Vol. 156, No. 3JOURNAL OF BACTERIOLOGY, Dec. 1983, p. 1214-12200021-9193/83/121214-08$02.00/0Copyright © 1983, American Society for Microbiology

Variation of (1-*3)-f3-Glucanases in Saccharomyces cerevisiaeDuring Vegetative Growth, Conjugation, and Sporulation

NGUYEN H. HIENt AND GRAHAM H. FLEET*

School ofFood Technology, University ofNew South Wales, Kensington, New South Wales, Australia, 2033

Received 28 Decemilber 1982/Accepted 24 August 1983

The total (1-+3)-,-glucanase activities associated with cell extracts and cellwalls of Saccharomyces cerevisiae were measured during vegetative growth,conjugation, and sporulation. Using a system of column chromatography, weresolved (1--*3)-13-glucapase activity into six different enzymes (namely, glucan-ases I, II, IIIA, IIIB, IV, and V). The contributions of the individual enzymes tothe total activity at the different stages of the life cycle were determined. Totalglucanase activity increased during exponential growth and decreased in station-ary resting-phase cells. Glucanase IIIA was the predominant enzyme in stationaryresting-phase cells. Glucanases I, II, IIIB, and IV were either absent or present atlow levels in stationary phase cells, but their individual activities (in particular4glucanase IIIB activity) increased substantially during exponential growth. Total(1-3)-3-glucanase activity did not change significantly during conjugation of twohaploid mating strains, S. cerevisiae 2180A and 2180B, and no notable changeswere detected in the activities of the individual enzymes, Sporulation wasaccompanied by a rapid increase and then a decrease in total glucanase activity.Most of the increase was due to a dramatic rise in the activity of glucanase V,which appeared to be a sporulation-specific enzyme. Glucanase activity was notderepressed by lowering the glucose concentration in the growth medium.

Glucans containing (1-*3)-p linkages are re-sponsible for the strength and integrity of yeastcell walls, and it has been proposed that endoge-nous (1--3)-A-glucanases effect controlled wallhydrolysis during cell expansion, budding, con-jugation, and sporulation (4). Evidence for this isbased on reports that yeast (1-*3)-f3-glucanaseactivity varies throughout the life cycle. In Sac-charomyces cerevisiae, budding and sporulationare accompanied by an increase in (1-*3)-1-glucanase activity (1, 10, 11). Cells from conju-gating cultures of Hansenula wingei and Schizo-saccharomyces versatilis exhibit much higher(1-÷3)-P-glucanase activities than cells fromnonconjugating cultures (2, 3).Recent studies have shown that several differ-

ent (1-*3)-3-glucanases may occur in one yeastspecies, and these observations have raised thepossibility that different enzymes may functionat different stages during the cell cycle (4). WithS. cerevisiae and Schizosaccharomyces pombe,for example, there is now some evidence tosuggest that certain (1-*3)-3-glucanases are spe-cifically associated with sporulation (8, 11, 12).Direct association of a particular glucanase ac-tivity with a particular morphogenetic event,

t Present address: C.S.R. Ltd., Research Division, Pyr-mont, New South Wales, Australia.

however, requires sound knowledge of the vari-ous glucanases that may occur in a yeast speciesand a means of measuring the individual en-zymes at different stages during the cell cycle.

In the accompanying paper we describe theisolition and characterization of six different(1-43)-P-glucanases from cell-free extracts andcell walls of haploid and diploid strains of S.cerevisiae 2180 (6). These enzymes were desig-nated glucanases I, II, IIA, IIIB, IV, and V. Wealso report a convenient column chromatograph-ic procedure for the separation and measure-ment of these individual enzyme activities (6).As part of an investigation into the function andregulation of P-glucanases in yeasts, in thispaper we describe the variation of the six p-glucanases in S. cerevisiae during the vegetativegrowth cycle and during the sexual phases ofconjugation and sporulation. Since the glucoseconcentration in the growth medium is known toregulate the production of ,B-glucanases in somefungi (9), the effect of this sugar upon (1--3)-,B-glucanase production in S. cerevisiae is alsodescribed.

MATERIALS AND METHODSYeast strains. Haploid mating type strains S. cerevis-

iae 2180A (mating type a) and S. cerevisiae 2180B(mating type a) and diploid strain S. cerevisiae 595 are

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the same strains described in the accompanying paper(6). Strain 2180B was used in vegetative growth stud-ies, and strains 2180A and 2180B were used forconjugation experiments. S. cerevisiae 595 was select-ed for sporulation studies due to its high sporulatingability.

