.) as well as by collected by filtration through a Type HA membrane and washed Postdoctoral Fellowship CA07901 (to M. K.). The costs of publication with 500 ml of Solution A. A small volume of Solution A was added of this article were defrayed in part by the payment of page charges. to thesurface of the membrane and thechitin was resuspended with This article must therefore be hereby marked "aduertisement" in the aid of a Pasteur pipette. The suspension was transferred to a 12ml disposable Poly-Prep column (Bio-Rad), andthe column was accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted drained of excess buffer. Chitin-protein conjugates were then stored in Solution A at 4 "C. For analysis, 50-mg (wet) samples of chitin to the GenBankTM/EMBL Data Bank with accession number(s) were suspended in 100 pl of sample buffer (2% SDS, 5% j3-mercapM74069 and M74070. To whom correspondence should be addressed. Current address: 'The abbreviations used are: SDS, sodium dodecyl sulfate; kb, Bldg. 700, Repligen Corp., One Kendall Square, Cambridge, MA kilobase pair(s); PCR,polymerase chain reaction. 02139.

19758

Chitinase Is Required for Cell Separation in Yeast

TABLE I
Yeast strains used in this study Yeast strains MATa ade2-101 suc2-215am MATa his4-38 MATa u r d - 5 2 lys2-801 leu2-3,112 MATa u r d - 5 2 his4-619 leu2-3,112 MATa sec7 MATa u r d - 5 2 sec6-4 secl8-1 MATa ade2-101 MATa u r d - 5 2 lys2-801 leu2-3,112 cts1::LEUZ MATa u r d - 5 2 his4-619 leu2-3,112 ctsl::LEU2 Source D. Botstein D. Botstein D. Botstein D. Botstein R. Schekman P. Novick P. W. Robbins This study This study

