JOURNAL OF BACTERIOLOGY, May 1990, p. 2360-23660021-9193/90/052360-07$02.00/0

Vol. 172, No. 5

Expression and Regulation of Glucoamylase from the YeastSchwanniomyces castelliit

T. M. DOWHANICK,l I. RUSSELL,1 S. W. SCHERER,lt G. G. STEWART,1 AND V. L. SELIGY2*Research Department, Labatt Brewing Company Limited, London, Ontario N6A 4M3,1 and Molecular Genetics Section,

National Research Council of Canada, 100 Sussex Drive, Ottawa, Ontario KIA OR6,2 Canada

Received 7 July 1989/Accepted 28 January 1990

Expression of the 146-kilodalton (kDa) extracellular glucoamylase by the budding yeast Schwanniomycescastellii is induced by maltose and starch. By use of antiglucoamylase antisera, we found that this expressionwas regulated at the level of the mRNA, taking place within 30 min after exposure of yeast cells to the respectivesugars. Polyacrylamide gel electrophoresis analysis of the in vitro-translated products of total RNA frommaltose-treated cells established that the glucoamylase precursor was approximately 120 kDa in size. Stableglucoamylase transcript was not produced in cells exposed to glucose, 2-deoxyglucose, and heat shock. Cellsexposed to these two sugars also degraded intracellular and extracellular glucoamylase. In the presence ofsugars such as cellobiose, galactose, lactose, and xylose or in the absence of any carbohydrate, a low-level,constitutive-like expression of this preglucoamylase occurred. The nascent glucoamylase underwent at least twoposttranslational modifications, resulting in a 138-kDa cell-associated form and the 146-kDa active form thatwas found free in the medium. These results suggest that glucoamylase expression is tightly regulated similarlyto expression of the enzymes responsible for maltose metabolism in Saccharomyces yeasts.

Organisms ranging from prockaryotes to humans produceenzymes capable of degrading starch (16). Many yeast andfungi possess amylolytic enzymes (17, 20). Glucoamylaseappears to be the most versatile of these enzymes, cleavingterminal a-1,4 glucosidic bonds from the nonreducing endsof starch, dextrins, and maltose to release glucose. In somecases, this enzyme also cleaves a-1,6 branch bonds adjacentto a-1,4 bonds (17, 29). However, complete degradation ofstarch to glucose requires a combination of enzymes, con-sisting of a-amylase for endo a-1,4 cleavage and glucoam-ylase with and without debranching activity.Only a few glucoamylases have been characterized to any

extent at the molecular level (2, 9, 22, 25, 33, 41-43). We andothers are interested in developing the genetics of membersof the budding yeast Schwanniomyces (7, 24), particularly inview of their ability to hydrolyze starch (3, 5, 6, 10, 15, 19,21, 23, 26, 28, 29, 31, 39, 40). The 146-kilodalton (kDa)extracellular glucoamylase of Schwanniomyces castellii thatwe have characterized is of particular interest because it hasboth a-1,4 and ot-1,6 activity and is thermally labile (29, 30).The general expression properties of this enzyme in relationto growth and carbon source were described by usingenzymatic and immunological assays (7). The level of mature146-kDa glucoamylase, expressed after glucose-adaptedcells were transferred (shifted) to maltose medium, wasabout 100-fold over the background produced by cells ex-posed to glucose. In this study, we used the induction andimmunodetection techniques to study glucoamylase expres-sion at the levels of stable mRNA and posttranslation.

MATERIALS AND METHODSGrowth of yeast cells. S. castellii 1402 (Labatt Breweries of

Canada Culture Collection) was maintained on 2% glucose-peptone-yeast extract agar slopes at 4°C. Cells were grown

* Corresponding author.t National Research Council of Canada publication no. 31215.t Present address: Hospital for Sick Children, Toronto, Ontario

M5G 1X8, Canada.

in minimal medium containing 0.67% (wt/vol) yeast nitrogenbase without amino acids (Difco Laboratories) and supple-mented with either 1% (wt/vol) glucose (Anachemia), solublestarch (Fisher Scientific Co. catalog number), maltose, 2-deoxyglucose, galactose, lactose, xylose, or cellobiose (Sig-ma Chemical Co.).Western (immunoblot) analysis of in vivo-translated pro-

teins. Procedures for performing the glucoamylase inductionexperiments, the isolation and electrophoretic separation oftotal cell-associated and extracellular proteins, and Westernimmunodetection studies with antisera made against the146-kDa extracellular glucoamylase have been described indetail (7, 34). However, in the study described here, thealkaline phosphatase-coupled immunoscreening system de-scribed by the manufacturer (ProtoBlot protocol of PromegaBiotec) was used instead of the 1251I-labeled protein Aprocedure. All experiments were performed at least threetimes, with results not varying by more than 10%.