Culture conditions for vegetative growth. Stock cul-tures were maintained on slants of malt extract agarand were subcultured into seed cultures containingYEG medium (5% glucose, 0.5% yeast extract) 48 hbefore an experiment was started. Liquid cultures forglucanase studies were grown in 3-liter flasks contain-ing 1 liter of YEG medium. A 1% inoculum was used,and the cultures were incubated with orbital shaking(800 rpm) at 30°C. At different times after inoculation,the yeast cells were harvested by centrifugation, andcell walls, cell extracts, and wall autolysates wereprepared as described in the accompanying paper (6).To investigate the effect of glucose concentration in

the growth medium on glucanase production, expo-nential phase cells were harvested, washed once withsterile distilled water and once with glucose-deficient(GD) medium (0.01% glucose, 0.5% yeast extract), andthen suspended in the same medium. The culture wasincubated, and samples of cells were harvested atregular intervals for glucanase analysis. After incuba-tion for 25 h, the remaining cells were harvested,washed once with sterile distilled water and once withfresh YEG medium, and suspended in YEG medium.The cells were incubated for an additional 10 h, andsamples were taken at regular intervals to check forrenewed growth and levels of glucanase activity. Cellconcentrations were estimated by measuring the opti-cal densities of the cell suspensions at 550 nm. Theglucose concentration in the growth medium wasdetermined by using glucose hexokinase reagents ob-tained from Boehringer Mannheim Diagnostica.

Yeast conjugation. S. cerevisiae 2180A and 2180Bwere conjugated under the conditions for mass hybrid-ization of S. cerevisiae described by Fowell (5). Thetwo opposite mating type strains were grown separate-ly in YEG medium to early stationary phase, harvest-ed by centrifugation at a low speed, and suspendedtogether in a glucose-enriched medium (10% glucose,0.5% yeast extract). The cultures were aerated byshaking for 2 h at 20°C. The cell mixtures werecompacted by low-speed centrifugation and left stand-ing in the form of cell pellets in the centrifuge tube for4 h at 20°C. During this period the cells underwentconjugation and zygote formation. Samples for analy-ses of glucanase activity were taken immediately afterthe cells were mixed and after 4 h in the centrifugetubes. The percentages of conjugation and zygoteformation were estimated by microscopic examina-tion.

Yeast sporulation. The conditions used for synchro-nous sporulation of S. cerevisiae 595 at high cellconcentrations were adapted from the method of Pe-terson et al. (7). Yeast cells were grown in YEGmedium to stationary phase until glucose was justexhausted (36 h). The cells were harvested, washedtwice with liquid acetate medium (1% potassium ace-tate, 0.05% glucose, 0.1% yeast extract), and resus-pended in 100 ml of the same medium in a 1-literconical flask to give a concentration of 1.0 x 10i cellsper ml. The cells were incubated at 25°C with vigorousorbital shaking (800 rpm), and samples were taken at

regular intervals to examine the glucanases. The ex-tent of spore development was followed by microscop-ic counting of samples in a Petroff-Hausser countingchamber (C. A. Hausser, City, State). Both the age ofthe presporulation culture and the cell concentration inthe sporulation medium influenced the percentage ofspores formed, and the conditions chosen were thosethat yielded maximum sporulation. More than 70% ofthe cells had sporulated after % h.Chromatographic separation and assay of (1--3)-j3-

glucanase activities. The (1--3)-p-glucanase activitiesin cell extracts and in cell wall autolysates wereseparated into the individual glucanase I, II, IIA,IIIB, IV, and V activities by a system of chromatogra-phy through columns of DEAE-Bio-Gel A, CM-Bio-Gel A, and HTP-Bio-Gel, as described in the accom-panying paper (6). Briefly, cell extracts or cell wallautolysates were dialyzed against 0.01M succinatebuffer (pH 5.0) and then fractionated into glucanases I,II, III, and IV by chromatograpy over DEAE-Bio-GelA. The unadsorbed effluent was collected and exam-ined as glucanase V on CM-Bio-Gel A. Fractionscontaining glucanase III were subsequently resolvedinto glucanases IIIA and IIIB by passage through acolumn of HTP-Bio-Gel. (1-*3)-,B-Glucanase assayson cell extracts, cell walls, or column fractions wereperformed by using laminarin as the substrate (6). The(1--3)-,3-glucanase elution profiles from the variouscolumns were plotted, and the total activities for theindividual (1- 3)-p-glucanase were measured by as-saying the pooled fractions containing each of theseparated enzymes.

