Eur. J. Biochem. 209,951 -959 (1992) 0 FEBS 1Y92

Characterization of the 56-kDa subunit of yeast trehalose-6-phosphate synthase and cloning of its gene reveal its identity with the product of CZFl, a regulator of carbon catabolite inactivation Walter BELL', Paul KLAASSEN ', Martin OHNACKER ', Thomas BOLLER ', Marga HERWEIJER', Peter SCHOPPINK', Peter VAN DER ZEE' and Andres WIEMKEN' ' Botanisches Institut, Univcrsitiit Bascl, Switzerland ' Gist-Brocades N. V. Research and Development, Delft, The Netherlands

(Received July 8, 1992) - EJB 92 0968

Trehalose-6-phosphate synthase is the key enzyme for biosynthesis of trehalose, the major soluble carbohydrate in resting cells of yeast. This enzyme was purified from a strain of Sacchurornyces cerevisiue lacking vacuolar proteases. It was found to be a multimeric protein of 630 kDa. Monoclonal antibodies were raised against its smallest subunit (56 kDa) and used for screening a yeast cDNA library. This yielded an immunopositive cDNA clone of 1.7 kb, containing an open reading frame of 1485 base pairs. Its sequence, called TPSl (for trehalose-6-phosphate synthase), was represented by a single gene in the yeast genome and was found to be almost identical with the recently sequenced CIFl, a gene important for carbon catabolite inactivation, believed to be allelic with FDPl . A mutant obtained by disruption of TPSl had a very low activity of trehalose-6-phosphate synthase, indicating that TPSl is an important component of the enzyme. The mutant also showed a growth defect when transferred from glycerol to glucose, a phenotype similar to that of the cifl and f d p l mutants deficient in carbon catabolite inactivation. Thus, the smallest subunit of the biosynthetic enzyme trehalose-6- phosphate synthase appears to have, in addition, a central regulatory role in the carbohydrate metabolism of yeast

The non-reducing disaccharide trehalose (a-D-glucopyra- nosyl( 1-1)a-D-glucopyranoside) accumulates in large amounts in resting cells of the yeast Sacchurornyces cerevisiae and has been proposed to be a storage form of carbohydrates together with glycogen (Lillie and Pringle, 1980; Thevelein, 1984). While the function of glycogen as a carbon and energy reserve is beyond doubt, trehalose has been proposed to function mainly as a stress protectant rather than a storage compound, being accumulated also in response to stresses such as ex- posure to heat, desiccation, or heavy metals (Hottiger et al., 1987a, 1989; Van Laere, 1989; Wiemken, 1990).

Biosynthesis of trehalose proceeds in two steps in the yeasts S. cerevisiuc, Cundidu utilis and in the bacterium Escherichiu coli (Cabib and Leloir, 1958; Elander, 1968; Giaever and Strom, 1988; Vicente-Soler et al., 1989). First, trehalose-6- phosphate synthase (TPS; UDP-glucose:u-glucose-6-phos- phate 1 -glucosyltransferase) transfers the glucosyl residue of UDP-glucose to glucose 6-phosphate to yield trehalose 6-

Correspondence to A. Wiemken, Botanisches Institut der L'niversi-

Fax: +41 61 261 53 18. Abhrevdutinns. TPS, trehalose-6-phosphate synthasc; TPP, trcha-

lose-6-phosphate phosphatase; YEP, yeast extractibactopeptone. Enzymes. Trehalose-6-phosphate synthase (EC 2.4.1.15); lreha-

lose-6-phosphate phosphatase (EC 3.1.3.12); neutral trehalase (EC 3.2.1.28).

Note. The nucleotide scqucnce TPSI has bccn deposikd in thz EMBL Data Library under accession number X68214.

tat Basel, Hebelstrasse 1, CH-4056 Basel, Switzerland

phosphate. Second, a specific phosphatase (trehalose-6-phos- phate phosphohydrolase; TPP) cleaves off the phosphate group producing trehalose. This pathway has first been identi- fied by Cabib and Leloir (1958), who also performed a partial purification of the two enzymes which co-purified. The en- zymes were further purified and their kinetic properties stud- ied in S. cerevisiue (Panek et al., 1987; Vandercammen et al., 1989). Recently, purification of a proteolytically modified form of the TPS/TPP complex has been reported by Londesborough and Vuorio (1991). Their purification resulted in a protein complex of about 800 kDa, composed of three polypeptides of 57, 86 and 93 kDa. In addition, a protein dimer of approximately 110 kDa was isolated which activated TPS but showed no effect on TPP. The proteolytic activation, the activator protein and the influence of physiological con- centrations of inorganic phosphate on TPS and TPP were discussed as mechanisms for the regulation of trehalose syn- thesis. FranGois et al. (1991) presented evidence that both TPS and TPP are subject to catabolite inactivation and repression.

A temperature-sensitive yeast mutant has been described (Piper and Lockheart, 1988) accumulating trehalose 6-phos- phate when shifted to temperatures above 3472, non-permis- sive for growth. This has been interpreted as a defect in TPP, the second enzyme of trehalose biosynthesis, but the corre- sponding gene has not yet been characterized.

In the present paper, we report on the purification of the TPS complex from S. cerevisiue and thc identification of the TPSl gene, encoding the 56-kDa subunit of the TPS complex.