Preparation and characterization of RNA. Total RNA wasisolated by modification of two previously published proce-dures (4, 12). Cells were harvested by centrifugation for 2min at 8,000 x g at room temperature (21 to 23°C). Thesupernatant was carefully decanted, and the cell pellet wasimmediately frozen in a dry ice-methanol bath. To each 0.5-to 1.0-g (wet weight) pellet offrozen cells, the following wereadded: an equal volume of glass beads (0.5-mm diameter), 20,ul of 100% Sarkosyl (CIBA-GEIGY Corp.; nl 30), 20 ,ul ofantifoam A (Sigma), 4.0 ml of 4 M guanidinium thiocyanatein 25 mM citrate, 1 mM EDTA (pH 7.0), and 40 ,ul of 14.1 Mmercaptoethanol. Each sample was vortexed at the highestspeed for 2 min and immediately placed on ice until allsamples had been similarly treated. Cell wall debris and glassbeads were removed by centrifugation at 8,000 x g for 10min at 4°C. In some cases, this step was repeated. Theresultant supernatant from each sample was layered onto a1.0-ml cushion of 5.7 M cesium chloride made up in 0.1 MEDTA (pH 7.0) in a Beckman SW50.1 ultracentrifuge tube.Total RNA was collected as a pellet after centrifugation for16 h at 30,000 x g and 18°C. Supernatants and residual

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cellular debris were removed from the RNA samples byaspiration and by careful draining after inversion of eachtube. The translucent RNA pellets were precipitated twice,using 2 volumes of 95% ethanol each time, with recovery bymicrocentrifugation. Each RNA pellet was dried and dis-solved in autoclaved water pretreated with 0.01% (vol/vol)diethylpyrocarbonate (Sigma). RNA concentration was de-termined by measuring optical density at 260 nm (45 ,g/ml =1 optical density unit). Intact RNA was confirmed by elec-trophoresis (65 V for 60 min) in a mini horizontal 2% (wt/vol)agarose gel containing 25 mM citrate (pH 3.5). RNA wasstained with 0.5 ,g of ethidium bromide and photographed,using type 52 Polaroid film with UV light.

In vitro Translation of RNA. Total RNA was translated invitro by using a rabbit reticulocyte lysate (AmershamCorp.). Each 20 ,ul of reaction mixture contained 16 ,ul (80%[vol/vol]) of lysate, 15 ,uCi of [35S]methionine (Amersham),20 p.g of RNA, 94 mM KCI, and 1.4 mM MgCl2. The reactionmixtures were incubated at 30°C for 1 h and then eitherstored at -70°C or immediately used. Immunoprecipitationof the "S-labeled preglucoamylase in vitro translation prod-uct was carried out by using 50% (10 ,ul) of the in vitrotranslation reaction in 330 ,ul of REPA buffer (7). Sampleswere analyzed by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis as described by Laemmli (13), using a 4%stacking gel and a 10o separating gel at 45 V for 16 h. Acomputing densitometer was used to quantitate radiolabeledproducts as well as those detected by Western blotting andCoomasie blue staining according to the programs of themanufacturer (Molecular Dynamics 300A).

RESULTS

Identification of the glucoamylase precursor. Expression ofthe 146-kDa glucoamylase occurs very rapidly in strain 1402cells when these cells are shifted from glucose- to maltose-supplemented media (7). The Western analyses using an-tiglucoamylase antiserum as a probe indicated that the146-kDa glucoamylase was found in the medium as well as inassociation with cell pellets, generally in about equalamounts. However, several minor products and one majorone measuring 138 kDa were found to be exclusively cellassociated. Since only the column-purified, 146-kDa extra-cellular enzyme was used to make the antiglucoamylaseantiserum, the cell-associated polypeptides may be eitherprecursors or degradation products of the glucoamylase orproteins that cross-react with the antibody probe. To obtainmore information about the early expression events of thismaltose induction system, the [35S]methionine-labeled poly-peptides made from the cell-free translation of RNA har-vested after cells were incubated for various time intervalswith medium containing either maltose or glucose wereanalyzed. A 120 + 5-kDa product was made only from RNAof maltose-treated cells, not from RNA of glucose-treatedcells (Fig. 1, lanes a to h). The concentration of the transcriptserving as the template for synthesis of this polypeptidechanged with time; it was first detected at 15 min; it thenreached a maximum by 60 min, decreased by 3 and 4 h, andincreased again at 5 h. The maximum reached within the firsthour is consistent with the earlier data indicating the appear-ance of the 138- and 146-kDa cell-associated glucoamylasepolypeptides (7).The immunoprecipitation results obtained by using the

cell-free products made from the glucose- and maltose-treated cells are presented in Fig. 2 (see also Fig. 3C and 5).Since only the 120-kDa polypeptide specific to maltose-

A

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a b c d e f g hFIG. 1. [35S]methionine-labeled in vitro translation products

made from total RNA of cells incubated in glucose (A) or maltose(B). Total RNA was isolated from cells incubated for various lengthsof time. Lanes: a, 15 min; b, 30 min; c, 45 min; d, 60 min; e, 2 h; f,3 h; g, 4 h; h, 5 h; i, 24 h. Arrow in panel A indicates position of the130-kDa protein. Arrows in panel B indicate 130-kDa and 120-kDa(±7%) proteins. Bars indicate molecular size standards (top tobottom: 200, 92.5, 68, 43, and 25.7 kDa). Fluorographs wereexposed for 20 h at -80°C.