19759

DBY939 DBY918 DBY1315 DBY2068 SF294-IC NY 17 PRY294 MKY1315 MKY2068

toethanol, 10%glycerol) and heated to 100 "C for 10 min. The samples E. coli shuttle vector YEp352(7). The resulting set of constructions were centrifuged and the supernatantsanalyzed by SDS-gel electro- were used to transform yeast strain MKY1315. Yeast cell lysates phoresis. The bulk of protein was eluted by heating at 65 "C for 1 h from individual transformants were screened by Western blot analysis 0-mercaptoethanol, 1% SDS. For immunization, the detergent with anti-chitinase antibody for the production of the truncated CTSl in 1% was removed by gel filtration on a Superose S-200 column (Pharmacia gene product. Two of the three constructions analyzed produced an immunologically active peptide of the expected size. One of these LKB Biotechnology Inc.) in 50 m NH4HC03, pH 8.0, and the M (designated pCT32) was chosen for the fusion experiments described preparation was then lyophilized. Approximately 1.0 mg of protein can be purified perliter of culture supernatant with the above below. Construction of pCT33 (SUC2-chitin Binding Domain Fusion&" technique. Purification of chitinase on a small scale for rapid analysis by SDS precise segment of the yeast invertase coding sequences with NotI gels was accomplished by placing 15 ml of YPD medium containing and XhoI cohesive ends was produced by in vitro DNA amplification. plasmid pRB576 (8)was linearized with EcoRI, chitinase or chitin binding domain fusions into 15-ml disposable The SUC2 containing centrifuge tubes followed by addition of 50 mg of chitin (wet). The which cut outside SUC2 coding sequences, and used as a template. tubes were mixed end-over-end at 4 "C for 3 h. The chitin was then The primers used were: 5'-GGGCGGCCGCACAAACGAAACTpelleted by centrifugation and washed three times with 15 ml of cold AGCGATAGACCTTTGGTC-3' and 5"GGCTCGAGTTTTACTSolution A (4"C). The washed pellet was suspended in 1.0ml of TCCCTTACTTGGAACTTGTCAAT-3'. The region of amplification Solution A and transferred tb a 1.5-ml Microfuge tube and centri- (1.5 kb) deleted the first 21 amino acids of the secreted message and fuged. Liquid was aspirated, and the pellet was suspended in 100 pl extends to and includes the last amino acid of the coding sequence (9). Secretion of the protein is therefore dependant on utilization of of SDS sample buffer, heated, and analyzed by electrophoresis. DNA Manipulations-Plasmids in E. coli were isolated by the rapid the signal sequence of CTSI. The DNA was concentrated from the minipreparation protocol according to standard methods (4). The amplification reaction by ethanol precipitation. The pellet was then suspended in restriction buffer and simultaneously digested with NotI lithium acetateprocedure was used for transformation of S. cerevisiae and XhoI. The invertase fragment was purified by electrophoresis in (5). E. coli was transformed using standard procedures (4). Plasmid low melting point agarose and ligated with pCT32 which had been constructions were performed according to Crouse et al. (6). Chitinase similarly digested and purified. E. coli tranformants were screened coding regions were subcloned into both M13 mp18 and mp19 to for incorporation of insert by restriction analysis of plasmid DNA. facilitate DNA sequencing. Overlapping clones were generated using Five clones containing insertswere transformed into MKY1315. Four a Cyclone I Biosystem (IBI) to create nested deletions of the insert. contained invertase activity and produced a protein product which DideoxyDNA sequencing was carried out using Sequenase (U. S. reacted with both antiinvertase and antichitinase antibodies on WestBiochemical Corp.) as specified by the manufacturer. Conditions and ern blots. One of these was chosen for further analysis and designated reagents used in polymerase chain reactions were included in the pCT33. GeneAmp DNA amplification reagent kit (Perkin-Elmer Cetus InRadiolabeling and Immunoprecipitation-Conditions for radiolastruments). Reactions were carried out in a Perkin-Elmer Cetus DNA beling yeast cells with [2-3H]mannose, isolation of radiolabeled chithermal cycler. The sequence of synthetic oligonucleotides used to tinase by chitin binding, immunoprecipitation, and analysis of radiamplify genomic CTSl sequences were 5"ATTGCTGTTTATTGGolabeled oligosaccharides have been described in detail (10). GGTCAAAACT-3' and 5'-AAAGTAATGCTTTCCAAATAAGAG-3'. Antibody Production-Polyclonal antisera production in rabbits Construction ofpCT32-A 2.5-kb BamHIIEcoRI fragment contain- was by peri-lymph nodal immunization using Freund's complete ing CTSl-2 was cloned into the coli vector, pUC19, yieldingplasmid E. adjuvant (BAbCO, Richmond, CA). Antisera were purified onan pCT3O (Fig. 3). The plasmid was digested with BstEII which cut affinity column prepared by linking purified chitinase to cyanogen within the CTSl-2 coding sequences between the signal sequence and bromide activated Sepharose (Pharmacia). Isolation of CTSl-&-The CTSl-disrupted strain MKY1315 was chitin binding domain (Fig. 4 . The linearized plasmid was used as a ) template for in uitro amplification by the polymerase chain reaction transformed with a YCp50 yeast genomic library prepared from strain which resulted in replication of vector sequences as well as deletion DBY918 (11). Transformants with restored chitinase activity were of the catalytic and Ser/Thr-rich regions. Restriction site "tails" identified as described previously (2). In brief, colony replicas for present on the ends of the primers were used to introduce two unique screening were made by pressing sterile filter paper discs onto the the restriction sites (NotI and XhoI) at the ends of the amplified frag- surface of agar plates containing transformants. The replicas were ment. NotI digestion followed by ligation joined the chitin binding then incubated in 0.1 M sodium citrate, 200 p~ 4-methylumbelliferylpH domain and signal sequence, retaining reading frame, and circularized 0-D-N,N',N"-triacetylchitotrioside, 3.0, for 30 min at 30 "C.After the plasmid. The sequence of oligonucleotides used were 5'- transfer to 0.5 M glycine, NaOH, pH 10.4, positive transformants GGGCGGCCGCTAGCAGACCTATCAAAGGCATCGGTTGG- were identified as blue fluorescent spots when viewed by a hand-held CAG-3' and 5'-GGGCGGCCGCCTCGAGTCAGACAGTACAGC- long wavelength UV lamp. CTSl-2 plasmids were then isolated from yeast (3) and characterized. TCGTACATTGGCTAAA-3'. I n Vivo Chitin Levels-Chitin levels were determined in stationary The amplified product was first ethanol-precipitated and treated with alkaline phosphatase (calf intestine). The digest was extracted phase cells according to Bulawa et al. (12). Cells were extracted with 6% KOH a t 80 "C for 90 min followed by sequential digestion with with phenol/chloroform and ethanol-precipitated. The pellet was Serratia chitinase and cytohelicase. Liberated N-acetylglucosamine suspended in restriction buffer and then digested with NotI. The was quantitated colorimetrically by the Morgan-Elson reaction. resulting fragment was purified by electrophoresis on low melting point agarose, excised, and ligated. Transformation of E. coli and RESULTS restriction analysis of plasmids obtained from transformants indicated that the majority of the clones had deleted the desired region Enzyme Purification and had incorporated the anticipated NotIand XhoI sites. The deleted Correa et al. (1) identifiedaperiplasmic chitinase in S. BamHIIEcoRI inserts isolated from several independent transformants were cloned individually into thepolylinker region of the yeast/ cereuisiae which could be extracted intact cells. In extenfrom

19760

Chitinase Is Required for Cell Separation in Yeast

m
m
m

sions of these experiments utilizing 4-methylumbelliferyl-~D-N,N,N-triacetylchitotrioseas a substrate, we found that the majority of chitinase activity produced in yeast cultures (90%) is secreted into the growth mediumwhen cells are grown in the nutrientrich medium, YPD. As shown in Fig. 1, chitinase secretion in YPD exactly parallels culture growth. Unlike radiolabeled chitin, the substrateused by Correa et al. (l), the fluorescent oligosaccharide substrate issoluble and can enter the wall, allowing direct assayof enzyme in the cell periplasm.Consistentwiththis,chitinaseactivitycan be detected in washed whole cells. Following washing and disruption of the cells with glass beads, most of the cell-associated chitinase activity remains sequestered in the cell wall (60%) and is roughly equal to the activity measured with intact cells. As described under Materials and Methods, the secreted enzyme activity can be quantitatively adsorbed from culture supernatants by addition of insoluble chitin. This interaction is stable inhigh salt (5 M NaCl), over a wide range of p H (3lo), and to many common denaturants, including 8 M urea. For the purposes obtaining protein antibody production of for or analysis on SDS the chitinase-chitin conjugate can gels, be dissociatedby heating in 1% SDS, 1% @-mercaptoethanol. The solubilized chitinase can be recovered as a single polypeptide of approximately 130 kDa. Yields in the range of 1 mg of purified protein per liter are typically recovered from a saturated stationary phase YPD culture. Less than 10% of this level is secreted into the medium when cells are grown in minimal SD medium.

