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THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 268, No. 7, Issue of March 5, pp. 4766-4774, 1993 Printed in U. 5’. A .

Molecular Analysis of the Neutral Trehalase Gene from Saccharomyces cerevisiae”

(Received for publication, September 10, 1992)

Meinrad Kopp, Hanne Muller, and Helmut Holzerz From the Biochemisches Institut, Uniuersitat Freiburg, Hermann-Herder-Strasse 7, 0-7800 Freiburg, Germany

Neutral trehalase (EC 3.2.1.28) is a trehalose hydrolyzing enzyme of the yeast Saccharomyces cerevisiae (App, H., and Holzer, H. (1989) J. Biol. Chem. 264, 17583-17588). The gene of neutral trehalase was cloned by complementation of a neutral trehalase-deficient yeast mutant which was obtained by ethylmethanesulfonate mutagenesis. Three mutants without detectable neutral trehalase activity were obtained and characterized by tetrad analysis and found to belong to the same complementation group. The mutants were transformed with a S. cerevisiae genomic library in YEp24. Two overlapping plasmids were isolated, containing the neutral trehalase gene NTHl with an open reading frame of 2079 base pairs (bp), encoding a protein of 693 amino acids, corresponding to a molecular mass of 79,569 Da. Several putative TATA boxes were found in the 5”nontranslated region of the NTHl gene. In positions -652 to -641 a possible binding sequence for the MIGl protein, a multicopy inhibitor of the GAL1 promotor, which also binds to the promotor sequences of the SUC2 and the FBP1 gene, was found. The start codon of the neutral trehalase is located about 2500 bp upstream of the centromere 4 consensus sequence elements I, 11, and I11 (Mann, C., and Davis, R. W. (1986) Mol. Cell. Biol. 6, 241-245). Vicinity to a centromere is known to have a depressing influence on the number of plasmid copies per cell. This probably explains why transformation with pNTH does not lead to overexpression of neutral trehalase. The four consensus sequences AATAAA contained in the centromeric elements and reconfirmed by our sequencing data might be polyadenylation signals for NTHl -mRNA transcription termination. Northern blot analysis yielded a single mRNA species of approximately 2.3 kilobase(s). The neutral trehalase protein has a putative CAMP-dependent phosphorylation consensus sequence RRGS from amino acid positions 22- 25. Therefore, the previously described activation of neutral trehalase by CAMP-dependent phosphorylation is probably due to phosphorylation of serine 25. Three potential N-glycosylation sites (Asn-X-Ser/Thr) occur in the open reading frame of the neutral trehalase gene. However, no evidence for glycosylation could be detected by Western blotting. The amino acid sequence of neutral trehalase from S. cerevisiae points to signif-

* This work was supported by the Deutsche Forschungsgemein- schaft, Sonderforschungsbereich 206, Bonn, and the Fonds der Chem- ischen Industrie, Frankfurt/Main. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in thispaper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) X65925.

$ To whom correspondence should be addressed Tel.: 49-761-203- 3220; Fax: 49-761-203-3331.

icant similarity in three domains with the osmoregu- lated treA gene encoding the periplasmic trehalase from E. coli K12 (Gutierrez, C., Ardourel, M., Bremer, E., Middendorf, A., Boos, W., and Ehmann, U. (1989) Mol. Gen. Genet. 217, 347-354) and in four domains to the trehalase gene of rabbit small intestine (Ruf, J., Wacker, H., James, P., Maffia, M., Seiler, P., Galand, G., Kiekebusch, A. v., Semenza, G., and Mantei, N. (1990) J. Biol. Chem. 265, 15034-15039). These domains might be part of the catalytic center of these trehalases. In order to study the biological function of the neutral trehalase, an isogenic disruption mutant, with the URA3 gene inserted, was constructed. It could be shown, that the steady state concentration of trehalose in the exponentially growing disruption mutant is three times higher than in the wild-type and that trehalose, accumulated at heat stress in the wild-type, must be degraded at return to normal growth temperature by neutral trehalase, not by acid trehalase.