treated cells reacted consistently with the antiserum and notany other products (e.g., the 130-kDa polypeptide in tracks ato d and a to h of Fig. 1A and B, respectively), these resultsestablished that there was only one polypeptide precursor ofthe 146-kDa glucoamylase. These data also show that max-imum production of intact glucoamylase mRNA occurred ataround 60 min of incubation of cells in maltose.The decrease of mRNA template at approximately 3 to 4 h

of incubation of cells in maltose (Fig. 1B, lanes f and g) mayhave been due to increased mRNA turnover or repression oftranscription. This repression could be explained by thetransient buildup of endogenous glucose that occurs at thisstage. The reappearance of the glucoamylase in mRNA againat 5 h may in turn have been due to a reinduction oftranscription by maltose. However, the level of inducedglucoamylase mRNA was lower than it was at 60 min,possibly because of the diminished amount of maltose

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FIG. 2. Immunoprecipitation of in vitro translation products oftotal RNA from cells incubated in either glucose- or maltose-containing medium. Lanes: a, b, d, and f, no RNA (lane a) and RNAfrom 0-min mineral medium salts-washed cells (lane b) and fromantiglucoamylase antiserum-precipitated glucose (lane d) and malt-ose (lane f) 35S-derived products; c and e, total RNA from cellsincubated for 60 min in glucose and maltose, respectively. Note the120-kDa glucoamylase precursor present in lanes e and f (arrow).Bars indicate molecular size markers (top to bottom: 200, 92.5, 68,43, and 25.7 kDa). Fluorographs were exposed for 20 h at -80°C.

present. In vitro translation of RNA from 24-h cells wasfound to be consistently poor because of RNA turnover atstationary phase.

Glucose represses glucoamylase expression induced by malt-ose. Amylolytic activity in S. castellii is repressed at glucoseconcentrations greater than 3 mM (30). Expression of intactglucoamylase mRNA was repressed in strain 1402 cells whenthese cells were exposed to medium containing 56 mMglucose (Fig. 1 and 2). However, although it has been shownthat glucose-treated cells can be rapidly induced to produceglucoamylase by a shift to maltose-containing medium, it isnot known whether glucose represses induced cells at thelevel of either transcription or translation. These alternativeswere examined in a reverse substrate shift experiment inwhich cells induced for 30 min with maltose (control) wereharvested after different time intervals with and withoutfurther addition of glucose (1% [wt/vol]). For analysis of theresults, cell samples were processed in two ways, usingantiglucoamylase antiserum to detect glucoamylase-relatedpolypeptides present in either total protein extracts or the invitro translation products of total RNA from these cells.The cell-associated 138- and 146-kDa glucoamylase prod-

ucts, initially found after 30 min of growth in maltose-supplemented medium, were easily detected throughout the180-min cell incubation with maltose (Fig. 3A). In contrast,the extracellular 146-kDa glucoamylase was first detected atabout 60 min (Fig. 3B, lane c), reaching a maximum level by120 min (lane i). The production of glucoamylase mRNA,measured by immunoprecipitation of the 120-kDa precursorfrom in vitro-translated proteins of RNA of maltose controlcells, indicated that intact glucoamylase mRNA was mostabundant at early time points (30- to 90-min samples; Fig.3C, lanes b, c, g, and i). The combined results are in contrastto those obtained from maltose cells shifted to glucose. Herethe Western analyses revealed that starting at 30 min afteraddition of glucose to maltose-induced cells, the level of allglucoamylase-related products decreased compared with therespective control levels (Fig. 3A and B, lanes d, e, f, and g).Even more striking was the notable absence of immunopre-cipitable glucoamylase precursor from any of the in vitrotranslations of the corresponding RNA samples (Fig. 3C,lanes d, e, f, and g). These results indicate that repression ofglucoamylase expression takes place very quickly, within at

a b c d e f g h i j k

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a b c d e f g h i j kFIG. 3. Analysis of glucoamylase production in cells incubated

in maltose before and after addition of glucose. Shown are Westernblots of total cellular (A) and extracellular (B) proteins probed withantiglucoamylase antiserum. (C) Immunoprecipitation of in vitro-translated [35S]methionine-labeled glucoamylase 120-kDa precursormade from RNA derived from cells assayed in panels A and B.Lanes: a, 0 min, no-sugar control cells; b, c, and h to k, maltose at30, 60, 90, 120, 150, and 180 min; d to g, maltose at 60 min shifted toglucose and incubated for 30, 60, 90, and 120 min. Arrows indicateglucoamylase derivatives (top to bottom: panel A, 146, 138, and 105kDa; panel B, 146 kDa; panel C, 120 kDa). Bars indicate positions of(top to bottom) 200-, 92.5-, 68-, 43-, and 25.7-kDa molecular sizestandards.