RESULTSVariation of the (1--3)-p-glucanases of S. cere-

visiae 2180B during vegetative growth. Cells from48-h stationary phase culture were inoculatedinto fresh YEG medium and cultured for up to 7days. At intervals during growth, cell sampleswere removed to prepare cell extracts and cellwalls. Figure 1 shows the variation in the total(1-)3)-,-glucanase activity in these fractions,along with the cell density and the concentrationof glucose in the medium. The glucanase activityin both fractions increased steadily during expo-nential growth (10 to 15 h) and, after reaching amaximum level at the end of exponentialgrowth, gradually declined. Low levels of(1--3)-,B-glucanase were still detected in cellsafter 7 days of culture. More than 80% of the cellpopulation was actively budding during expo-nential growth, whereas in stationary phase cul-tures more than 90% of the cell population wasunbudded.Samples of cell extracts and wall autolysates

prepared from cells harvested at different timesduring the growth cycle were fractionated intotheir individual glucanase components by col-umn chromatography. Chromatographic profilesshowing resolution and the relative proportionsof the different glucanases are shown in Fig. 2.Data on the activities of the different glucanasesare summarized in Table 1. In a 48-h stationary

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FIG. 1. Variation in (1-+3)-3-glucanase activity inS. cerevisiae 2180B during vegetative growth. Sym-bols: *, 01--3)-3-g ucanase activity in cell extracts; E,(1--3)-p-glucanase activity associated with isolatedcell walls; 0, cell density; 0, glucose concentration inculture medium. A550m, Absorbance at 550 nm.

phase culture (used as an inoculum for a freshculture) glucanase IIIA predominated and ac-counted for more than 80%o of the total glucanttseactivity in both extracts and wall autolysates.Small amounts of glucanases I, II, and V werepresent, and glucanases IIIB and IV were nota-bly absent. As the culture entered the exponen-tial phase, the proportion of glucanase lIIAactivity decreased, and increasing amounts ofglucanases I, II, and IIIB were found. Theproportion of glucanase V activity progressivelydecreased during exponential growth.By late log phase, glucanases IIIA and IIIB

were of equal predominance in cell extracts, andeach accounted for about 38% of the total activi-ty. These were followed by glucanase 11 (15% ofthe total activity) and glucanase I (9o). Glucan-ase V accounted for only 4% of the total activityat this time, and glucanase IV was never detect-ed in cell extract fractions.

In cell wall autolysates, glucanase IIIA activi-

ty decreased as exponential growth continued,and by late exponential growth it accounted foronly 7% of the total activity. At this time glucan-ase IIIB was the predominant enzyme, account-ing for 67% of the total activity. Glucanases IIand I contributed 14 and 8%, respectively, of thetotal activity. Glucanase IV became detectablein the wall autolysates of exponential phase cellsbut at most accounted for only 0.1% of the totalactivity, whereas glucanase V activity droppedto 3% of the total activity.As the cells entered the stationary phase,

glucanase IIIA once again became the predomi-nant enzyme in both cell fractions. This wasmainly at the expense of glucanase IIIB and, tolesser extents, glucanases II and I, whose activi-ties also decreased. Glucanase V activity in thecell wall fractions showed a significant increaseand accounted for about 11-% of the total activity(Table 1).

It is important to distinguish between therelative proportions of the individual enzymesand the absolute activity levels, as shown inTable 1. Of the six glucanases, the activity ofglucanase IIIB showed the greatest variation. Inboth cell extracts and cell wall autolysates, thisenzyme activity increased from virtually zero inresting cells to a maximal level during exponen-tial growth and then decreased during the sta-tionary phase. The actual level ofglucanase IIIAactivity in cell extracts remained almost con-stant despite the fact that its contribution to totalactivity decreased during exponential growth.However, the absolute values for glucanase IIIAactivity in wall autolysates did decrease duringexponential growth.