952

We show that TPSI expression is induccd by hcat shock, concomitantly with TPS. We further show that the DNA sequence of TPSl is nearly identical with CIPl (Gonzales et al., 1992) a yeast gene essential for catabolite inactivation of fructose-1,6-bisphosphatase (Navon et al.. 1979). thought to be allelic with F D P l (van de Poll and Schamhart, 1977; Banuelos and Fraenkel, 1982). Disruption of TPSI gives rise not only to a deficiency in TPS activity, but also to an inability to grow on glucose, a well known phenotype ofjdpl (van de Poll and Schainhart, 1977) or cifr mutants (Navon et al., 1979). Thus, TPSl is an important part of TPS and at the same time a regulator of glucose metabolism in yeast. Taken together, this points to a central role of the TPSl (CIFI, F D P l ) gcnc product in yeast carbon metabolism.

MATERIALS AND METHODS

Strains and media

S. cerevisiae strain C13-ABYS 86 (MATa hi.~?-ll,15 leu2- 3,112 ura3 canr prnl-1 p r b l - l prcl-1 cps 1-3) was provided by D. H. Wolf, University of Freiburg, FRG. Wild-type strain D 273-10B (MATE) and strain GRD 11 -21, a diploid strain derived from the parental strains X 2180-1A (MATu SUC2 mu1 gal2 C U P / ) and MC 333 (MATE leu2 t rp l met8) wcre obtained from Gist-Brocades. Strain BKpl1 was constructed by disruption of the T P S l gene in strain GRD 11-21. S. carlshergensis strains DFY 333 (MATE lys2 MAL6 FDPI) and DFY 334 (MATu lys2 MAL6, fdpl ) were froin D. Fraenkel, Harvard Medical School, Boston, USA.

E. coli strain Y 1090 F', A(lacU169) proA+ A(1onJ araU139strAsupFtrpC22: :TnlO(teP) (pMC9) hsdR ( r i ,ma was used as a host for bacteriophage I-gl 11. The cloning procedures were carried out in E. coli strain WK 6 Ajlac- proAB) galE strA Flue Ia Z AM15 proA+B+ (Zell and Fritz, 1987).

Yeast cells were grown on a rotary shaker (125 rpm) at 27 'C in 1 % yeast extract, 2% bactopeptone (YEP) containing either glucose, maltose or glycerol (2%) as carbon source. Selective medium was the respective YEP medium supplied with 300 pg/ml G-418 sulphate.

E. coli were grown in Luria-Bertani medium (Sambrook et al., 1989) supplied with the indicated antibiotics.

Chemicals

Ethyl-agarose and enzymes werc from Sigma, DEAE- Trisacryl M from 1BF Biotechnics, Sephacryl S-400 HR froin Pharmacia, and Fractogel EMD TMAE 650-(S) from Merck.

Assays

TPS and neutral trehalase were determined as described (De Virgilio et al., 1990; De Virgilio ct al., 1991). Protein was measured according the method of Bradford (1976) with bovine serum albumin as standard. Trehalose was extracted froin yeast with trichloroacctic acid and determined by thc anthrone procedure described by Lillie and Pringle (1 980). The identity of the sugar was verified by thin-layer chromato- graphy.

Purification of TPS

S. ccrcvisiae strain C13-ABYS 86, lacking proteases A and B and carboxypeptidases Y and S, was employed. Cells were

grown in rich medium (1 % yeast extract, 2% peptone and 1 YO glucose) to stationary phase. For each preparation, 60 - 80 g cells were harvested by centrifugation, washed twice with cold double-destilled water containing 1 mM EDTA and sus- pended in 50mM imidazole buffer adjusted with HCl to pH 6.3 (buffer A), containing 1 mM each of phenylmethylsul- fonyl fluoride, EDTA and 1,4-dithiothreilol. All procedures were performed at 0-4'C. The cell suspension was treated with glass beads (0.45 mm diameter) in a cell disrupter ('Bead beater') in several bursts of 45 s with 1-min breaks inbetween to avoid warming up of the suspension. The extract was com- bined with the solution resulting from the washing of the glass beads with the homogenization solution and centrifuged for 15 min at 30000 g. A protaminc sulfate solution (2% in buffer A) was added dropwise to a final concentration of 0.05 gig protein. After 30 min of stirring and removal of precipitate (I5 min, 30000 g), solid ammonium sulfate was added in small portions to bring the supernatant to 45% saturation. After 1 h stirring, the precipitate was collected (10 min, 12000 g) and resuspended in 60 ml30% saturated ammonium sulfate in buffer A, containing 1 mM EDTA. After 30 min, undissolved material was removcd by centrifugation (10 min 12000 g). The clear Supernatant was applied to an ethyl-agarose column (2.5 cm x 15 cm) equilibrated with buffer A containing 1 mM EDTA and 16.2% (mass/vol.) ammonium sulfate. After wash- ing with the same solution, a linear gradient of 30 - 0% satu- rated ammonium sulfak in buffer A was applied. Active frac- tions werc pooled, dialyzed twice against 2 120 mM imidazole adjusted to pH 6.3 with IIC1, and applied to a DEAE- Trisacryl column (2.5 cm x 14 cm), equilibrated with the same buffer. After a 150-ml wash. a linear gradient of 0-0.5 M KCl in 0.5 1 dialysis buffer was applied. The active fractioiis were pooled and concentrated in a stirred ultrafiltration cell (Amicon) over a YM 100 membranc. The concentrated pro- tein solution was Ioadcd onto a Sephacryl S-400 H R column (2.5 cm x 96 cm) equilibrated with 50 mM Tricine adjusted to pH 7.0 with KOH conlaining 0.1 M KC1 and 1 mM EDTA. Fractions containing TPS activity were pooled and loaded onto a Fractogel EMD TMAE 650-(S) column (I cm x 15 cm) without further treatment. The column was equilibrated with 20 mM Bistris adjusted to pH 6.3 with HC1 containing 0.1 M KC1, and the enzyme was eluted by a 40-ml gradient of 0.1 - 0.6 M KC1 in the same buffer. Active fractions were pooled, dialyzed against 1 mM Bistris, pH 6.3, and lyophilized for storage at - 20 "C. In initial isolations, the purification was stopped after the gel-filtration step. In this case, the pooled enzyme fractions were put into a dialysis tube, embedded in solid sucrose and thus concentrated to a few millilitres. The resulting solution was almost saturated with sucrose, and the enzyme activity was found to be stable for sevcral months when stored on ice.