130 KD

FIG. 2. SDS-polyacrylamide gel electrophoresis of secreted chitinase from various strains of S. cerevisiae. Secreted chitinase wasisolatedfrom the medium of saturatedcultures of the indicated strains by chitin binding. Bound protein was eluted by boiling in sample buffer, and samples were then analyzed on 6% polyacrylamide gels. Theamount loaded correspondstomaterial bound from 5 ml of culture medium.

slightly higher molecular weight chitinase was isolated from a CEN plasmid bank (DBY918). The two clones have only slightly different restriction maps (Fig. 3) and differ only by about 5% in nucleotide sequence (Fig. 4). T o confirm the fact that the restrictionsitepolymorphismsfoundinthe two chitinase plasmids are not due to cloning artifacts, oligonucleotides (see MaterialsandMethods) were synthesized corresponding to conserved regions at the 5 and 3 ends of the CTSl gene. These primers were then used inpolymerase chain reactions to amplify the chitinase coding regions from Strain Variation of Chitinase genomic DNA isolated fromlibrarystrains DBY918 and Analysis of the secreted chitinase from haploid strains used DBY939. Both amplifications yielded the anticipated DNA in our laboratory revealed measurable variations in chitinase product of 1.6 kb. Restrictionsitesunique to each clone size on SDS gels even though all strains have an S288C predictedto be presentineach amplified fragment were genetic background. Analysisof several representative strains observed (Fig. 5). The DBY918 PCR fragment was resistant is shown in Fig. 2. Chitinase clones were isolated from two toHindIII digestion butcutwithSphI yieldingamajor strains displaying size variation in the secreted protein using product of1.32 kbaspredictedfrom Figs. 4 and 5. The a n activity based filter screen method described previously amplified fragment from DBY939 cut with HindIII yielding as (2). the expected 1.30-kb product. Digestion of the DBY939 PCR fromtheCarlson The original CTSl genewasisolated product with SphI, however, yielded a resistant fragment and YEp24 yeast genomic library (13). The source of DNA for the 1.46-kb fragment corresponding to the second clone. The this librarywas DBY939, a strain producing a low molecular resistant fragment may represent a gene duplication in the weight chitinase. A second genomic clone correspondingto a DBY939 strain. The two chitinase genomic clones isolated from DBY939 and DBY918 by activity screens likely represent allelic variants of a unique chitinase locus. DBY939 chitinase sequences lC2 e l o 3 can beused to direct disruption of residentchitinase sequences in a diploid strain (DBY1315 x DBY2068) which contains only chitinase sequences characteristic of DBY918 by PCR analysis.Crosses withhaploidstrains DBY2068 (DBY918 chitinase) andDBY939 followed by tetrad analysis reveals a 4:O segregation of a chitinase plus to chitinase minus phenotype, whereas crosses of both haploid parents with a strain disrupted for chitinase gave a 2:2 segregation. This indicates that eachof these strains containsa distinct allele of a single chitinase gene locus termed CTSl. We have since designatedthechitinase genomicclonefrom DBY939 as 10-1 CTS1-1 and the cloneisolatedfromDBY918 as CTSI-2. 0 5 10 15 CTSl-2 is larger thanCTSl-1 as a result of two 15-base pair Time (h) direct nucleotide repeats which flank a serine/threonine-rich FIG.1. Growth and chitinase secretion from S. cereuisiae region in the protein (Fig. 4). Both alleles have equal specific growing on YPD. Cells froman exponentially growing yeast culture activities with either chitin or 4-methylumbelliferyl oligosac(DBY2068) were centrifuged, washed with fresh YPD medium, and charide substrates.
then diluted to a final OD,, of 0.25 in 200 ml. The culture was incubated a t 30 C with agitation. At the indicated times 1-ml samples Domain Structureof Chitinase of theculture were takenandthe OD6m determined. Cells were The deduced amino acid sequence of the Saccharomyces removed by centrifugation, and secreted chitinase activity was measchitinase clearly suggests that the protein divided into four is ured as described under Materials and Methods.

CTS1-1

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Chitinase Is Required for Cell Separation in Yeast

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FIG. 3. Restriction endonuclease maps of CTSl-1and CTSl-2. Chitinase coding regions and direction of transcription are indicated for pCT12 and pCT2O by arrows. The CTSl-1 plasmid pCT12 was constructed from the 7.0-kb BarnHI-SalI fragment of previously described pCT2 (2). CTSI-2 (pCT2O) was isolated from a YCp50based yeast genomic library with the same activity-based filter screen used to clone CTSI-1. pCT18 and 30 are subclones of these plasmids constructed by standard cloning methods. Disruption of CTSl was performed using the 5.5-kb BarnHI-Hind111 fragment of pCT19 which contains a LEU2 insertion that blocks chitinase expression. pCT32 encodes a site-specific deletion that results in the secretion of only the COOH-terminal chitin binding region of CTSI-2. pCT33 contains yeast invertase sequences fused in frame to the chitin binding domain. B , BarnHI; Bg,BglII; RZ, EcoRI; H , HindIII; K , KpnI; N , NotI; P, PstI; S , SalI; Sp, SphI; X , XhoI.