Variation in 13-glucanases during con,jugation.S. cerevisiae mating type strains 2180A and2180B were mixed and incubated under conju-gating conditions. After 2 h most of the cells had

FRACTION NUMBERFIG. 2. (1- 3)-p-glucanase profiles of cell wall autolysates of S. cerevisiae 2180B at stages during vegetative

growth. (A) Profiles of glucanases I, II, III, and IV on DEAE-Bio-Gel. (B) Resolution of glucanases lIA andIIB on HTP-Bio-gel. (a) 48-h inoculum culture. (b) 6-h culture. (c) 12-h culture. (d) 20-h culture. (e) 36-h culture.

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agglutinated, and about 5% were actually conju-gating or forming tubelike extensions. By 4 habout 30% of the cells had conjugated, and a fewzygotes had appeared. Approximately 50% ofthe cells were conjugating after 8 h. The totalglucanase activities in either cell extracts or cellwalls did not show any substantial increasesduring conjugation, up to 8 h. The proportions ofthe individual enzymes before and after conjuga-tion were also very similar and resembled thoseof early stationary phase cultures (Table 1, 36-hdata). However, although glucanase I contribut-ed only a very minor percentage to the totalextract and wall activities, this enzyme almostdoubled its activity during conjugation. The con-tribution of glucanase IIIA to the total activityincreased by 5 to 10% during conjugation, andthis small change appeared to be at the expenseof glucanase IIIB. Overall, the small enzymechanges noted during conjugation were reminis-cent of a culture moving further into the station-ary phase. The chromatographic profiles of thep-glucanases before and after conjugation wereessentially the same as the 36-h data for vegeta-tive cultures.

Variation in j-glucanases during sporulation.The diploid strains obtained from S. cerevisiae2180A and 2180B matings did not produce highyields of sporulating cells (less than 10%) whenthey were tested on a variety of liquid and solidsporulation media. On the other hand, S. cere-visiae 595 readily produced sporulating cells inamounts that could be examined for glucanaseactivity, and since the P-glucanase system ofthis yeast strain was the same as that of S.cerevisiae diploid strain 2180D (6), it was con-sidered suitable for our studies.

Cells of S. cerevisiae 595 were incubatedunder sporulation conditions, and the total glu-canase activities in cell extracts and cell wallswere measured at intervals during incubation.Figure 3 shows the development of sporulatingcells during culture and the associated variationin (1-*3)-P-glucanase activity. Just before theappearance of spores (36 to 48 h), there was asharp increase in total glucanase activity in thecell extracts, followed by a sharp decrease inthis activity (48 to 60 h). Wall-associated glucan-ase activity also increased after 36 h, but thisincrease was more gradual than the increase forcell extracts and did not peak until 60 h. After 60h, this activity also decreased; by this time only25% of the cells showed the presence of asco-spores. The percentage of sporulating cells con-tinued to increase after this time, although theglucanase activities in cell extracts and cell wallshad decreased. By 96 h, approximately 75% ofthe cell population had sporulated. This se-quence of events, during which glucanase activi-ty peaked first in the cytoplasm and then in the

.00 2 4 6 24 36 i8 60 72 4 96

TINE (hours )FIG. 3. Variation in total (1--3)-,B-glucanase activi-

ties in cell extracts and cell walls of S. cerevisiae 595during sporulation. Symbols: A, percentage of sporu-lating cells; 0, (1- 3)-p-glucanase activity in extracts;O, (1- 3)-P-glucanase activity in cell walls.

cell walls and then decreased well before amajority of the cells had formed spores, wasvery reproducible.Table 2 shows the 0-glucanase compositions

of cells before transfer to sporulation mediumand of cells after 48 or 60 h in the sporulationmedium when the extract and wall-associatedglucanase activities, respectively, had peaked.Before sporulation the cells exhibited a P-glu-canase composition typical of early stationaryphase cultures, when glucanase IIIA was thepredominant enzyme (46% of the total activity),followed by glucanases IIIB (29%), II (12%), I(8%), and V (6%). After sporulation, the totalglucanase level increased by almost threefold.However, with the exception of glucanase V,whose activity increased by about 30-fold, all ofthe other glucanases showed either no change orreductions in activity. Glucanase IIIB showedthe greatest reduction. Glucanase V was by farthe most prevalent enzyme in sporulating cellsand accounted for more than 70% of the totalglucanase activity in both cell extracts and wallautolysates. Glucanase IIIA was the next mostprevalent enzyme, accounting for 20% of thetotal activity in both cell fractions. The contribu-tion of glucanase IIIB decreased from around 28to 30% to 1 to 2% after sporulation.The glucanase V found after sporulation had

the same properties on DEAE-Bio-Gel, CM-Bio-Gel, and Sephadex G-200 as the enzymeobtained before sporulation. Figure 4 shows thechromatographic resolution of glucanases I, II,IIIA, and IIIB of S. cerevisiae 595 before andafter sporulation and the behavior of glucanaseV (after sporulation) on CM-Bio-Gel and Sepha-dex G-200. These chromatographic properties ofS. cerevisiae 595 glucanases are similar to thosereported for S. cerevisiae 2180B (6).