The disaccharide a-glucosido-1,l-glucose, now known as trehalose, was discovered in 1832 by Wiggers (1). An enzyme hydrolyzing trehalose was first found in Aspergillus niger by Burquelot (2) and then in Saccharomyces cerevisiae by Fischer (3). Since then, trehalase has been detected in many other organisms of the plant and animal kingdom (4). The yeast enzyme was reported by van Solingen and van der Plaat ( 5 ) to be activated by cyclic AMP-dependent phosphorylation. In 1982 Wiemken and coworkers (6,7) demonstrated that the phosphorylatable trehalase was localized in the cytosol, whereas a second permanently active, trehalase was found in the vacuoles. Londesborough and Varimo (8) separated these two activities and found different pH optima for the two enzymes. The permanently active vacuolar trehalase, exhibits its maximal activity at pH 4.5 and is therefore designated as “acid trehalase.” It was purified and characterized by Mitten- buhler and Holzer (9). The cytosolic neutral trehalase which after phosphorylation exhibits its maximal activity at pH 7.0 has been studied by several groups (8, 10, 11) and purified to homogeneity by App and Holzer (12). Trehalose functions in yeast as an energy source in spore germination and as pro- tecting agent for maintaining structural integrity under en- vironmental stress such as heat and desiccation (13-15). According to what we know at present the concentration of trehalose in yeast is the result of the activities of the synthe- sizing bifunctional enzyme trehalose-6-phosphate-synthase/ trehalose-6-phosphate phosphatase (16) on the one hand and the trehalose hydrolyzing enzymes, e.g. cytosolic neutral trehalase (12) and vacuolar acid trehalase (9). As far as the regulation of trehalose synthase is concerned, phosphorylation/dephosphorylation ( 1 6 ~ 7 ) a n d also catabolite inactiva- tion and repression (18) have been discussed. The regulation

4766

Neutral Trehalase Gene from S. cerevisiae 4767

TABLE I Strains used

Strain Genotype Source S. cereuisiae strains

YHH65 Mata his3-11,15 leu2-3,112 urdA5 canR pral AEN1::HISJ D. H. Wolf YS18 Mata his3-11,15 leu2-3,112 ura3A5 canR gal- D. H. Wolf YMN3 Mata his3-11,15 leu2-3,112 ura3A5 canR gal- nthl::URA3 This laboratory YMN4 Mata his3-11,15 leu2-3,112 urdA5 canRpralAENl::HIS3 nth1::URAJ This laboratory DF5a Mata lys2-802 leu2-3, 2-112 urd-52 his2A200 trpl-1 (am) S. Jentsch YMN5 Mata lys2-802 leu2-3, 2-112 urd-52 his2A200 trpl-1 (am) nth1::URAJ This laboratory

DH5a F- endAl recAl hsdRl7(rk- mk+) deoR supE44 thi-1X- gyrA96 D. Klionsky E. coli strain

relAlA(lucZYA-argF)U196 @801acZhM15

of the synthase activity is affected by the substrate concentrations, i.e. glucose 6-phosphate and UDP-glucose. This has been demonstrated in vitro (16) and in uiuo (19). On the other hand, cyclic AMP-dependent phosphorylation/dephosphorylation has been shown by van Solingen and van der Plaat to control neutral trehalase activity (5). Very little is known about the biological function and possible control mechanisms for vacuolar acid trehalase, whereas neutral trehalase is con- sidered to be the key enzyme responsible for trehalose degradation in intact yeast cells (13). It seemed therefore worth- while to get more information on the biological function and the regulation of this important enzyme in yeast. For that purpose we have constructed mutants deficient in neutral trehalase activity and have used complementation of one of these mutants for cloning and sequencing of the neutral trehalase gene.

MATERIALS AND METHODS

Reagent~-[a-~~P]dCTP (110 TBq/mmol) and [a-35S]dATP (>37 MBq/mmol) were from Amersham Buchler, Braunschweig, Germany. Restriction endonucleases and other modifying enzymes were pur- chased from Boehringer Mannheim, Amersham Buchler, Braun- schweig, Germany, and United States Biochemicals Corp. Random priming materials and additional enzymes were from United States Biochemical Corp. Biochemical reagents were from Sigma, Deisen- hofen, Germany.