CTSl-2 secreted gene products have the same oligosaccharide profiles. Whenthe enzyme is expressed ina conditional mutant in which synthesis of dolichol-P-mannose (the donor of the first 0-linked mannose residue) can be blocked, the protein migrates with the predicted molecular weight for the unglycosylated protein (60 kDa) (10) on SDS gels. Assuming Asn-Thr-Asn-Ile-Ala-Val-Tyr-Trp-Gly-Gln-Asn-Ser-Ala) each oligosaccharide adds roughly 600 daltons (a trisacthat which exactly corresponds to the deduced sequence for CTSl- charide) to the molecular mass of the protein, it follows that I starting at amino acid 21. most if not all of the serines and threonines (approximately Catalytic Domain (Amino Acids 21-327)Three lines of 90) in this region must carry sugar chains to account for the evidence support the hypothesis that this region contains the apparent 130 kDa size observed on SDS gels. It should be hydrolytic domain of the enzyme. pointed out, however, that this is probably an overestimate, 1)A HindIII deletion of CTSl-1 (Fig. 3), pCT18, eliminat- since carbohydrate chains bind SDS per unit weight than less ing 80 amino acids from the COOH terminus of the protein does the protein portionof the molecule and additionally may results in secretion of a smaller chitinase protein that retains interfere with SDS binding to the polypeptide. A single pofull catalytic activity toward both chitin and oligosaccharide tential site for N-glycosylation (Asn-Phe-ser) is present near substrates, yet displays an apparent decreased affinity toward the COOH terminus which is apparently not utilized since the mobility of the protein is insensitive to digestion with insoluble chitin (Fig. 2 , lane ACTS1-1 ). 2 ) The proposed catalytic domain is homologous with a either endoglycosidase H or N-glycanase. plant chitinase. The CTSI-2 sequence was compared with High Affinity Chitin Binding Domain (Amino Acids 481available chitinase, cellulase, and lysozyme sequences from 562)The last 80 amino acids of CTSl encodes a noncatalytic Genbank and Protein Identification Resource data bases as peptide capable of high affinity binding to chitin. This hywell as those from the recent literatureby dot plot comparison, pothesis is supportedby the following data. Most significant was the relationship between CTS1-2 and a 1) The carboxyl-terminal deletion of CTSI-1 mentioned cucumber chitinase (14) produced in response to pathogen above does not bind with high affinity to chitin or to cell invasion (Fig. 6). The roughly 30% similarity at the protein walls (Fig. 2). The truncated enzyme expressed and secreted level extends over the entire length of the plant sequence. in a chitinase-disrupted strain cleaves both soluble 4 MU Interestingly, the similarity is limited to the first 300 amino oligosaccharides and radiolabeled chitin. Also, cell wall prepacid residues of CTSl-2 and ends at the start region rich arations produced by disruption of cells carrying this mutant of a in serine and threonine. have less than 10% of the wild-type levels of activity when 3) Two small regions are conserved in the Saccharomyces assayed with 4-methylumbelliferyl trisaccharide. Surprischitinase, the cucumber chitinase (14), several bacterial chi- ingly, the COOH-terminal deletion shows an enhanced rate tinases (15),endoglycosidase H (16), and a mammalian lyso- of chitin hydrolysis (Fig. 8). somal chitobiase. All cleave the @l-4 glycosidic bond between 2) Controlled proteolysis of the wild-type enzyme bound to adjacent N-acetylglucosamine residues. The conserved re- chitin resulted in the production of an undigested chitin gions are highlighted in Fig. 6 and are shown in detail in Fig. bound fragment. This peptide had an apparent molecular 7. These common residues could obviously play an important mass of 18 kDa (Fig. 9) and gave a clean amino-terminal role in the catalytic function of these enzymes. analysis (Ser-Asp-Ser-Thr-Ala-Arg-Thr-Leu-Ala-Lys-GluSerlThr-rich Domain (Amino Acids 328-480)Greater Leu-Asn-Ala-Gln-Tyr) which corresponds exactly to the sethan 50% of the amino acid residues in this region are serine quence which starts at amino acid 480 of the CTSl-2 sequence or threonine which potentially can act as acceptor sites for 0- (Fig. 4). glycosylation. Mannose labeling of the protein followed by 3) A precise deletion was constructed of CTSI-2 using treatment with mild alkali to facilitate @-elimination demon- synthetic oligonucleotidesto remove amino acids 21-481. This strates that the enzyme carries the typical array of short resulted in directfusion of the signal sequence to the proposed mannose oligosaccharides containing 2-5 mannose residues chitin binding domain. Expression of this construction in a (see Chitinase Secretion Pathway). Both the CTSl-1 and chitinase-deficient strain resulted in secretion of an 18-kDa domains. We describe these below and suggest a function for each. Amino acid numbers refer to the CTSl-2 sequence. Signal Sequence (Amino Acids 1-2O)The first 20 amino acids serve as a typical cleavable signal sequence. Aminoterminal analysis of the secreted CTSl-1 gene product gives a clean sequence for 18 amino acids (Phe-Asp-Ser-Ser-Ala

19762
CTSI-1

Chitinase Is Required for Cell Separation in Yeast

A

CTSI-2

TATGTGTTTCAGATGTTW\GTIWLCATTI\GUTTGGTGTARGARCT~CC~GATffiTTC~~TTC~ARC~ARTA~AC~TATTT~CAT
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H S L L Y I I L L F T +1 C T G ......................... CACMTTCTTACTACTGCCARCCGATGCCTTTGATAGGTCTGCTMCAC~TATT~GTTTATTG~C~~C~C~ARC~~TC~T~~TTACTGT~TCTT 150

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S
140

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160

511

TAAGAACTTTGTTTGCCGARGGT~IWLGCARTATTACCTTTCTGCCGCACC~CARTGTCCATACCC~T~GTT~TGACTTG~T~~ATTGAT~T~G~CA ""-""-""-""-""-""~""-""-""-""t""""""+""+""+""+""+""+""+""*""~""""+""+ 630