In a control experiment, cells of haploid strain

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TABLE 2. Variation in the activities and proportions of individual (1-*3)-p-glucanases in S. cerevisiae 595during sporulation in acetate medium

Presporulation culture Sporulation culture

Prepn Enzyme Activity % of Activity % of(U/g of total (U/g of totalwalls) activity walls) activity

Cell extracts Total activity 1.44 100 3.26a 100Glucanase I 0.10 6.9 0.08 2.6Glucanase II 0.16 11.1 0.16 4.8Glucanase IIIA 0.67 46.5 0.61 18.7Glucanase IIIB 0.43 29.9 0.08 2.4Glucanase IV 0 0 0 0Glucanase V 0.08 5.6 2.34 71.4

Cell wallsb Total activity 0.67 100 1.79C 100Glucanase I 0.05 7.5 0.02 1.1Glucanase II 0.08 11.9 0.03 1.7Glucanase IIIA 0.31 46.2 0.36 20.1Glucanase IIIB 0.19 28.5 0.03 1.7Glucanase IV 0 0 0 0Glucanase V 0.04 5.9 1.35 75.4

a The glucanase composition of a 48-h culture was analyzed.b Cell walls were subjected to 12 h of autohydrolysis before fractionation into the glucanase components.c The glucanase composition of a 60-h culture was analyzed.

S. cerevisiae 2180B were incubated under sporu-lation conditions. There was no cell growth orascospore formation under these conditions, asexpected. Total glucanase activity did not in-crease during incubation, and the glucanase pro-files before and after incubation were similar.Therefore, we conclude that glucanase V pro-duction is specifically due to sporulation and isnot a result of changes in culture conditions.

Variation in 1-glucanases under conditions ofglucose starvation. When cells from exponentialphase cultures were transferred to a glucose-deficient medium, growth stopped almost imme-diately, and the level of glucanase activity asso-ciated with the cell wall fraction decreased by50o within 5 to 10 h in the new medium. Incontrast, glucanase activity in cell extractsshowed little change during this period, but haddecreased by about 13% after 25 h. The compo-sition of the wall-associated (1-*3)-P-glucanasesystem was analyzed after 5 h in GD medium,and the activities of glucanases I, IT, and IIIBwere found to have decreased by 50, 50, and65%, respectively, whereas glucanase IV com-pletely disappeared (Table 1, 12-h data). Howev-er, the level of glucanase IIIA remained con-stant, and glucanase V activity increased by27%. After 25 h, the cells were transferred fromGD medium to fresh YEG medium. Growth andbudding started after an initial lag phase, andwall-associated and extract activities increasedto their original levels. After 10 h, this cultureentered the stationary phase, and the levels of(1-.3)-3-glucanase activities in cell extracts andwall preparations decreased in line with theobservations presented in Table 1.

0 20

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FIG. 4. (1-3)-,B-glucanase profiles of cell wall au-tolysates of S. cerevisiae 595. (a and b) Glucanases I,II, and III on DEAE-Bio-Gel before and after sporula-tion, respectively. (c and d) Glucanases IIIA and IIIBon HTP-Bio-Gel before and after sporulation, respec-tively. (e and t) Glucanase V after sporulation on CM-Bio-Gel and Sephadex G-200, respectively. The elu-tion conditions used are described in the accompany-ing paper (6).