Electrop horcsis

SDSjPAGE was performed as described (Laemmli, 1970) on 135 x 80 x 10 mm gels or in a Bio-Rad Mini Protean I1 device. For immune blotting. the separated protcins wcre transferred onto nitrocellulose membranes, probed with 1 : 50- diluted cell-culture liquid of a monoclonal antibody producing cell line and detected with a goat anti-(mouse Ig) ~ alkaline- phosphatase conjugate (1 : 3000) from Bio-Rad. Nitroblue tetrazolium and 5-bromo-4-chloro-3-iiidolyl phosphate were the colour rcagentb.

953

on ice, the high-molecular-mass bands shifted to molecular masses of 97 kDa and 94 kDa, respectively (data not shown).

As a tool for further investigations, we attempted to pre- pare monoclonal antibodies directed against the three main

the immunization of mice. No monoclonal antibodies were obtained against TPS2 and TPS3. One of the monoclonal antibodies against TPSl showed good sensitivity and low background on Western blots and hence, we concentrated our studies on TPSI.

Monoclonal antibodies

Monoclonal antibodies were prepared at the Rescarch and Development department of Gist-Brocades, according to

was separated on preparative SDS/PAGE and stained with Coomassic blue R 250. Single polypept~des were excised, ground in a mortar after addition of physiological saline solu- tion, emulsified 1 : 1 with Freund’s Incomplete Adjuvant and injected into male Balb/c mice.

procedures and Lane, 9p8)’ Purified TPS bands of the purified protein which were separately used for

Cloning of the TPSl gene

A yeast cDNA library (Clontech) in ig t 11 was screened according to the immunological technique described by Young and Davis (1983), using the monoclonal antibody against the 56-kDa protein of the TPS complex. 200000 plaques were screened with the antibody, resulting in 41 positive plaques. Seven of these plaques, chosen at random, were purified, and their inserts were amplified by polyrnerasc-chain reaction in a Perkin-Elmer Cetus DNA thermal cycler according the in- structions of the manufacturer, using primers cornplcmentary to the Agt 11 sequence beside the EcvRI cloning site of the inserts [primer a = 5’-d(GGCGACGACTCCTGGAGCC- CG)-3’, primer b = 5’-d(CACCAGACCAACTGGTAAT-

Isolation and manipulation of DNA was performed using standard procedures as described (Sanibrook et al., 1989). The yeast DNA sequences were integrated into the EcoRI sitc of the vector pSNENS (Gist-Brocades). This vector is basically plasmid pTZ19R (Pharmacia), but the polylinker is replaced by another linker which contains symmetrical sites for S’iI and Not1 on both sides of the unique EcoRI site. Southern- blot analysis was performed under high stringency conditions, essentially as described (Southern, 1975) using [K-~’P]~ATP from Amersham.

GG)-3’].

DNA sequencing

Sequencing was performed with the plasmids pWR 111-1 and pWB 111-2 (Fig. 4), prepared by CsCl gradicnts, using thc chain-termination method (Sanger et al., 1977) with Sequen- ase (United States Biochemical Corporation), and [35S] dATP[S] from Amersham. Primers were used according to the DNA sequence of Nclson et al. (1989).

Sequence analyses were carried out with the GCG package (Devereux et al., 1984).

RESULTS

Purification of TPS

Table 1 summarizes the purification of TPS from S. cerct.isiue, C13-ABYS86, a yeast strain lacking vacuolar pro- teases (Achstetter et al., 1984). The enzyme was purified 300- fold with an overall yield of 34%. The molecular mass of the native protein, estimated by gel filtration on a Sephacryl S- 400 HR column, was approximately 630 kDa (data not shown). SDSIPAGE analysis of the purified enzyme revealed three main bands with apparent molecular masses of 55: 100 and 105 kDa, and occasionally a minor contaminating band of 27 kDa (Fig. 1). The three main polypeptides were designated TPS1, TPS2 and TPS3, respectively. In some preparations, the two larger polypeptides displayed signs of degradation, probably due to remaining protcases. After extended storagc

Induction of TPSl by heat shock

It is well known that the activity of TPS in yeast is induced by a heat shock (Hottiger et al., 1987b). We therefore exam- ined whether heat shock also induced the synthesis of TPSl. Immune blots indeed demonstrated that the amount of TPSl increased during heat shock (Fig. 2). It should be noted that the protein is found also in normal growing cells, even though at a lower level.