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a b c d e f g h i j k I mn p q r s t u v w x y z a'b'c' de' f g ht j j"FIG. 4. Western analysis of glucoamylase in cellular (A) and extracellular (B) extracts of exponentially growing cells incubated with

various sugars. Experiments were carried out as for Fig. 3. Experiments: no-sugar control (lane b, 0 min; lane c, 30 min; lane d, 60 min; lanee, 90 min); 1% maltose (lane f, 30 min; lane g, 60 min; lane h, 90 min); 1% maltose plus heat shock (lane i, heat shock from 30 to 60 min at39°C); 1% soluble starch (lanej, 30 min; lane k, 60 min; lane 1, 90 min); 1% lactose (lane m, 30 min; lane n, 60 min; lane o, 90 min); 1% galactose(lane p, 30 min; lane q, 60 min; lane r, 90 min); 1% xylose (lane s, 30 min; lane t, 60 min; lane u, 90 min); 1% cellobiose (lane v, 30 min; lanew, 60 min; lane x, 90 min); 1% glucose (lane y, 30 min; lane z, 60 min; lane a', 90 min); 1% glucose plus 50 puM cAMP (lane b', 30 min; lanec', 60 min; lane d', 90 min), 1% 2-deoxyglucose (lane e', 30 min; lane f, 60 min; lane g', 90 min); 1% 2-deoxyglucose plus 50 ,uM cAMP (laneh', 30 min; lane i', 60 min; lane j', 90 min). Lanes a to j' corresponds to original gel slots; lane a corresponds to (top to bottom) 146-kDaglucoamylase and 78-kDa degradation product. Bars indicate molecular size standards (top to bottom: 200, 94.7, 68, 43, and 25.7 kDa). Arrowsindicate positions of glucoamylase-related products (top to bottom: panel A, 146, 138, 82, and 78 kDa; panel B, 146 and 78 kDa).

least 30 min after glucose addition, occurring mainly at thelevel of glucoamylase transcript expression.

Low-level glucoamylase expression without either glucose ormaltose. Experiments were performed to determine whetherremoval of glucose alone from cells is sufficient to induceexpression of glucoamylase. After pregrowth in minimalmedium plus 1% glucose, cells were quickly washed inminimal medium without a carbohydrate supplement, toremove exogenous glucose, and then incubated in the samemedium before harvesting for total protein and RNA andanalysis as described for Fig. 3. Cell-associated 146-kDaglucoamylase was detected in equal abundance at all timepoints (Fig. 4A, lanes c, d, and e), although the relativelevels of this cell-associated enzyme were about three timesgreater than those of the corresponding glucose controls(lanes y, z, and a') and only about 1/10 those observed in the

maltose controls (lanes f, g, and h). Extracellular glucoam-ylase was detected in the 60- and 90-min samples (Fig. 4B,lanes d and e) at about 1/20 the amount observed in therespective maltose controls. As observed in previous exper-iments, the glucose controls produced no extracellular glu-coamylase (Fig. 4B, lanes y, z, and a'). Data on the immu-noprecipitation of glucoamylase-related in vitro-translatedpolypeptides further indicated that only trace amounts (lessthan 5%) of the 120-kDa precursor were made with RNAfrom 30- and 60-min samples compared with those of maltosecontrols (Fig. 5, lanes b and c). Since, as observed previ-ously, the 120-kDa precursor was not detected in the glucosecontrols (Fig. 5, lanes x, y, and z; see also above), theseresults indicate that glucoamylase is expressed at significantbut low levels within 30 min of removal of exogenousglucose from glucose-adapted cells.

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abcde fghij kImnopqrst uvwxwyzab'c'd'ef'g'h'FIG. 5. Comparison of immunoprecipitable 35S-labeled products made in vitro from RNA from cells treated with various sugars. Samples

of total RNA were harvested from cells at 30, 60, and 90 min. Experiments: No-sugar control (lane a, 0 min; lane b, 30 min; lane c, 60 min;lane d, 90 min); 1% maltose (lane e, 30 min; lane f, 60 min; lane g, 90 min); 1% maltose (lane h, heat shock from 30 to 60 min); 1% solublestarch (lane i, 30 min; lane j, 60 min; lane k, 90 min); 1% lactose (lane 1, 30 min; lane m, 60 min; lane n, 90 min); 1% galactose (lane o, 30min; lane p, 60 min; lane q, 90 min); 1% xylose (lane r, 30 min; lane s, 60 min; lane t, 90 min); 1% cellobiose (lane u, 30 min; lane v, 60 min;lane w, 90 min); 1% glucose (lane x, 30 min; lane y, 60 min; lane z, 90 min); 1% glucose plus 50 ,M cAMP (lane a', 30 min; lane b', 60 min;lane c', 90 min); 1% 2-deoxyglucose (lane d', 30 min; lane e', 60 min; lane f', 90 min); 1% 2-deoxyglucose plus 50 ,M cAMP (lane g', 30 min;lane h', 60 min; lane i', 90 min). Lanes a to i' correspond to original gel slots; bars indicate molecular size standards (top to bottom: 200, 94.7,68, 43, and 25.7 kDa). Arrows indicate positions of glucoamylase-related products (top to bottom: 120, 88, 78, 52, and 48 kDa).