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DISCUSSION

In the accompanying paper (6) we report theoccurrence of six different (1--3)-P-glucanasesin S. cerevisiae. In this study we demonstratedthat these enzymes have different functions andindependent patterns of variation during the cellcycle.The increases in extract and wall-associated

(1-*3)-P-glucanase activities during exponentialgrowth and their decreases during the stationaryphase are consistent with other reports (1, 10)and support the proposal that (1--3)-P-glucan-ases act as wall modifiers and plasticizers duringcell expansion and budding.Of the two exoglucanases, only glucanase II

activity increases during exponential growth andappears to be involved in vegetative division. Itmay be significant that this exoglucanase caninitiate action on glucan substrates at pointsother than the nonreducing terminal residue (6).Moreover, this enzyme seems to exhibit somesynthetase or transferase activity in addition toits hydrolytic action (6), so that it may specifical-ly function in cell wall expansion through theinsertion of new glucosyl units. This interestingpossibility certainly warrants further study. Theactivity of exoglucanase IIIA remains relativelyconstant or even decreases during exponentialdivision and, therefore, appears to have nodirect function at this stage of growth. This isconsistent with a previous report that mutantslacking this enzyme exhibit normal growth andbudding (13). However, the striking predom-inance of exoglucanase IIIA in stationary phaseand older cultures suggests some functional roleat this stage of the growth cycle.

Endoglucanases I, IIIB, and IV all show vary-ing increases in activity during exponentialgrowth and, consequently, may play importanthydrolytic roles during cell wall expansion andbudding. Specific definition of these roles re-quires further study. The behavior of glucanaseIIIB is particularly noteworthy since this en-zyme activity is completely absent in stationaryor resting-phase cells but is rapidly synthesizedor activated to become the predominant glucan-ase during exponential growth. Since glucanaseIIIB hydrolyzes laminarin to produce mainlylaminaripentaose (6), it is possible that this en-zyme randomly cleaves cell wall glucans atwidespread points to allow insertion of newglucosyl units as needed for wall expansion. Thedecreasing activity of endoglucanase V duringexponential growth suggests that this enzyme isnot required for vegetative division.

It was surprising to find little alteration in totalglucanase activity or in individual glucanasecomposition during cell conjugation, when con-siderable localized wall expansion and dissolu-

tion are expected to occur (2). These findings areconsistent with reports by Rey et al. (10) but arecontrary to the increased glucanase activitiesnoted during the conjugation of H. wingei (2)and Schizosaccharomyces species (3, 8). Thus,the role of glucanases in yeast conjugationseems to be more complicated than originallythought and demands closer examination.

Sporulation in S. cerevisiae was accompaniedby a large increase in total (1--3)-p-glucanaseactivity, and this was mainly due to a dramatic(30-fold) increase in the activity of glucanase V.This enzyme accounted for around 75% of thetotal glucanase activity in sporulating cultures,and our data strongly indicate that this is asporulation-specific enzyme. It is not possible tostate the exact role of this endoglucanase in thesporulation process, but since its developmentoccurs largely before the majority of ascosporeshave actually formed, it may function in themobilization and organization of vegetative cellwalls into asci and ascospore walls. Precedentsfor such glucanase function have been reportedpreviously for Aspergillus nidulans (14). It isunlikely that glucanase V is involved in the lysisof ascus walls to release ascospores since thespores were still encased within the asci longafter the level of glucanase V activity had in-creased and then decreased. Glucanase activityhas been implicated in ascus lysis for some otheryeast species (4). We found no evidence for theinvolvement of exoglucanase activity in thesporulation of S. cerevisiae as recently de-scribed by Rey et al. (12).The production (1-+3)-f-glucanases by some

fungi is repressed by the presence of glucose inthe culture medium and is derepressed underconditions of glucose limitation (9). Repressionof glucanase activity by glucose was not ob-served for S. cerevisiae, and, in fact, glucoselimitation had the effect of switching off expo-nential growth and causing a reduction in thelevels of those glucanases (namely, glucanases I,II, IIIB, and IV) suspected of acting duringvegetative division. Glucose limitation did causea slight increase in glucanase V activity, and thismight be interpreted as a response in preparationfor sporulation, which in S. cerevisiae is encour-aged by conditions of glucose deprivation.

It is pertinent to point out certain limitationsassociated with the measurement of glucanaseactivity when laminarin is used as the substrateand reducing sugar formation is used as an indexof activity. First, an enzyme that produces largeamounts of glucose (e.g., glucanase IIIA) orlow-molecular-weight oligosaccharides appearsto be more active than an enzyme which pro-duces large products (e.g., laminaripentaoseproduction by glucanase IIIB). Thus, for exam-ple, glucanase IIIA appears to be more active

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than glucanase IIIB when, in reality, there couldbe more molecules of the latter. Second, oneglucanase may have totally different levels ofactivity on different (1-*3)-P-glucan substrates.For example, although glucanase I activity ap-peared to be a relatively minor component oftotal glucanase complex activity (Table 1), thisconclusion was based on the use of laminarin asthe substrate. This might not have been the caseif a more natural substrate (e.g., a wall glucan)had been used. However, the use of laminarin asthe substrate did permit valid comparisons of thesame enzyme at different stages during the lifecycle.