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FIG. 4. Nucleotide and derived amino acid sequences of CTSI-Iand CTSl-2. entire DNA sequence The of CTSI-2 is shown with the corresponding amino acid sequence printed below it. Differences in the CTSI-1 allele are printed above the C T S l - 2 sequences. Nucleotides deleted in CTSI-1 are indicated by asterisks. The common BglII site near the 5' end of both genes is underlined. HindIII, BstEII, and SphI sites within the coding regions are also indicated. Positions corresponding to PCR primers ("Materials and Methods") used to amplify genomic

Chitinase Is Required for Cell Separation in Yeast

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FIG. 5. PCR amplificationof CTSl from librarystrains used in this study. Genomic DNA from DBY918 and DRY939 was
used as templates for PCR reactions. Primers ("Materials and Methods") directed amplification of greater than 90% of the C T S I coding sequence. Reaction products were analyzed on 1% agarose gels and visualized by staining with ethidium bromide. Restriction site polymorphisms indicated in Figs. 3 and 4 are illustrated.

peptide which displayed high affinity binding to chitin (Fig. 10, lane 3 ) . 4)Expression of a portion of the yeast invertase gene, SUC2, fused in frame with the CTSI-2 signal sequence and chitin binding domain resulted in secretionof invertase activity into the culture medium. This material was glycosylated, enzymatically active, and bound efficiently to chitin (Fig. 10, lanes 4 and 5). The chitin-invertase fusion complex retains the ability tohydrolyze sucrose. It is striking thatanalogous noncatalytic high affinity cellulose bindingdomainshave been found in bacterialand fungal cellulases (17). It hasbeen demonstrated recently that a synthetic peptide (34-mer) from the binding domain of a Trichoderma reesi cellulase binds with high affinity to cellulose (18).Comparison of this 34-mer with the chitin binding domain reveals conservation of many amino acids including a n exact match of a block of 7 amino acids flanked by 2 cysteines (Fig. 7). The chitin binding domain, however, does not display significant affinity for cellulose. The Chitinase SecretionPathway-A series of temperaturesensitive mutants have been used to define the pathway of protein secretion in yeast (19).Previously, transit through this pathway hasbeen defined in terms of events that occur in Asn-linked glycoprotein assembly. It was therefore of interest to examine the glycosylation state of chitinase, an 0mannosylated protein, blocked a t comparable points in the secretion pathway. Strainscontainingsecl8, sec7, or sec6 mutations were initially examined for chitinase secretion a t the permissive temperature (24 "C). All strains produced a chitinase of similar size as determined SDS-gel electrophoby resis. On shift to the nonpermissive temperature (37 "C), chitinase secretion was blocked and the enzyme accumulated withinthe cells. Thetrappedchitinase wasmetabolically DISCUSSION labeled with [2-'H]mannose and isolated by immunoprecipitation. Secreted enzyme was isolated by chitin binding. The The CTSIgene encodes an endochitinase which hydrolyzes intracellular and secreted forms were resolved by SDS-gel both insoluble chitin and soluble substrate analogs. We deCTSI sequences are indicated by >>>>. Amino acid residue numbers for the CTSI-2 sequence as well as proposed domain junctionsare printed helow the deduced amino acid sequence.

electrophoresis, blotted to nitrocellulose, and visualized by autoradiography. All three sec mutants accumulated a form of the enzyme which was clearly different than the one that isnormally secreted.Awild-type strain producedacellassociated chitinase identical to the secreted form (Fig. 11A). Sections of the blot corresponding to radiolabeled bands were excised and treated with mild base to remove the oligosaccharides which were subsequently resolved by paper chromatography (10). The size of the accumulated sec/chitinase intermediates on SDS gels correlates with the size distribution of attached 0linked oligosaccharides (Fig. 12). A wild-type strain produces a 130-kDa enzyme which contains a series of oligosaccharides ranging in size from Mann to Mans. The sed8 strain accumulates a 110-kDa intermediate containing only mannobiose and mannose. Sec6 and strains produce a 180-kDa intersec7 mediate which contains a significantly higher proportion of Mann than does the secreted form of the enzyme. The same biosynthetic intermediates with similar oligosaccharide profiles are observed in wild-type strains following a short pulse labeling with [2-:'H]mannose (Fig. 11R). Chitinase Disruption-To investigate thepossible function of the chitinase, a plasmid was constructed with the Saccharomyces LEU2 gene insertedinto CTS1-1 170 base pairs upstream from the chitinase start codon a t a convenient RglII site (Fig. 4). A purified restriction fragment containing the auxotrophicmarkerandflankingchitinase sequences was used to transform a diploid strain (DBY1315 x DBY2068) resulting in disruption of the resident CTSI allele by homologous recombination (20). In these experiments, both haploid parents were found to secretea similar size chitinase (Fig. '2) and to contain restriction sites characteristic of the CTSI-2 allele as determined by PCR analysis. Diploid transformants were sporulated. Dissection followed by germination generally produced four viable spores perascus. As expected, growth on leucine-deficient media co-segregated with a lackof chitinase activity in a 2:2 distribution in analyzed asci. Integration at the CTSl locus wasconfirmedinseveral chitinaseminus strains by Southern blot analysis (data not shown). The lack of chitinase activity resultsin a clear inability of the cells to separate normally. This block in cell separation was manifest in large aggregatesof cells attached by their cell septum regions. Two patterns of cell aggregation were observedin the chitinase-disrupted strains which were later traced to an independently segregating gene contributed by DBY1315 which affected bud placement.As shown in Fig. 13, a disrupted strain which displayed an axial budding pattern typical of haploid cells grew as tightly packed clusters, whereas cells with an apparently random budding pattern formed larger branched arrays of cells. Sites of attachment were confirmed by stainingwith Calcofluor to be at the septum region of the cells. It is striking that Sakuda a1. (21) et have recently shown that dimethylallosamidin, a potent inhibitor of yeast chitinase, produces the same defect in cell separation displayed by chitinase deletion strains. The separation phenotype can becomplementedwitha plasmid containing either CTSl-1 or CTSl-2. The COOHterminal deletion without the chitin binding region, however, only partially complements the defect on a multicopy plasmid.