2-Deoxyglucose represses expression of glucoamylase. Theglucose analog 2-deoxyglucose is transported into but isbelieved not to be metabolized by yeast cells (1, 30, 35, 44).This analog has been used with various regimens of muta-genesis to select carbon catabolite-derepressed mutants thatproduce amylolytic activity during growth on glucose (32). Afew mutants of this type have been isolated from differentSchwanniomyces species (6, 20, 30, 39). To determinewhether 2-deoxyglucose affects glucoamylase expression inthe same way as glucose, cells adapted to minimal mediumwith 1% glucose were shifted to minimal medium containing1% (wt/vol) 2-deoxyglucose, harvested at 30, 60, and 90 min,and analyzed for glucoamylase expression as describedearlier (Fig. 4A and B, lanes e', f', and g'; Fig. 5, lanes d', e',and f'). In all cases, 2-deoxyglucose was as effective inrepressing glucoamylase expression as glucose.

Effects of various sugars on glucoamylase expression. Apartfrom the carbohydrates already studied, four additionalsugars (cellobiose, galactose, lactose, and xylose) weretested on glucose-adapted cells for glucoamylase expression.The effectiveness of soluble starch as an inducer of gluco-amylase expression was examined as well. Cell-associated146-kDa glucoamylase produced by cells shifted to mediumcontaining one of the four sugars was about 5 to 10 times thelevels produced by cells incubated in glucose or 2-deoxyglu-cose, about 2 times the levels produced by cells incubatedwithout carbohydrate, and about S times less than the levelsproduced by respective maltose cell controls (Fig. 4A and B,lanes j to x; Fig. 5, lanes i to w). Related polypeptides suchas the 138-kDa polypeptides accounted for no more thanapproximately 5% of the amounts detected in the respectivemaltose cell controls and were comparable to levels pro-duced by cells lacking exogenous sugar. The extracellular146-kDa glucoamylase was produced at levels comparable tothose of cells incubated in the absence of added sugar andabout 10 to 20 times less than the levels produced by themaltose control cells. As expected, no extracellular 146-kDaglucoamylase was detected in cells grown in either glucoseor 2-deoxyglucose (Fig. 4B, lanes y to a' and e' to g').

Comparison of the levels of immunoprecipitated 120-kDaprecursor glucoamylase produced from in vitro-translatedRNA isolated from the respective cell samples and controlsindicated no more than about 5% of the amount producedfrom the maltose-induced controls.As shown previously, the glucoamylase precursor was not

detected in the glucose- or 2-deoxyglucose-grown controls(Fig. 5, lanes x to z and d' to f'). In contrast, the resultsobtained with samples incubated with soluble starch werecomparable to those measured for other sugar-treated cellsat early time points (Fig. 4A and B, lanes j to 1; Fig. 5, lanesi to k). However, by 90 min, an increase in glucoamylaseexpression was evident, even though this increase was stillsignificantly less than that observed for maltose-treated cellsat any of the time points assayed. These observations areconsistent with our earlier work showing that starch-inducedproduction of glucoamylase was generally low and lagged incomparison with that induced by maltose (7).Heat shock blocks glucoamylase transcription. In Saccha-

romyces cerevisiae, a shift in growth temperature (25 to30°C) to 39°C results in the expression of heat shock proteinsand the repression of many other proteins through rapiddegradation of their transcripts and blocking of transcriptionand posttranscriptional events (14). Since the pattern of heatshock induction differs also according to the carbon sourceused for growth (14), we used this procedure to assess thestability of maltose-induced glucoamylase expression at thelevels of transcription and translation before, during, andafter a 30-min heat pulse (i.e., a shift from 30 to 39°C andthen back to 30°C).

Strain 1402 cells responded positively to this procedure,since in vitro translation of the 30-min (heat shock) RNAproduced an additional five polypeptides measuring 41, 76,86, 97, and 101 kDa, similar in size to heat shock proteins ofS. cerevisiae (14) (data not shown). Also, the 30-min heatshock significantly reduced the level of 138-kDa cell-associ-ated product by about 75% compared with the control levelbut did not alter the size or amount of extracellular gluco-amylase (Fig. 4A and B, lanes i). Further analysis of the in

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vitro translation products after immunoprecipitation (Fig. 5,lane h) indicated that the amount of immunoprecipitatedglucoamylase precursor was reduced to less than 5% of thelevel made from RNA of the 60-min control sample. Theseresults establish that heat shock, unlike glucose, preferen-tially blocks glucoamylase expression at the levels of tran-scription and translation and not at the levels of posttrans-lational processing and secretion.