Regulation or control of glucanase activitywas not considered in this study. Here and in theaccompanying paper (6), we show that the(1--3)-P3-glucanase system of S. cerevisiae is farmore complex than previously thought and thatthe individual enzymes of the system vary inde-pendently throughout the life cycle. It is notknown whether these variations are due to en-zyme synthesis or enzyme activation or both,and these questions, along with questions con-cerning possible control mechanisms, remainopen for further investigation.

LITERATURE CITED1. Cortat, M., P. MatlIe, and A. Wiemken. 1972. The isola-

tion of glucanase-containing vescicles from budding yeast.Arch. Microbiol. 82:189-205.

2. Crandall, M., R. Egel, and V. L. Mackay. 1977. Physiolo-gy of mating in three yeasts. Adv. Microb. Physiol.15:307-398.

3. Fleet, G. H., and H. J. Phaff. 1975. Glucanases in Schizo-saccharomyces. Isolation and properties of an exo-,B-glucanase from cell extracts and culture fluid of Schizo-

saccharomyces japonicus var. versatilis. Biochim.Biophys. Acta 401:318-332.

4. Fleet, G. H., and H. J. Phaff. 1981. Fungal glucans-structure and metabolism, p. 416-440. In W. Tanner andF. A. Loewus (ed.), Plant carbohydrates. II. Extracellularcarbohydrates. Encyclopedia of plant physiology, Newseries, vol. 13B. Springer-Verlag, Berlin.

5. FoweU, R. R. 1969. Sporulation and hybridisation ofyeasts, p. 303-385. In A. H. Rose and J. S. Harrison (ed.),The yeasts, vol. 1. Academic Press, Inc., London.

6. HlIen, N. H., and G. H. Fleet. 1983. Separation andcharacterization of six (1-*3)-p-glucanases from Saccha-romyces cerevisiae. J. Bacteriol. 156:T30.

7. Peterson, J. G. L., L. W. Olson, and D. Zickler. 1978.Synchronous sporulation of Saccharomyces cerevisiae athigh cell concentrations. Carlsberg Res. Commun.43:241-253.

8. Relchelt, B. Y., and G. H. Fleet. 1981. Isolation, proper-ties, function, and regulation of endo-(1-.3)-0-glucanasesin Schizosaccharomyces pombe. J. Bacteriol. 147:1085-1094.

9. Rey, F. J., I. Garcia-Acha, and C. Nombela. 1979. Theregulation of 3-glucanase synthesis in fungi and yeast. J.Gen. Microbiol. 110:83-89.

10. Rey, F. J., I. Garcia-Acha, and C. Nombela. 1979. Synthe-sis of 1,3-,-glucanases in Saccharomyces cerevisiae dur-ing the mitotic cycle, mating, and sporulation. J. Bacte-riol. 139:924-931.

11. Rey, F. J., T. Santos, I. Garcda-Acha, and C. Nombela.1979. Synthesis of ,-glucanase during sporulation in Sac-charomyces cerevisiae: formation of a new, sporulation-specific 1,3-0-glucanase. J. Bacteriol. 143:621-627.

12. Rey, F. J., T. G. Villa, T. Santos, I. Garcia-Acha, and C.Nombela. 1982. Purification and partial characterization ofa new sporulation specific, exoglucanase from Saccharo-myces cerevisiae. Biochem. Biophys. Res. Commun.105:1347-1353.

13. Santos, T. F., F. J. Rey, J. R. Code, J. R. Villanueva, andC. Nombela. 1979. Saccharomyces cerevisiae mutant de-fective in exo-1,3-0-glucanase production. J. Bacteriol.139:333-338.

14. Zonneveld, B. J. M. 1972. Morphogenesis in Aspergillusnidulans. The siginficance of a-(1-+3)-glucan of the cellwall and oL-(1-.3)-glucanase for cleistothecium develop-ment. Biochim. Biophys. Acta 273:174-181.

VOL. 156, 1983

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