Strains, Plasmids, and Growth Media-Escherichia coli DH5a was used for amplification of all plasmids. The yeast strains that were used for EMS' mutagenesis, screening of the genomic plasmid library, and gene disruption were YHH65, deficient in proteinase A (20) and YS18 (21), a gift from D. H. Wolf (University of Stuttgart) (Table I). Wild-type yeast strains were grown in YEPD medium containing 2% glucose, 2% bactopeptone, and 1% yeast extract. Selection media consisted of 0.67% of YNB, 2% glucose, and the appropriate supple- ments. Bacterial strains were grown on TYE medium (0.5% yeast extract, 0.5% NaC1, 1% Bactotryptone) and TYE-ampicillin medium (TYE containing 100 pg/ml ampicillin), respectively.

EMS Mutagenesis-4 ml of a YHH65 yeast cell culture was grown to stationary phase, centrifuged at 3000 X g for 5 min and washed twice in 0.1 M potassium phosphate, pH 8.0. After resuspending in 5 rnl of 0.1 M potassium phosphate solution, EMS treatment was carried out as described (22). After incubation, cells were collected by centrifugation and washed twice with sterile water, and cell numbers were counted in a Neubauer chamber before spreading the cells out on a YEPD-plate. Based on a survival rate of about 20% a 1:104 dilution was calculated to yield approximately 1,500 cells/ml. With a sterile Pasteur pipette 100 p1 were spread out onto a YEPD plate and incubated for 2 days at 30 "C. The colonies were attached to sterilized Whatman filters and placed onto minimal medium, containing fructose as the carbon source. After an additional incubation for 2 days cells were assayed by the enzymatic overlay test.

Enzymatic Overlay Test-Because neutral trehalase, in contrast to acid trehalase, is not secreted into the periplasm when the enzyme is overproduced, the yeast cells had to be lysed for 5 min in chloroform

The abbreviations used are: FBPase, fructose-1,6-bisphosphatase; EMS, ethylmethanesulfonate; kb, kilobase pair(s); bp, base pair(s); kbp, kilobase pair(s).

(23-25). After lysis, Whatman filter papers were separated from chloroform and air-dried. Because neutral trehalase is only active when phosphorylated, the lysed colonies were incubated for 30 min at 30 "C with 3.6 mM ATP, 0.18 mM CAMP, and 3.6 mM MgC12 in 50 mM imidazole-HC1 (8). One ml of the mixture was spread on each Whatman filter and incubated for 20 min at 30 "C. The remaining supernatant was removed with a Pasteur pipette and the overlay- assay-mix was immediately poured onto the sheets. To prepare 100 ml of final voiume of the overlay-assay-mix 18.9 g of trehalose was dissolved in 80.0 ml of 50 mM imidazole-HCl, pH 7.0, 1 g of agarose was added, and the mixture was melted in a microwave oven and then cooled down to 50 "C. Immediately before pouring the mixture onto the filter-attached colonies, 2 ml of N-ethylmaleimide (2.5 mg/ml), 985 units of horse radish peroxidase (EC 1.11.1.7), 800 units of glucose oxidase (EC 1.1.3.4), and 4.8 ml of o-dianisidin (10 mg/ml) were added. The overlay-assay-mixture was incubated for 30 min at 30 "C. Colonies with trehalase activity at pH 7 developed a brown color, whereas mutants devoid of neutral trehalase activity remained white.

Tetrad Analysis of the Yeast Mutants-The haploid mutant strains were mated with the haploid wild type strain YS18. After induction of sporulation the tetrads were separated with a micromanipulator according to the technique described in Ref. 26. With the intention of further characterizing the mutants, the spores from mating experiments, which had a neutral trehalase-negative phenotype, were crossed to determine the complementation groups. The results demonstrated that the mutants YMN2/1 and YMN2/2 belong to the same complementation group (27). In cases werepralAENl::HIS3 X pralAENl::HIS3 genotypes of the parental strains were mated, the diploids did not sporulate properly, as described previously for other pral X pral crossings (28).

Western Blots with the Spores Lacking Neutral Trehnlase Actiuity- Crude extracts were prepared from stationary yeast cells, and protein concentration was measured according to the method of Lowry (29). About 100 pg of protein per lane were loaded onto SDS-polyacrylamide gel electrophoresis and run with 100 V constantly at room temperature. After the gel run was complete, proteins were blotted onto a nitrocellulose membrane (Amersham Buchler, Braunschweig, Germany), and neutral trehalase was detected using polyclonal anti- neutral trehalase antiserum as described (12, 30).