19764

Chitinase Is Required for Cell Separation in Yeast

scribe here the domain structure of the CTSl protein which and disaccharides were also found to accumulate in s e d 8 is summarized schematically in Fig. 14. Since most of the when bulk cell mannan was examined (23). This result is at endochitinase activity in yeast cultures is found free in the variance with the prediction that 0-mannosylated proteins cell medium or localized to the wall, the enzyme must traverse accumulating in the endoplasmic reticulum should contain the usual cellular secretion pathway. The signal sequence is mannose but not oligosaccharide moieties. One explanation cleaved, and theserine/threonine-rich domain is glycosylated for accumulation of disaccharide would be leakiness in the with sugar chains containing from 2 to 5 mannose residues. s e d 8 mutation. However, we did not observe an intermediate Addition of the first mannose residue probably occurs in the which might correspond to chitinase glycosylated with only endoplasmic reticulum by a transfer of sugar from dolichol- single mannose residues. The possibilities that the second P-mannose. Subsequent elongation of the mannose chains is mannose residue is added co-translationally in the endothought to occur in the Golgi with GDP-mannose serving as plasmic reticulum or that the s e d 8 block defines a compartdonor (22). ment that includes limited post-endoplasmic reticulum With respect to this scheme we have made some intriguing processing must also be considered. observations by examining chitinase that accumulates in sec A secretion block at the Golgi stage (sec7) or in post-Golgi mutants blocked at different stages in the secretion process. secretion vesicles (sec6) results in accumulation of an interTwo distinct biosynthetic intermediates were identified. One mediate that appears to be Uover-mannosylated. compared As had a lower apparent molecular weight than the fully proc- with the secreted enzyme, a higher proportion of Man5 is essed secreted enzyme and was found to accumulate in as e d 8 found in this intermediate with a corresponding reduction of strain, whereas the other had a higher molecular weight and Man4. These data suggest that outer chain mannose residues accumulated in both sec7 and sec6 strains. These forms are are removed concurrent to secretion of the enzyme. This probably physiologically relevant since they can also be ob- reaction could occur in the periplasm since the same high served in wild-type cells at short labeling times (15 min). proportion of Man5 is observed even as late as thesec6 block. Greater than 50% of the carbohydrate label found on the A minor band of overmannoslyated material also appears to low molecular weight s e d 8 chitinase intermediate was found accumulate in the s e d 8 strain (Fig. 1lA). This may represent to be mannobiose with the remainder being mannose. Mono- protein just transferred the to Golgi at the time of temperature shift where it is further elongated with labeled mannose. None Sional Sequence Ser, Thr Rich ReOiDn of this material, however, is further converted to the final H secreted form during this period, suggesting that the s e d 8 mutation may have secondary effects on the later stages in secretion. These observations will require further investigation before a definitive interpretation is possible. Following fusion of secretion vesicles with the plasma membrane, chitinase has two potential fates: incorporation into the cell wall or release into thegrowth medium. That portion of the enzyme that becomes associated with the cellwall apparently functions in cell separation as evidenced by the phenotype of the chitinase disruption. This association with the cell wall is dependent upon the COOH-terminal chitin binding domain. Deletion of this domain from a CTSl plasmid results in a decreased ability of the gene to complement the separation defective phenotype of a CTSl disruption. These YEAST CHlTlNASE data suggest that thechitin binding region functions in localFIG. 6. Sequence similarity of Saccharomyces and cucum- izing the enzyme to cell wall chitin where it hydrolyzes chitin ber chitinases. The deduced amino acid sequences of Saccharomyces cells. Analysis of eight and Cucurnis satiuis (14) chitinases are compared using dot matrix fibers which join mother and daughter computer analysis. Points represent identity in 8 out of 30 amino chitinase minus and eight chitinase plus segregants from the gene disruption experiment, however, reveals chitin levels to acids.

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FIG. 7. Sequence comparisons of Saccharomyces chitinase with other chitinases and glycosidases. Regions 1 and 2 of the yeast chitinase shown in Fig. 6 are aligned with other chitinases or related sequences. Identities with the yeast enzyme are enclosed by rectangles. Also shown is similarity between the COOH-terminal chitin binding domain of the yeast enzyme and a portion of a fungal cellulase reported to display high affinity binding to cellulose. Sequences forrat liver chitobiase, Streptomyces plicatw, and Vibrio chitinases represent unpublished data from Dr. N. N. Aronson, Jr., Dr. Janice Pero, and Dr. Rodger Laine, respectively.