Cloning of a cDNA encoding TPSl

We screened a yeast cDNA expression library with mono- clonal antibodies against TPSl and found seven immunoposi- tive clones with insert sizes over 0.6-1.7 kb. Southern-blot analysis of genomic yeast DNA was performed with each of the 7 igt 11 inserts (Fig. 3). All probes hybridized to a BurnHl band of approximately 12 kb and a Hind111 fragment of 9 kb. In EcoRI digests, a 2.6-kb band hybridized with all clones, and an additional band of 3.2 kb hybridized with three of the larger Igt 11 inserts, indicating that these inserts harbour an internal EcaRI site. This analysis strongly indicated that all seven clones represent a single gene in the yeast genome, designated here TPSI (trehalose-6-phosphate synthase 1). A restriction map of the largest insert is shown in Fig. 4, along with thc start and stop sites of the open reading frame encoded (see below).

Disruption of the TPSl gene

To disrupt TPSI, EcoRI fragments, prepared from iLgt 11 inserts, were cloned into the EcvRI site of the plasinid pSNENS (from Gist-Brocades). This yielded two plasmids containing inserts of 0.55 kb and 1.2 kb, called pWB 111-1 and pWB 111-3 (see Fig. 4). G-418 resistance was introduced into pWB 111-1 as a dominant selective marker between the StuI and H p I sites using a Bg.111 fragment of plasmid pRRz (from Gist-Brocades) containing the ADH I promotor of S . cuerisiue coupled to the G-418-resistance gene derived from transposon Tn 5 . The genc replaccment cassette was cut out by ZjiI prior to transformation of the yeast. The haploid strain D 273-10B and the diploid strain GRD 11 -21 were transformed with the gene-replacement cassette and plated on YEPlglucose agar containing the antibiotic (3-41 8. Interest- ingly, we found only diploid transformants and no haploids, suggesting that an essential gene had been mutated. The dip- loid transformant carrying an intact and a disrupted copy of TPSI was called strain BKp11.

Characterization of the transformants

Haploid transformants were prepared by sporulation of strain BKpll and subsequent dissection of the asci (Sherman et al., 1986). After two days. each tetrad showed only two

954

Table 1. Purification of TPS from S. eerevisiae. Strain C13-ABYS86 was employed for the protein purification. The table shows the results of a single purification. Similar data were obtained in three other preparations.

Step Volume Activity Protein Specific Purification Yield activity

Crude homogenate Protamine supernatant Ammonium sulfate fractionation Ethyl-agarose DEAE-Trisacryl Scphacryl S-400 HR Fractogel TMAE pool

ml pkat

177 163 41 88

107 62

3

10.1 11.6 7.1 5.9 5.7 5.5 3.4

3440 2070

347 64.4 26.0 7.3 3.9

m t / g protein

2.94 5.58

20.6 91.4

21 9 7 60 887

-fold Yo

1 100 2 105 7 71

31 59 75 57

260 55 300 34

Fig. 1. SDS/PAGE. of purified TPS. Lanes A -C, 700,280 and 140 ng purified enzyme, respectively, subjected to SDSjPAGE and visuali~ed by silver staining. Lane M, marker proteins with the molecular masses indicated (in kDa).

colonies on YEP/glucose medium, indicating that disruption of the gene was lethal (Fig. 5). However, a microscopic check of the ascospores that did not yield colonies showed that the single spores actually had started to grow but stopped growing after five or six divisions, pointing to a kind of imbalancc in metabolism rather than to a fully lethal deletion.

A second tetrad dissection was performed on YEPlagar with maltose as the carbon source. In this experiment, four viable spores at most were obtained from the asci. Apparently, sensitivity to glucose was responsible for the growth stop on Y EP/glucose agar. Replica plating on Y EPinialtose contain- ing G-418 yielded a 2 : 2 segregation of resistance, indicating a single-gene inheritance.

The protein formed in cells carrying the gene disruption was examined on immune blots with the monoclonal antibody against TPSl (Fig. 6). Thc two G-418 resistant strains of each tetrade had a truncated version of TPSl which was 2 - 3 kDa smaller than the normal protein, explained by the fact that the gene-replacement cassette had been integrated into the yeast genome close to the end of the coding sequence of TPSl (Fig. 4).

Tetrad 11 was selected for the determination of TPS and neutral trehalase (Table 2). The two wild-type haploids, strain A and C, showed normal activities of TPS. Strains €3 and D, possessing the truncated version of TPSI, displayed very low

Fig. 2. Heat-shock-induced accumulation of the small subunit of the TPS complex in yeast. Cells of wild-type yeast (strain D 273 - 10B), exponentially growing in YEP/glucose medium were shifted from 27°C to 40°C. Protein was extracted as described (De Virgilio et al., 1991) after a heat shock for the times indicated, blotted to nitrocellu- lose after SDS/PAGE and probed with the monoclonal antibody against the 56 kDa protein of the TPS complex. The molecular mashes of the prestained markers in lane M are 106, 80, 49.5. 32.5. 27.5 and 18.5 kDa.

trehalose-6-phosphate activity, confirming the hypothesis that the 56-kDa protein indeed is a part of the TPS complex, being important for TPS activity. As expected, the activity of neutral trehalase, the enzyme for trehalose degradation, was not af- fected by the mutation.