Amplification of Plasmid Library-A YEp24 library was kindly provided by D. Botstein (Stanford). One liter of TYE medium (0.5% NaCI, 1% Bacto-trypton, 0.5% yeast extract), containing 100 pg/ml ampicillin was inoculated with E. coli cells harboring the genomic YEp24 library to an OD578 of 0.15. Cells were grown for two doubling times until they reached an OD578 of 0.6, and then chloramphenicol was added to a final concentration of 170 pg/ml. Plasmids were prepared according to Ref. 31 by scaling up to the required volume.

Preparation of Competent Yeast Cells, Transformation, and Com- plementation Screening-Preparation of competent yeast cells by the lithium-acetate method and transformation was carried out as de- cribed by Ito et al. (32). After cell lysis the same overlay assay as described above was performed, and the lysed cells were screened for reconstitution of neutral trehalase activity. Eight colonies were obtained, exhibiting neutral trehalase activity. Two plasmids with overlapping inserts at 6 and 10 kb, respectively, were isolated. An aliquot of the isolated DNA was transferred to competent E. coli DH5a, and cells were selected for ampicillin resistance in TYE medium containing 100 rg/ml ampicillin. Plasmid preparation from E. coli was done according to standard procedures (33).

RNA Isolation and Northern Analysis-YS18 cells were grown in YEPD medium to stationary phase. Cells were washed twice with

4768 Neutral Trehalase Gene from S. cerevisiae

destilled water, and total RNA was isolated by subsequent ultracen- trifugation at 250,000 X g for 24 h according to (34). For Northern blot analysis about 10 pg of total RNA were separated on a 0.7% formaldehyde-agarose gel and transferred onto a Hybond N+ nylon membrane (Amersham). The hybridization procedure was according to Ref. 35. As a radioactively labeled probe the 300-bp EcoRI/XhoI fragment from the neutral trehalase gene was used.

Preparation of Genomic Yeast DNA and Southern Analysis-YS18 wild type and YMNB (YS18 nthl::URA3) yeast cells were grown in YEPD until glucose could no longer be detected. The cells were converted to spheroblasts as described (36), lysed by SDS and potassium acetate, and the DNA was precipitated finally with isopropanol after RNase digestion. About 20 pg of YS18 wild type and YMN3 (YS18 nth1::URAJ) genomic DNA was digested separately in a total volume of 400 r l with 50 units of EcoRI for 12 h. DNA was precipitated with ethanol and separated on a 1% agarose gel and blotted onto a Hybond N+ membrane in 0.4 M NaOH after subsequent incubation in 0.25 M HCl. Random priming of about 0.1 pg of EcoRI/XhoI fragment from the neutral trehalase was performed with a random priming kit according to the manufacturer's instructions (United States Biochemical Corp.).

Heat Stress and Assay of Trehalose with Acid Trehalase-Yeast cells of the wild-type strain YHH65 and mutant strains YMN2/1 and YMNB were grown to exponential phase at 30 "C and shifted subsequently to 39 "C for 40 min. After the heat stress, the culture was shaken for additional 40 min at 30 "C. An aliquot was removed after each incubation period, and the cells were collected by centrifugation. Cells were washed free from glucose and resuspended in imidazole- HCI, pH 7.0 to yield a final suspension (w/v) of 50% and were heated for 20 min at 95 "C. After centrifugation, trehalose concentrations were measured in the supernatant by use of acid trehalase as described before (19). Neutral trehalase activity was measured in crude extracts according to Ref. 19.

Restriction Site Mapping-Cleavage sites for BstEII, EcoRI, EcoRV, HindIII, HpaI, KpnI, StuI, and XhoI were localized on the insert derived from the YEp24 library, which was able to reconstitute the enzyme defect of the mutants. A 6.0-kb SalI fragment containing 276 bp of the YEp24 shuttle vector was excised from the insert. It represented nearly the entire insert of the clone except for 885 bp at the 5' side and was able to reconstitute enzyme activity in the mutant. An EcoRI digest yielded a 1.5-kb fragment which proved to be an insert sequence. This DNA fragment was used as probe for Southern blots of YS18 wild-type strain and the YS18 nthl::URA3 disruption mutant. An additional 3.5-kb EcoRI fragment contained about 2.0 kb of vector sequence (URA3 gene and part of the tetracycline-resistant gene) and 1.5 kb of insert sequence. A third fragment was isolated and subcloned by an EcoRIISalI digest, which revealed a 3.5-kb fragment containing 276 bp of the YEp24 tetracycline-resistant gene. A 300-bp EcoRI/XhoI fragment which is located within the 3.5-kb EcoRIISalI was used for Northern analysis with total RNA. A XhoI digest released a 1.4-kb fragment from the complete insert. This fragment is located within the 3.5-kb EcoRIISalI fragment. After religation of the remaining fragment and transformation into the mutant, the residual construct was no more able to express enzyme activity.