Effect of cAMP. In yeasts and fungi, the role of cyclic AMP(cAMP) in catabolite repression is not clear (1, 18, 27, 36).Afanas'eva et al. (1) have studied the effect of glucosecatabolite repression on glucoamylase biosynthesis in theyeast Saccharomycopsis (Endomycopsis) fibuligera. Usingeither glucose (up to 2% [wt/vol]) or 2-deoxyglucose (up to 5mM), the addition ofcAMP (to 50 ,M) resulted in about 95%derepression of glucoamylase activity. We tested the effectof cAMP on glucoamylase expression by growing S. castelliicells in minimal medium containing either glucose or 2-deoxyglucose supplemented with cAMP (50 ,M final con-centration). Cells were harvested and assayed for gluco-amylase as usual after 30, 60, and 90 min of growth.Exogenously added cAMP did not derepress glucoamylaseexpression (Fig. 4A and B, lanes b' to d' and h' toj'; Fig. 5,lanes a' to c' and g' to i'). 3H-labeled cAMP does not appearto be incorporated into these cells under the conditionsspecified here (T. M. Dowhanick, unpublished data), whichthis may explain why cAMP failed to derepress glucoam-ylase expression.

DISCUSSION

Glucoamylase expression by S. castellii 1402 was studiedby exposing mid-log, glucose-grown cells to a variety ofcarbon substrates and under different conditions in order todevelop a more comprehensive understanding of the mech-anism of its regulation. This expression was monitored at thesteady-state level by using the Western blot technique tomonitor cell proteins and at the transient level of intactglucoamylase mRNA production by using in vitro translationof total RNA followed by immunoprecipitation of the gluco-amylase precursor product. The combined information fromthese experiments allows us to hypothesize the genesis ofthe 146-kDa extracellular glucoamylase. As in previous workon the a-amylase of Aspergillus oryzae (8, 11) and cellulaseof Schizophyllum commune (37, 38), there is no doubt thatexpression of glucoamylase is highly regulated in response toits source of carbohydrate and that this regulation is primar-ily at the level of mRNA transcription and stability. Theimmunoprecipitation of the glucoamylase-related translationproduct made in a rabbit reticulocyte cell-free system re-vealed that the nascent glucoamylase precursor is approxi-mately 120 kDa (±+7%) in size. Similar immunoprobing ofcell-associated and extracellular products detected in vivofurther indicated that the 120-kDa precursor must be rapidlymodified into at least two stable forms. The first may be thecell-associated 138-kDa polypeptide. Further modificationslead to the formation of the 146-kDa mature, active extra-cellular enzyme, most of which is released into the medium.The difference in the size of the 120-kDa glucoamylase

precursor from strain 1402 compared with its final 146-kDaproduct is about 17.8%. The additional 26 kDa is most likelythe result of posttranslational modifications such as glyco-sylation, which can account for 3 to 30% of the totalmolecular weight of the enzyme, depending on the producingorganism (17). Proteolysis likely occurs as well for removalof the glucoamylase secretion signal peptide as in other

glucoamylases (22) and for turnover during glucose repres-sion (see below). Additional information may be obtained bystudying the biosynthesis of glucoamylase in the presence ofan inhibitor of glycosylation such as tunicamycin (37, 38).

It was reported that in Saccharomycopsis (Endomycopsis)fibuligera, a buildup of cAMP during glucose repression ofglucoamylase expression was responsible for derepressionof the enzyme (1). However, we found that exogenouslyadded cAMP neither affects glucoamylase expression nor isincorporated into strain 1402 cells. The detection of low-level production of glucoamylase in media lacking anycarbohydrate was further shown to be the result of alow-level production of glucoamylase transcript that occursin the absence of glucose and its analog (2-deoxyglucose)and known inducers such as maltose and starch. Further-more, we found that the repressive effect of glucose canoverride even the strong inducer action of maltose. Thisrepressive effect also extends to the turnover of intracellularand extracellular glucoamylases, which are presumably de-graded by a protease(s) expressed by cells in the presence ofglucose (Fig. 5, lanes x to c').

Expression of glucoamylase by cells in the presence ofstarch is not as high as in the presence of maltose. The levelof starch-induced glucoamylase expression is probably mod-ulated by changing levels of inducer and glucose breakdownproduct. The inhibition of glucoamylase expression by glu-cose likely occurs at two levels. At one level, it appears to besimilar to the action of heat shock, which rapidly blocks bothglucoamylase transcription and translation. However, heatshock promotes expression of heat shock proteins and onlygradually affects posttranslational levels of cell-associatedand extracellular glucoamylase, suggesting that proteolysisis the other level of glucose action which is involved. Theseresults suggest that glucoamylase expression is tightly regu-lated in a manner similar to that of the enzymes responsiblefor galactose and maltose metabolism in Saccharomycesyeasts (7, 9, 26).

ACKNOWLEDGMENTS

This work was supported by NRCC-PILP contract CA910-3-003-576 and National Research Council funds to V.L.S.We gratefully acknowledge the technical support provided by M.

Dove and critical review of the manuscript by T. D'Amore, C. A.Bilinski, and K. Mussar.