Disruption of TPSl disturbs the association of TPSl with the TPS complex

In a wild-type yeast strain, most of the TPSl was found to co-fractionate with the TPS complex on a gel-filtration column (Fig. 7A, B). The disruption mutants harbouring a gene encoding a truncated version of TPSI, having only a very low TPS activity (Table 2), showed a totally different pattern of the immunopositive, truncated TPSI product (Fig. 7C). It was distributed over the whole column effluent, showing no peak in the fractions where the TPS complex would be expected.

955

Fig. 3. Southern-blot analysis of two Agt 11 inserts hybridized to genomic yeast DNA. Genomic yeast DNA was digested with BurnHl (B), EcoRI (E) or Hind111 (11). A 1.5-kb insert (a) and a 0.6-kb insert (b) were used as probes.

E

G 418 resistance

II BS B E F) E

- Start 100 bp

t Stop

plasmid pWB 111-3 f * (1.2 kb) plasmid pWB In-1

(0.55 kb)

Fig. 4. Restriction map of the cDNA encoding TPSI, and strategy to disrupt the gene. The abbreviations for the restriction endonucleases are: B, BglII; E, EcoRI; H, HpaI; S, Sun; St, StuI. The length of the whole sequcncc is 1719 bp, the opcn reading frame contains 1485 nucleotides. The cDNA was subcloned into two plasmids, and pWB 111-1 was uscd for inscrtion of a cassette containing neomycin phosphotransferase under the control of an ADHl promoter (G-418 resistance). This construct was employed for gene disruption.

Fig. 5. Analysis of ascospore tetrads with regard to growth on glucose. Asci of strain GRD 11-21 wild-type (A) and strain BKpl1 (B) harbouring a disruption in TPSl wcrc dissected, and ascospores were placed on YEfimedium with 2% glucose. The growth after 2 days at 30'C is shown.

The nucleotide sequence of TPSl

The cDNA containing TPSI had a length of 1719 bases and contained an open reading frame of 1485 nucleotides, coding for 495 amino acids (Fig. 8). The molecular mass of the resulting gene product, calculated according to the predicted

Fig. 6. Immune-blot analysis of three tetrads derived from the diploid strain BKpll harbouring one disrupted and one intact copy of TPSI. Total yeast protein (5 pg/lanej was separated by SDS/PAGE, trans- ferred to a nitrocellulose filter and probed with the antibody against the 56-kDa polypeptide of the TPS complex. tpsl, truncated version of TPS1; wt, wild type.

Table 2. TPS and neutral trehalase in the progeny of spores from a tetrad of S. cerevisiue strain BKpll, harbouring a deleted copy of the TPSl gene and in S. curlsbergensis, strains DFY 333 (wild-type) and DFY 334 (&I). Cells were grown at 27°C in YEP medium with 2% glycerol as sole carbon source. Cells in the exponential growth phase were harvested at an Ahoo of 0.3-0.5, cells in the stationary phase after 3 days. Activities of TPS and neutral trehalase were determined in Triton-X-100-pcrmeabilized cells (De Virgilio et al., 1991) using the methods described by De Virgilio et al. (1990, 1991).

Strain Trehalose-6-phosphate Neutral trebalase growth synthase growth phase phase

exponential stationary exponential stationary

pkat/g protein

A (TPSI) 0.19 3.85 11.2 44.5 B ( A ~ p s l ) 0.03 0.12 18.7 36.5 C (TPSI) 0.52 1.93 17.2 37.0 D ( d f p s l ) 0.04 0.05 29.4 40.2 FDPl 0.26 1.13 45.8 58.6 fdP1 0.03 0.28 65.8 100.9

amino acid sequence, is 56.2 kDa. This value is in agreement with the molecular mass of TPSI determined on denaturing gels. The codon bias index (Bennetzen and Hall, 1982) of TPSl is 0.23, which means that the codon usage is little biased. The gene is terminated by two opal (TGA) codons. Both facts suggest that TPSl is not as highly expressed as the glycolytic enzymes (Sharp and Cowe, 1991).

A hydrophobicity plot of the deduced amino acid sequence according to Kyte and Doolittle (1982) gave no additional information about the possible structure or function of the protein (data not shown).

l h e DNA sequence of TPSl has already been determined as part of a genomic clone of 5 kb encoding a yeast vacuolar ATPase (Nelson et al., 1989). Although this clone contained the full sequence encoding TPSI, the open reading frame was missed, probably because of some frameshifts due to sequencing artifacts (Fig. 8). Recently, the CIFl gene has been identified by functional complementation of a yeast strain carrying the cifl mutation and sequenced by Gonzales et al. (1992). This sequence is almost identical with T P S I ; the open reading frame of TPSl showed only seven changes of amino acids compared to CZFI (Fig. 8). Originally, the cis] mutant has been isolated by Navon et al. (1 979) in a screen for glycoly- sis mutants. Howcver, no defect in the glycolytic enzymes was found, only the gluconeogenetic enzyme fructose-I ,6-bis- phosphatase was impaired in the inactivation by glucose which occurs in wild-type yeast after a shift from gluconeogenesis to glycolysis. The c f l mutant grows well on non-fermentable