Strategy of Gene Disruption-The 1.5-kb EcoRI fragment, subcloned in pTZlSR, was used to disrupt the neutral trehalase gene. The EcoRI fragment was excised from the pTZ18R 1.5-kb EcoRI construct and cloned in pTZ18R without a HindIII restriction site. The 1.1-kb HindIII URA3 fragment was then ligated into the remaining HindIII site within the 1.5-kb EcoRI fragment. The disrupted gene fragment was subsequently isolated after EcoRI cleavage and used for transforming, with approximately 30 pg of DNA (32), the yeast strains YS18 and YHH65, yielding the mutants YMN3 (nth1::URAS) and YMN4 (nthl::URA3 pralAENl::HIS3), respectively.

RESULTS

Isolation and Characterization of Neutral Trehalase Mu- tants-Before treatment of the yeast cells with the chemical mutagen EMS, no other trehalase activity than that from neutral trehalase should be detectable after cell lysis at pH 7. Because acid trehalase exhibits some minor activity at pH 7, the yeast strain YHH65, defect in the gene for proteinase A

and consequently ath- was used? as PRAl is necessary for activation of acid trehalase in yeast vacuoles. No acid trehalase activity at pH 4.5 was detected in crude extracts prepared from this strain. EMS mutagenesis yielded three mutants deficient in neutral trehalase activity named YMN2/1, YMN2/2, and YMN138, and detected in the enzymatic overlay assay, developed for neutral trehalase (see "Materials and Methods"). Tetrad analysis showed that all three mutants carried single recessive mutations for neutral trehalase. In mating experiments with the wild-type strain YS18 a 2:2 segregation was observed, indicating a single gene mutation (37). The diploid from the mating of YMN138 x YS18 however did not sporulate. As control, ATH activity-segregation was examined. The tetrads of the resulting two mutants showed every conceivable segregation phenotype for ATH/ ath and NTH/nth. Mating experiments with the spores (nth-/ ATH+ and nth-/ath-) were performed to confirm the complementation group of the mutants. After mating type determi- nation of the spores, a and a cells were crossed. The spores were tested after dissection for neutral trehalase activity. Crude extracts were prepared from 12 tetrads, resulting in a 4:O ratio in all cases. Thus, the two mutants do belong to the same complementation group. These mutants were screened for the neutral trehalase structural gene. To exclude that a false neutral trehalase negative phenotype appeared as a consequence of an inhibitory effect, crude extracts from wild- type YHH65 and the mutant cells were mixed (50% each) and neutral trehalase activity was measured. About half of the wild-type activity was detectable, indicating that the nth- phenotype could not be due to an inhibitory effect.

Isolation of the Structural Gene for Neutral Trehalase (NTH1)"The mutants were cultivated in YEPD medium to an OD57s = 0.7 and transformed with a genomic yeast DNA library in YEp24. After growth on MV-fructose medium and subsequent cell lysis, the same overlay assay was performed as used in the screening procedure. Eight colonies out of about 30,000 exhibited a reconstituted neutral trehalase activity. The plasmids were isolated from the corresponding yeast cells and propagated in E. coli DH5a. Two different, but overlapping plasmids were isolated from the yeast clones with inserts of 6 kb (designated p44bII) and 10 kb (designated p5bI), respectively. Each plasmid reconstituted the nth- defect in both the EMS-treated mutants and the tetrads. Responsible for the reconstitution was a 6-kb fragment in the two plasmids detected by means of a SalI restriction digest. After subcloning of this 6-kb SalI fragment in pSEY8, which was a gift from D. Klionsky (Davis, CA), yeast transformants had a reconstituted neutral trehalase activity as shown in Table 11. Retrans- formation into the nth- spores confirmed their ability to complement the mutation.