Chitinase Is Required for Cell Separation in Yeast

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FIG.8. ['HIChitin solubilization by CTSZ-2, CTSZ-2, by and the CTSZ-1 deletion enzyme. Medium from a CTSI-disrupted
strain (MKY1315) transformed with the chitinase plasmids pCT12 (intact CTSZ-I),pCT18 (CTSZ-I deletion), pCT20 (intact CTSI-2), or YEp352 (vector control) were used for sources chitinase. Medium of containing 300 units of enzyme (4-methylumbelliferyl substrate) was concentrated to approximately 0.1 ml in a Centricon 30 microconcentrator (Amicon). For the vector control (disruption) a volume of medium equal to the CTSI-2 sample was processed. Concentrated samples were assayed and volumes were adjusted to give 2000 units/ ml. 0.1 ml of chitinase preparation was then added to 0.9 ml of 0.1 M citrate buffer, pH 3.0, containing 3 mg of['HI chitin. Radiolabeled chitin was prepared by acetylation of chitosan with tritiated acetic anhydride according to Molano et al. (31). Reactions were continuously mixed by rotation at 30 "C. At the indicated times 0.1-ml samples of the suspensions were removed, centrifuged, and assayed for radioactivity.

transformed with pCT2O (CTSI-2 lune I ) , YEp352 (vector control: lune 2), pCT32 (CTSI-2 deletion expressing only the chitin binding domain: lane 3 ) ,and pCT33 (invertase chitin binding domain fusion: lune 4 ) . Protein was isolated from the culture medium by chitin binding and analyzed on 12% polyacrylamide gels. Lune 5 shows the elutedinvertase-chitinase fusion aftertreatment with endo-0-Nacetylglucosaminidase H.

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FIG. 11. Synthesis of chitinase in secretion mutants. chiA, tinase was isolated from wild-type (DBY2068), secl8, sec7, and sec6 strains by immunoprecipitation after labeling with [2-"H]mannose for 90 min at 37 "C. Chitinase secreted into the growth medium was isolated by chitin binding. The labeled samples were separated by SDS-PAGE, transferred to nitrocellulose by electroblotting, and then visualized by autoradiography using tritium-sensitive film (10). B, chitinase was isolated from DBY2068 by immunoprecipitation after a 15-min labeling with [2-'H]mannose at 30 "C and processed as above. Mobility of chitinase forms observed in A are indicated.

2214FIG. 9. Partial proteolysis chitin-chitinase complexes. 25 of mg of chitin-chitinase complex (wet) was suspended in 50 pl of 50 mM Tris, pH 8.0, and the indicated amount of proteinase K was added to each reaction. Digests were then incubated for 16 ha t 24 "C. The samples were centrifuged, supernatants removed (S), and 1.25 pl of phenylmethylsulfonyl fluoride (40 mM in isopropyl alcohol) was added. After 30 min a t room temperature a second aliquotof phenylmethylsulfonyl fluoride was added followed by another 30-min incubation. The samples were dryed under vacuum and suspended in 30 pl of sample buffer, boiled, and loaded on a 12% polyacrylamide gel. Material still bound to chitin( C B )was eluted with sample buffer and loaded on the polyacrylamide gel following three washes with 50 mM Tris, pH 8.0.

be relatively constant at 0.2% (wet weight) with a variation of less than 20% in all strains analyzed. It is possible that only internal chain cleavage reactions occur in vivo, yielding a net conservation of total cellular chitin but a decrease in average chain length. A suggestion has been made that yeast chitinase may under some circumstances "overdigest" and damage thecell wall and that this damage can repaired by be chitin synthaseI (24). Beforethis hypothesis can accepted be or even interpreted in detail we must obtain further information concerning localization of chitinase and the normal restrictions that are made on its action the cell wall. in Available data indicate that several classes of chitinases For have exist in nature. example, bacterial chitinases (15,25) been characterized which are secreted anddegrade chitin for have been use as a carbon source. Numerous other chitinases studied from plant sources (14,26-28) which exhibit antibacterial or anti-fungal activity and areproduced in response to

19766
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Chitinase Is Required for Cell Separation in Yeast

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FIG. 12. Profiles of mannooligosaccharides attached tochitinase blocked at different stages in secretion. Regions of the nitrocellulose filter corresponding to the major bands seen in Fig. 11B were excised usingtheautoradiogram as a template.Filter segments were treated with 0.1 N NaOH overnight a t room temperature. The released oligosaccharides were neutralized with acetic acid and were resolved by descending paper chromatography on Whatman FIG. 13. Phase contrast microscopy of CTSI-disruptedcells. No. 1 with ethyl acetate/butanol/acetic acid/water (3:4:2.5:4) as the A: chitinase plus, axial budding pattern (DHY206X); 13: chitinase solvent. Lanes were cut into 1 X 2-cm strips, added to the bottom of minus, axial budding pattern(MKYZOfX);C: chitinase minus, random 10-ml scintillationvials, and radioactive oligosaccharides were eluted budding pattern (MKY1315). by addition of 1 ml of H20. Scintillation fluid was added prior to counting.