956

A

4 6 S 10 12 14 16 18 20 22

Fig. 7. Association of 'IPS and TPSl analyzed by gel filtration. (A) Distribution of TPS (--I) and total protcin (a) or S. cermiszue strain D 273 -- 1OB (wild type) aftcr gel filtration over a Superose 6 column. A yeast culture, growing exponentially in YEP/glucose, was incubated a1 40 'C 75 min prior to analysis. The TI'S activity of non-induced cells in the cxponential growth phase is too low for a reliable determi- nation in the column eluatc. Thc arrows above the elution profile indicate the position of marker proteins (T : thyroglobin, 669 kDa; F = ferritin. 440 kDa; C = catalase, 240 ItDa; B = bovine serum albumin, 66 kDa). (B) Immune-blot analysis of the TPSl protein in the eluate of the Superose 6 column using a monoclonal antibody. For each lane 25 111 of a column Fraction were used. (C) as (Rj but with the strain BKpl1 - l l B ( d t p s l j growing in YEP/glycerol medium.

sugars but tolerates only- low concentrations of glucose (Navon et al., 1979). A very similar phenotype is known from two other mutations, thcfdpi (van de Poll el al., 1974) and sstl (Operti et al., 1982) mutations. All three strains are known to possess only a very low activity of TPS (Charlab et al.. 1985). Since most previous investigations have been performed with the .fdpl mutant in Saccharomyes cnrlsber- gensis (van de Poll et al., 1974; van de Poll and Schamhart, 1977; Banuelos and Fraenkel, 1982; Charlab et al., 1985; Beullens and Thevelein, 1990), we used this strain for a com- parison with our tpsl strain (Table 2). Both the,fdpl strain and our 1psJ strain showed a diminished activity of TPS.

Disruption of TPSZ gives rise to glucose sensitivity

In a first attempt to produce haploid transformants, two out offour spores from the diploid transformant BKp 11 were

not able to survive on YEP/glucose (Fig. 5). However, all four segregants grew well on maltose and glycerol as carbon source (data not shown). In order to test if disruption of TPSJ con- ferred sensitivity to glucose, the progeny of tetrad 11 of strain BKp 11 was grown in liquid Y EP/glycerol. In the exponential growth phase, glucose was added, and the growth of the cells was monitored spectrophotometrically. The growth curvcs clearly demonstrated that the two tpsl segregants ceased growing upon addition of glucose (Fig. 9). The,fdpI mutant behaved very similarly.

Thus, the TPSl gene product reprcscnts a component of the TPS complex, and is involved in the use of glucose as carbon source. This points towards a central role for the TPSl gcnc product in the yeast carbon metabolism.

DISCUSSION

TPS has been purified recently by Londesborough and Vuori (1991). Since thcir data indicated problems with proteol- ysis, we used the ABYSl strain (Achstetter et al.. 1984) devoid of the four major vaculoar proteases. Like Londesborough and Vuori (1991), we found TPS to be composed of three subunits. The smallest subunit, called TPS1, had the same size as previously reported. However, the two larger subunits, TPSZ and TPS3, were considerably larger than reported pre- viously (Londesborough and Vuorio, 1991) which might be due to the diminished protease activity of the ABYSl mutant.

When TPSl WBS disrupted, TPS activity was strongly diminished (Tablc 2). The fact that the enzyme activity was not completely destroyed by the disruption of TPSl allows seVera1 explanations. The truncated version of TPSl could still partially fulfill its function or there might be other non- related polypeptides with the same or a similar function as TPS1, or TPSl might be a regulatory protein. In the latter case, the other polypeptides, TPSZ andlor TPS3, are likely to perform the catalytical function of the enLyme. According to this model, the residual activity would represent part of en- zyme activity, present constitutively, independent from TPSl .

The activity of TPS is low in yeast during their exponential growth phase on glucose as carbon source, but strongly in- creases upon a temperature shift to 40°C (Hottiger et al., 1987b). Contrary to results with Schizosacrharo~~~ces ponibe (De Virgilio et al., 1990), heat-induced activation of TPS in S. cerevisiue depends at least partially on protein synthesis, sincc it is inhibited by cycloheximide (Hottiger, T.. Ohnacker. M. and Bell, W., unpublished results) or in a mutant tempera- ture sensitive for protein synthesis (De Virgilio et al., 1991). Western blots of total ycast protein prepared after a shift to 40 C demonstrated that TPS1 was also induced by heat shock (Fig. 2). Thus: de-izovo synthesis of TPSl appears to be im- portant for the induction of TPS activity. In addition. post- translational mechanisms may play a role in TPS regulation (De Virgilio et al., 1991; Winkler et al., 1991). Howevcr, regu- lation by CAMP-dcpendcnt phoshorylation and dephosphory- lation, as proposed by Panek et al. (1987) does not appear to play a role in TPS regulation (Vandercammen et al., 1989).