Restriction digests with EcoRI, HindIII, BarnHI, XhoI disclosed identical fragments in p5bI and p44bII now designated pNTH (Fig. 1).

Deletion of a 1.4-kb XhoI fragment in the 6-kb SalI fragment cloned in pSEY8 lead to a plasmid that was no longer able to express an enzymatically active neutral trehalase protein. The same result was obtained when the XhoI fragment was deleted either in p44bII or p5bI (data not shown).

Northern analysis was carried out with total RNA, isolated according to Ref. 34. As a radioactively labeled probe, a 300- bp EcoRI/XhoI DNA fragment (cf. Fig. 1) was used. One single band of approximately 2.3 kb was observed (Fig. 2). Considering the 2079 bp of the open reading frame and the extension for possible polyadenylation, this value corresponds to the expected size of the mRNA for the neutral trehalase

D. Klionsky, personal communication.

Neutral Trehalaqe Gel

TABLE 11 Activity of neutral trfhalase in wild-type. mutants, and

complemented mutants Crude extracts were prepared from yeast cells with glass heads and

assayed for NTH as descrihed under “Materials and Methods.” First column ( I ) , activity without preincuhation with ATP/cAMP; second column (11). assav after areincuhation with ATP/cAMP.

Strains I 11, ATP/cAMP rnilliunits/rnE

YHH65 wild type 11 29 YS18 wild type 14 34 YMN2/1 eo. 1 YMN2/2

<o. 1 ND“ eo. 1

YMNZ/l pNTH 10 YMN2/2 pNTH

31 13 26

YMN3 (nthl::URA3) ND ND YMN4 (nthl::URA3) ND ND

ND, not determined.

EcoRl

I h l l S l l l

FIG. 1. Restriction map of the plasmid pNTH. pNTH was screened hy transformation of chemically prepared EMS mutants with a high copy lihrary in YEp24. The hox within the insert represents the structural gene for N T H I , encoded hy 2079 hase pairs. Restriction sites were as indicated on the map. The CEN4 sequence (40) extends from the first Xhol restriction site to the second one in the insert part of the plasmid.

3.4 kt> -+ 1.7 kt>-+ (I)

FIG. 2. Northern blot with total RNA. Total RNA from strain YS18 was extracted from cells growing overnight in YEPD medium. 20 pg were separated on a 1 1 agarose/MOPS/formaldehyde gel in the presence of ethidium hromide. The RNA was transferred to a nylon memhrane and hyhridized to the 300-hp EcoRI/XhoI fragment of the NTHI gene.

ae from S. cerevisiae 4769

36,500 lkl

26.600 Ikl

1 2 . 1 4 5 . 0 7 8 ‘ )

FIG. 3. Western blot analysis. Lane 1, prestained SDS molecular weight marker standard mixture; lanp 2, fructose-6-phosphate kinase from rahhit muscle (molecular mass 84,000 daltons); lono 3 ,

crude extract from the wild-type strain S H H G ; lonrs 4-6. Cnlde extracts from the mutants, prepared hy EMS mutaEenesis (SMX2/ 2, YMN2/1, and YMNlXR); lancs 7-9, c n ~ l r extracts from the mutants complemented with pNTH (YMN2/:! pS’I’H. Y M S 2 / 1 pNTH. YMN138 pNTH). Crude extracts were prepared as descrihed under “Materials and Methods” and separated on lor; SDS-polyacrylamide gel electrophoresis. Each lane contained ahout 100 p~ of protein as determined according to Lowry et 01. (29). The proteins were transferred to a nitrocellulose memhrane. and neutral trehalase protein was detected as descrihed under “Materials and Methods.’‘ The 86- kDa hand represents neutral trehalas~. The other hands could result from contaminations of the neutral trehalase preparation used for immunization.

gene (Fig. 2). Western hlots (Fig. 3) with the mutants showed no antigenic material at 86,000 daltons neither for the spores nor the disruption mutant YMNB (not shown) with the ex- ception of the mutant spore 2/1 4C. This mutant disclosed antigenic material at 86,000 daltons after reaction with polv- clonal antiserum (12), although no enzymatic activity could he detected. Similar observations had heen made hefore ( 3 7 ) . After transformation with pNTH the mutants had a reconstituted activity and showed a hand a t 86 kDa which reacted with the polyclonal antiserum.