LITERATURE CITED1. Afanas'eva, V. P., T. V. Gridneva, 0. E. Zaborina, and G. I.

Burd. 1978. Glucose catabolite repression of glucoamylasebiosynthesis in the yeast Endomycopsis fibuligera. Appl. Bio-chem. Microbiol. 14:676-681.

2. Boel, E., I. Hjort, B. Svenson, F. Norris, and K. D. Norris. 1984.Glucoamylases Gl and G2 from Aspergillus niger are synthe-sized from two different but closely related mRNA's. EMBO J.3:1097-1102.

3. Calleja, G. B., S. Levy-Rick, F. Moranelli, and A. Nasim. 1984.Thermosensitive export of amylases in the yeast Schwanniomy-ces alluvius. Biotechnol. Lett. 4:543-547.

4. Chirgwin, J. M., A. E. Pryzbyla, R. J. MacDonald, and W. J.Rutter. 1979. Isolation of biologically active ribonucleic acidfrom sources enriched in nuclease. Biochemistry 19:5294-5299.

5. Clementi, F., J. Rossi, L. Costamagna, and J. Rossi. 1980.Production of amylase(s) by Schwanniomyces castellii andEdomycopsis fibuligera. Antonie van Leeuwenhoek J. Micro-biol. Seral. 46:399-405.

6. Dhawale, M. R., and W. M. Ingledew. 1983. Starch hydrolysisby derepressed mutants of Schwanniomyces. Biotechnol. Lett.5:185-190.

7. Dowhanick, T. M., S. W. Scherer, G. Wilick, I. Russell, G. G.

VOL. 172, 1990

on March 30, 2019 by guest

http://jb.asm.org/

Dow

nloaded from

2366 DOWHANICK ET AL.

Stewart, and V. L. Seligy. 1988. Differential glucoamylaseexpression in Schwanniomyces castellii induced by maltose.Can. J. Microbiol. 34:262-270.

8. Erratt, J. A., P. E. Douglas, F. Moranelli, and V. L. Seligy. 1984.The induction of a-amylase by starch in Aspergillus oryzae:

evidence for controlled mRNA expression. Can. J. Biochem.Cell Biol. 62:678-690.

9. Erratt, J. A., and A. Nasim. 1986. Cloning and expression of aSaccharomyces diastaticus glucoamylase gene in Saccharomy-ces cerevisiae and Schizosaccharomyces pombe. J. Bacteriol.166:484 490.

10. Frelot, D., G. Moulin, and P. Galzy. 1982. Strain selection forthe purpose of alcohol production from starch substrates. Bio-technol. Lett. 4:705-708.

11. Gines, M. J., M. J. Dove, and V. L. Seligy. 1989. The taka-amylase of Aspergillus oryzae is coded by two functional geneseach containing eight introns. Gene 79:107-117.

12. GIlsin, V., R. Crkvenjkov, and C. Byus. 1974. Ribonucleic acidisolation by cesium chloride centrifugation. Biochemistry 13:2633-2637.

13. Laemmli, U. K. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London)227:680-685.

14. Lindquist, S., and E. A. Craig. 1988. The heat-shock proteins.Annu. Rev. Genet. 22:631-677.

15. Lusena, C. V., C. C. Chapagne, and G. B. Caileja. 1985.Secretion and export of amylolytic activities in Schwanniomy-ces alluvius. Can. J. Biochem. Cell Biol. 63:366-371.

16. MacKay, R. M., S. Baird, M. J. Dove, J. A. Erratt, M. Gines, F.Moranell, A. Nasim, G. E. Willick, M. Yaguchi, and V. L.Seligy. 1985. Glucanase gene diversity in prokaryotic and eu-karyotic organisms. BioSystems 18:279-292.

17. Manjunath, P., B. C. Shenoyk, and M. R. Raghavendra-Rao.1983. Fungal glucoamylases. J. Appl. Biochem. 5:235-260.

18. Matsumoto, K., I. Uno, and T. Ishikawa. 1985. Genetic analysisof the role of cAMP in yeast. Yeast 1:15-24.

19. McCann, A. K., and J. A. Barnett. 1984. Starch utilization byyeasts: mutants resistant to carbon catabolite repression. Curr.Genet. 8:525-531.

20. McCann, A. K., and J. A. Barnett. 1986. The utilization ofstarch by yeasts. Yeast 2:109-120.

21. Moranelli, F., M. Yaguchi, C. V. Lusena, C. Champagne, I.Veliky, A. Nasim, T. Walker, B. Y. Yoo, S. Levy-Rick, and G. B.CafleJa. 1982. Schwanniomyces: yeasts of potential importance,p. 521-526. In B. A. Summers (ed.), Proceedings of the 4thBio-energy R&D Seminar. NRC Publications Office, Ottawa,Ontario, Canada.

22. Nunberg, J. H., J. M. Meade, G. Cole, F. C. Lawyer, P.McCabe, V. Scheikart, R. Tal, V. P. Wittman, J. E. Flatgaard,and M. A. Innis. 1984. Molecular cloning and characterization ofthe glucoamylase gene ofAspergillus awamori. Mol. Cell. Biol.4:2306-2315.