Surprisingly, the sequence of TI'S1 turned out to be nearly identical to the CIFI sequence (Gonziles ct al.. 1992). We found only seven differences in thc deduced amino acid se- quence. Thesc differences may be due to sequencing errors, differences between yeast strains, or artifacts introduced dur- ing cloning which included, in our case, amplification by the polymerase chain reaction. At any rate, since TPSI occurs

958

lack of ATP has been considered as the primary reason for the growth stop of fdpl and cifl strains on fermentable sugars (Banuelos and Fraenkel, 1982; Alonso et al., 1984).

With the gel-filtration experiments shown in Fig. 7, we demonstrate that TPSl is present predominantly in the region of the TPS complex. In contrast, the shortened TPSl gene product present in the tpsl mutant strain shows no preferential association resulting in a wide distribution over many frac- tions of the column eluate. The enzyme activity of TPS in the tpsl strain was below the limit of detection aftcr the separation by gel filtration.

The fact that in thc wild-type yeast (Fig. 7B) a small part of TPSl was found to be not associated with the TPS-complex, raises a question regarding the function of the free protein. One could speculate that TPSl is able to interact with different enzymes of the carbohydrate metabolism and that the free form of TPSl is the part which is needed for purposes other than TPS regulation, e.g. growth on glucose.

We wish to thank D. H. Wolf (University of Freiburg, FRG) and D. G. Fracnkel (Harvard Medical School, Boston, USA) for kindly providing yeast strains. We thank N. Biirckcrt (University of Basel, Switrerland) for sequencing and J.-M. Neuhaus (University of Bascl, Switzcrland) for support in the analysis of sequence data and helpful discussions. This work was supported by the Swiss National Science Foundation.

REFERENCES Achstetter, T., Emter, O., Ehmann, C. &Wolf, D. H. (1984) Proteol-

ysis in eucaryotic cells. J . Bid. Chem. 259, 13334- 13343. Alonso, A,, Pascual, C., Herrera, L., Gancedo, J. M. & Gancedo, C .

(1984) Melabolic imbalance in a Saccharamyces cereviiiae mutant unable to grow on fermentable hexoses, Eur. J. Biochem. 138,

Banuelos, M. & Fraenkel, D. G. (1982) Saccharomyces carlsbergensis fdp mutant and futile cycling of fructose-6-phosphate, Mol. Cell. Biol. 2, 921 -929.

Bcnnctzcn, J. L. & Hall, B. D. (1982) Codon selection in yeast, J . Bid. Cheni. 257, 3026- 3031.

Beullens, M. & Thevelein, J. M. (1990) Tnvestigation of transport- associated phosphorylation of sugar in yeast mutants (snf3) lack- ing high affinity glucose transport and in a mutant (fdpl) showing deficient regulation or initial sugar metabolism, Curr. Microbiol.

Bradford, M. M. (1976) A rapid and sensitive method for the quantita- tion of microgram quantities of protein using the principle of protein-dye binding, Anal. Bioehem. 72, 248 -254.

Cabib, E. & Leloir, F. L. (1958) The biosynthesis of trehalose-6- phosphate, J . B i d . Chem. 231,259-2275.

Charlab, R., Oliveira, D. E. & Panek, A. D. (1985) Investigation of the relationship between sstl and fdp mutations in yeast and their effect on trehalose synthesis, Bum. J . Med. Biol. Res. 18, 447.

De Virgilio, C., Simmen, U., Hottiger, T., Boller, T. & Wiemken, A. (1990) Hcat shock induces enzymes of trehalose metabolism, trehalose accumulation, and thermotolerance in Schizosaccharo- myces pombe, even in the presence of cycloheximide, FEBS I,ett.

Dc Virgilio, C., Piper, P., Boller, T. & Wiemken, A. (1991) Acquisition of thermotolerance in Saccharomyces cerevisiae without heat shock protein hsp 104 and in the absence of protein synthesis, FEBS Lett. 288, 86 - 90.

Devereux, J., Haeberli, P. & Smithies, D. (1984) A comparative set of scquencc analysis programs, Nucleic Acids. Res. 12, 387- 395.

Elander, M. (1968) Trehalose-6-Phosphat-Synthase aus Backerhefe, Arkiv fo r Kemi31. 17-30.

Franqois, J. Neves, M.-J. & Hers, H.-G. (1991) The control of trehal- ose biosynthesis in Saccharomyces cerevisiae: Evidence for catabolite inactivation and repression of trchalose-6-phosphate phosphatase, Yeast 7, 575 - 587.

407 - 41 1.

21,39-46.

273,107-110.

Giaever, H. M. & Stroni, A. R. (1988) Biochemical and geneticcharac- terization of osmoregulatory trehalose biosynthesis in Escherirhia coli, J . Bacteriol. 170, 2841 -2849.

GonzLles, M. I., Stuck, R., Blazquez, M. A,, Feldmann, H. & Gancedo, C. (1992) Molecular cloning of CIF1, a yeast gene necessary for growth on glucose. Yeast 8, 183- 192.

Harlow, E. & Lane. D. (1988) Antibodies: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor.

Hottiger, T., Roller, T. & Wiemken. A. (1987a) Rapid changes of heat and desiccation tolerance correlated with changes of trehalose content in Saccharomyces cerevisiae cells subjected to ternperaturc shifts, FEBSLeft . 220, 113-115.