wounding or pathogeninfection. The Saccharomyces chitinase described here is functionally unique in that it apparently acts physiologically to selectively modify the yeast cell wall. FIG. 14. Schematic representationof yeast chitinase. Interestingly, thehydrolytic domain is clearly homologous to a pathogenesis-related plant chitinase. Recently, it has been dissociation constants shown that a Kluyveromyces yeast killer toxin shares homol- the polymer. Wehave not measured the ogy with chitinases from both plant and bacterial sources (29, for these complexes, but in practical terms they can only be 301. As shownin Fig. 7 thisrelationshipextendstothe disrupted by denaturation of the protein. It is noteworthy Saccharomyces enzyme and other chitinase-like glycosidases. that a number of cellulose and starch-degrading enzymes have Perhaps the secreted Saccharomyces chitinase plays a dual recently been found to carry noncatalytic affinity binding high role by also suppressing the growth of other microorganisms domains (17) for their respective substrates. One hypothesis through hydrolysis of cell wall chitin or related polysaccha- is that these domains play roles in increasing the catalytic rides. efficiency of these glycosidases. In Saccharomyces we have Detailed physicalstudy of the association the small chitin shown that the presence of the chitin binding domain does of binding domain with the essentially hydrophobic surface of not increase the rate of chitin hydrolysis but rather may efficiency. On the other hand, the bindinsoluble chitin fibers should be extremely interesting. As decrease its catalytic mentioned above, we have already shown that other peptides ing domain clearly is required for localization of the enzyme fused to the chitin binding domain develop high affinity for to the yeast wall. Apparently, this cell general structural motif

Chitinase Is Required for Cell Separation Yeast in

can be used to either enhance or limit the action of glycosidases toward insoluble carbohydrates.

19767

14. Metraux, J. P., Burkhart, W., Moyer, M., Dincher, S., Middlesteadt, W., Williams, s., Payne, G., Carnes, M., and Ryals, J. (1989) Proc. Natl. Acad. Sci. U. A. 86,896-900 S. 15. Jones, J., Grady, K., Suslow, T., and Bedbrook, J. (1986) EMBO Acknowledgments-Wewould like to thank C. E. Bulawa, S. C. J. 5,467-473 Hubbard, P. Orlean, and K. D. Kuranda for useful discussions and 16. Robbins, P. W., Trimble, R. B., Wirth, D. F., Hering, C., Maley, suggestions. We also wish to thank E. Bothwell for assistance preF., Maley, G. F., Das, R., Gibson, B. W., Royal, N., and paring the figures. Biemann, K. (1984) J. Biol. Chem. 259,7577-7583 17. Knowles, J., Lehtovaara, P., and Tuula, T. (1987) Trends BioREFERENCES technol. 5,255-261 18. Johansson, G., Stahlberg, J., Lindeberg, G., Engstron, A., and 1. (1982) J. Biol. Chem. 267,1392-1397 Pettersson, G. (1989) FEBS Lett. 2 4 3 , 389-393 2. Kuranda, M. J., and Robbins, P. W. (1987) Proc. Natl. Acad. Sci. 19. Schekman, R., and Novick, P. (1982) Metabolism and Gene U.S.A. 84,2585-2589 Expression, pp. 361-398, Cold Spring Harbor Laboratory, Cold 3. Sherman, F., Fink, G.R., and Hicks, B. J. (1986) Methods in Spring Harbor, NY Yeast Genetics, Cold Spring Harbor Laboratory, Cold Spring 20. Rothstein, R. J. (1983) Methods Enzymol. 1 0 1 , 202-211 Harbor, NY 21. Sakuda, S., Nishinato, Y., Ohi, M., Watanabe, M., Takayama, S., 4. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Isogai, A., and Yamada, Y. (1990) Agric. Biol. Chem. 54,1333Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1335 Cold Spring Harbor, NY 22. Tanner, W., and Lehle, L. (1987) Biochim. Biophys. Acta 9 0 6 , 5. Ito, H., Fukuda, Y., Murata, K., and Kimura, A. (1983) J. Bac81-89 teriol. 163,163-168 23. Haselbeck, A., and Tanner, W. (1983) FEBS Lett. 168,335-338 6. Crouse, G . F., Frischauf, A., and Lehrach, H. (1983) Methods 24. Cabib, E., Sburlati, A., Bowers, B., and Silverman, S. J. (1989) J. Enzymol. 101,202-211 Cell Biol. 108,1665-1671 7. Hill, J. E., Myers, A. M., Koerner, T. J., and Tzagoloff, A. (1986) 25. Robbins, P. W., Albright, C., and Benfield, B. (1988) J. Biol. Yeast 2,163-167 Chem. 263,443-447 8. Kaiser, C.A., Preuss, D., Grisafi, P., and Botstein, D. (1987) 26. Parsons, T. J., Bradshaw, H. D., and Gordon, M. P. (1989) Proc. Science 236,312-317 Natl. Acad. Sci. U.S. A. 8 6 , 7895-7899 9. Taussig, R., and Carlson, M. (1983) Nucleic Acids Res. 11,194327. Shinshi, H., Mohnen, D., and Meins, F. (1987) Proc. Natl. Acad. 1954 S C ~U. A. 84.89-93 . S. 10. Orlean, P., Kuranda, M. J., and Albright, C. F. (1991) Methods 28. Broglie, K. E., Gaynor, J. J., and Broglie, R. M. (1986) Proc. Natl. Enzymol. 194,682-697 Acad. Sci. U. A. 83,6820-6824 S. 11. Rose, M., Novick, P., Thomas, J., Botstein, D., and Fink, G. 29. Bradshaw, H. D. (1990) Nature 345,299 (1987) Gene (Amst.) 6 0 , 237-243 30. Stark, M. J. R., Mileham, A. J., Romanos, M. A., and Boyd, A. 12. Bulawa, C. E., Slater, M., Cabib, E., Au-Young, J., Sburlati, A., (1984) Nucleic Acids. Res. 12, 6011-6030 Adair, W. L., and Robbins, P. W. (1986) Cell 4 6 , 213-225 31. Molano, J., Duran, A., and Cabib, E. (1977) Anal. Biochem. 8 3 , 13. Carlson, M., and Botstein, D. (1982) Cell 2 8 , 145-154 648-656