23. Oteng-Gyang, K., G. Moulin, and P. Galzy. 1981. A study of theamylolytic system of Schwanniomyces castellii. Z. Allg. Mikro-biol. 21:537-544.

24. Prakash, K., and V. L. Seligy. 1988. A temporally expressedgene from Schwanniomyces alluvius and detection of homolo-gous sequences in other yeasts. Gene 73:131-140.

25. Pretorius, I. S., D. Modena, M. Vanoni, S. Englard, and J.Marmur. 1986. Transcriptional control of glucoamylase synthe-sis in vegetatively growing and sporulating Saccharomycesspecies. Mol. Cell. Biol. 6:3034-3041.

26. Rossi, J., and F. Clementi. 1985. Protein production by Schwan-niomyces castellii on starchy substrates, in liquid and solidcultivation. J. Food Technol. 20:319-330.

27. Schlanderer, G., and H. Dellweg. 1974. Cyclic AMP and catab-olite repression in yeasts. Eur. J. Biochem. 49:305-316.

28. Sills, A. M., I. Russell, and G. G. Stewart. 1983. The productionand use of yeast amylases in the brewing of low carbohydratebeer, p. 377-384. In P. A. Martin (ed.), Proceedings of the 19thEuropean Brewing Convention Congress. IRL Press, Oxford.

29. Sills, A. M., M. E. Sauder, and G. G. Stewart. 1984. Isolationand characterization of the amylolytic system of Schwanniomy-ces castellii. J. Inst. Brew. 90:311-314.

30. Sills, A. M., P. S. J. Zygora, and G. G. Stewart. 1984. Charac-terization of Schwanniomyces castellii mutants with increasedproductivity of amylases. Appl. Microbiol. Biotechnol. 20:124-128.

31. Simoes-Mendes, B. 1984. Purification and characterization of theextracellular amylases of the yeast Schwanniomyces alluvius.Can. J. Microbiol. 30:1163-1170.

32. Stewart, G. G., R. M. Jones, and I. Russell. 1985. The use ofderepressed yeast mutants in the fermentation of brewery wort,p. 243-250. In P. A. Martin (ed.), Proceedings of the EuropeanBrewing Convention Congress. IRL Press, Helsinki.

33. Svenson, B., T. G. Pederson, I. B. Svendsen, T. Sakai, and M.Ottesen. 1983. Characterization of two forms of glucoamylasefrom Aspergillus niger. Carlsberg Res. Commun. 47:55-69.

34. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretictransfer of proteins from polyacrylamide gels to nitrocellulosesheets: procedure and some applications. Proc. Natl. Acad. Sci.USA 76:4350-4354.

35. van Uden, N., C. Cabeca-Silva, A. Madiera-Lopes, and I. Spen-cer-Martins. 1980. Selective isolation of derepressed mutants ofan a-amylase yeast by the use of 2-deoxyglucose. Biotechnol.Bioeng. 22:651-654.

36. van Wik, R., and T. M. Konijn. 1971. Cyclic 3',5'-AMP inSaccharomyces carlsbergensis under various conditions of ca-tabolite repression. FEBS Lett. 13:184-186.

37. Willick, G. E., R. Morosoli, V. L. Seligy, M. Yaguchi, and M.Desrochers. 1984. Extracellular proteins secreted by the basid-iomycete Schizophyllum commune in response to carbonsource. J. Bacteriol. 159:294-299.

38. Willick, G. E., and V. L. Seligy. 1985. Multiplicity in cellulasesof Schizophyllum commune derives in part from transcript andglycosylation heterogeneity. Eur. J. Biochem. 151:89-96.

39. Wilson, J. J., and W. M. Ingledew. 1982. Isolation and charac-terization of Schwanniomyces alluvius amylolytic enzymes.Appl. Environ. Microbiol. 44:301-307.

40. Wilson, J. J., G. G. Khachatourians, and W. M. Ingledew. 1982.Protoplast fusion in the yeast Schwanniomyces alluvius. Mol.Gen. Genet. 186:95-100.

41. Yamashita, I., and S. Fukul. 1983. Molecular cloning of aglucoamylase-producing gene in the yeast Saccharomyces.Agric. Biol. Chem. 47:2689-2691.

42. Yamashita, I., and S. Fukui. 1985. Transcriptional control of thesporulating-specific glucoamylase gene in the yeast Saccharo-myces cerevisiae. Mol. Cell. Biol. 5:3069-3073.

43. Yamashita, I., T. Itoh, and S. Fukul. 1985. Cloning and expres-sion of the Saccharomyces fibuligera glucoamylase gene inSaccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 23:130-133.

44. Zimmerman, F. K., and I. Scheel. 1977. Mutants of Saccharo-myces cerevisiae resistant to carbon catabolite repression. Mol.Gen. Genet. 154:75-82.

J. BACTERIOL.

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