Hottiger, T., Schmutz, P. & Wiemken, A. (1987b) Heat-induced ac- cumulation and futile cycling of trehalose in Sacchuromyces cerevisiae, J . Bacteriol. 169, 551 8 - 5522.

Hottiger, T., Boller, T. & Wiemken, A. (1989) Correlation between trehalose content and heat rcsistance in yeast mutants altered in the RAS/adenylate cyclase pathway - is trehalose a thermopro- lectant? FEBS Lett. 255,431 -434.

Kyte, J. & Doolittle, R . F. (1 982) A simple mcthod for displaying the hydropathic character of a protein, J . Mol. Biol. 157, 105- 132.

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

Lillie, S. JT. & Pringle, J. R. (1980) Kescrve carbohydrate metabolism in Saccharomyces cerevisiae: responses to nutrient limitation, J. Bacteriol. 143, 1384- 1394.

Londesborough, J. & Vuorio, 0. (1991) Trehalose-6-phosphate synthaseiphosphatase complcx from baker's yeast: Purification or a proteolytically activated form, J. Gen. Microbiol. 137, 323 - 330.

Navon, G., Shulman, R. G., Yamane, T., Eccleshall, T. K., Lam, K.- B., Baronowsky, J. J. & Marmur, J . (1979) Phosphorus-31 nuclear magnetic resonance studies of wild type and glycolytic pathway mutants of Saccharomyces cerevisiae, Biochemistr-v 18, 4487 - 4498.

Nelson, H., Mandiyan, S. & Nelson, N. (1989) A conserved gene encoding the 57-kDa subunit of the yeast vacuolar H+ATPase, J. Biol. Chem. 264, 1775.

Operti, M. S., Oliveira, D. E., Freitas-Valle, A. B.. Ocstreichcr, E. G., Mattoon, J. R. & l'anek, A. D. (1982) Relationships betwccn trehalose metabolism and maltose utilization in Sacchnromyces cerevisiae, Curr. Genet. 5, 69 - 76.

Panek, A. C., de Araujo, P. S., Moura-Neto, V. & Panek, A. D. (1987) Regulation of the trehalose-6-phosphate synthase coniplex in Sac- charomyces, Curr. Genet. 11,459-465.

Piper, P. W. & Lockheart, A. (1988) A temperature sensitive mutant of Saccharomyces cerevisiae defective in the spccific phosphatase of trehalose biosynthesis, FEMS Microbiol. Lett. 49, 245 - 250.

Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular cloning: a laboratory manual, 2nd edn, Cold Spring Harbor Laboratory Prcss, Cold Spring Harbor.

Sanger, F., Nicklcn, S. & Coulson, A. R. (1977) DNA sequencing with chain termination inhibitors, Proc. Natl Acad. Sci. USA 74,

Sharp, P. M. & Cowe, E. (1991) Synonymous codon usage in Sac- charomyces cerevisiae, Yeast 7, 657 -678.

Sherman, F., Fink, G. R. & Hicks, J. B. (1986) Methods in yeast genetics, Cold Spring Harbor Laboratory Prcss. Cold Spring Harbor.

Southern, E. M. (1975) Detection of spccific sequences among DNA fragments separated by gel elcctrophorcsis, J . Mol. Biol. 98, 503 - 517.

Thevelein, J. M. (1984) Regulation of trehalosc mobilization in fungi, Mierobiol. Rev. 48,42 - 59.

van de Poll, K. W. Kerkcnaar, A. & Schamhart, D. H. J. (1974) Isolation of a regulatory mutant of fructose-l,6-diphosphatase. J. Bacteriol. 117, 965-970.

van de Poll, K. W. & Schamhart, D. H. J. (1977) Characterization of a regulatory mutant of fructose-1 ,6-bisphosphatase in Saccliaro- mycrs carlsberensis, Mol. Gen. Genet. 154, 61 -66.

Van Laere, A. (1989) Trehalose, reserve and/or stress metabolite'? FEMS Mierobiol. Rev. 63, 201 -210.

Vandercammen, A,, Franqois, J. & Hers, H. G. (1989) Characteriza- tion of trehalose-6-phosphate synlhase and trehalose-6-phosphate

5463 - 5467.

959

phosphatase in Saccharomyces cerevisiae, Eur. J . Biochem. 182,

Vicente-Soler, J., Argucllcs, J. C. & Gacto, M. (1989) Presence of two trehalosc-6-phosphate syiithase enzymes in Candida utilis, FEMS Microbiol. Lett. 61, 273-278.

Wiemkcn, A. (1990) Trehalose in yeast, stress protectant rather than reserve carbohydrate, Ant. Leeuwenhoek Int. J. Gen. Microbiol.

613-620.

58.209-217.

Winklcr. K., Kicnle, I., Burgert, M., Wagner, J. C. &Holzer, H. (1991) Metabolic regulation of the trehalose content of vegetative yeast,

Young, R. A. & Davis, R. W. (1983) Efficient isolation of genes by using antibody probes, Proc. Natl Acad. Sci. USA 80,1194- 11 98.

Zell, R . &Fritz, H.-J. (1987) DNA mismatch-repair in Escherichia coli counteracting the hydrolytic determination of 5-methyl-cytosine residues, EMBO J . 6. 1809-1815.

FEBS Lett. 251, 269 - 272.