Nucleotide Sequence of the Neutral Trpholaw G n P with 5 ’ - and 3‘-Flanking Regions-EcoRI restriction fragments of p N T H were suhcloned into pTZ1RR and sequenced. An open reading frame was found throughout a 1.5-kh F h R I fragment heginning at hp position 10s. Each suhcloned fragment was sequenced on both strands. The sequencing strategy is shown in Fig. 4. DNA connection was computed with the programm DNASIS (Pharmacia, Freihurg, Germany) leading to an open reading frame with a total size of 2079 hp. To confirm the correct DNA connection, several sequence runs throughout the EcoRI restriction sites on the 6-kh Sal1 DNA fragment were done. Further fragments were sequenced in the upstream and downstream region of the open reading frame (see Fig. 5 ) . The 5”nontranslated region of the NTH1 gene contains three possible TATA boxes at nucleotides -300, -246, and -162. In the 3’-nontranslated region 4 putative polvadenvl- ation signals are encoded in the DNA sequence, at nucleotides 2480, 2484, 2S10, and 2S17. The translated nucleotide sequence resulted in a protein with a calculated molecular mass of 79,569 Da based on the predicted amino acid sequence. A CAMP-dependent phosphorylation consensus sequence, RRGS, at, amino acid position 22 with a phosphorylatahle serine in position 25 is in agreement with the biochemical data reported previously (S, 12). At amino acid sequenre

4770 Neutral Trehalase Gene from S. cereuisiae

I l l I I I

, c a 4 kb < FIG. 4. Sequencing strategy used for the NTHl gene. The

6.0-kb Sal1 fragment, which was shown to complement the mutagenic defect, was cloned into the polylinker region of the plasmid pTZ18R (Pharmacia, Freiburg). For sequencing reactions EcoRI and EcoRI/ XhoI digests were prepared for formation of smaller fragments. Each of the subclones was sequenced according to Ref. 56. The arrows

ing was done on both DNA strands with double stranded DNA. represent the region sequenced in each sequencing step. The sequenc-

positions 210, 264, and 290 three putative N-glycosylation sites were observed. No carbohydrate residues however could be removed by treatment with endoglycosidase F (data not shown) indicating, that the Asn residues are not glycosylated. The complete sequence of the NTHl gene with flanking regions is shown in Fig. 5 .

Search for homologous proteins in the SWISS-PROT protein sequence data base Release 21 revealed that the amino acid sequence of neutral trehalase from yeast shows homologies to the periplasmic trehalase from E. coli (38) and to the rabbit small intestinal trehalase (39) (Fig. 6, E and R, respectively, for details see ''Discussion"). Sequence comparison in the 3"nontranslated region with the EMBL nucleotide sequence data base Release 30 brought to light identity with a previously published nucleotide sequence by Mann and Davis (40). These authors found two large open reading frames near the centromere-CEN4 consensus sequences. One of these two open reading frames can now be assigned to be part of the neutral trehalase gene from yeast. Thus, we conclude, that the neutral trehalase gene is located in close proximity to the centromere of chromosome 4 (cf. legend to Fig. 1). The centromere linkage of the NTHl gene was reconfirmed by South- ern blot with SalIIPuuII-digested genomic DNA from YS18 and the corresponding disruption mutant YMN3. The genomic DNA was hybridized independently with a radioactively labeled probe either from the CEN4 sequence or the 1.5-kb EcoRI fragment from the NTHl gene. Signals in the same migration distance were obtained after exposure to x- ray film and subsequent detection (27).

Disruption of the NTHl Gene-NTH1 gene disruption in YS18 and YHH65, yielding YMN3 and YMN4, respectively, was performed with the intention of ascertaining that the structural gene of NTHl has indeed been cloned and further- more to introduce an exactly defined mutation in the yeast genome. This should be helpful in evaluating the physiological role of cytoplasmic neutral trehalase. The 6-kb Sal1 fragment was digested with EcoRI, and a 1.5-kbp fragment was used for the insertion of the URA3 gene in a Hind111 site. After transformation of YS18 and YHH65 with the URA3-disrupted NTHl gene fragment, uracil prototrophic yeast cells were tested with respect to neutral trehalase activity. Out of

12 colonies examined, none had a detectable neutral trehalase activity (Table 11).

Southern blot analysis (Fig. 7) showed the correct integra- tion of the disrupted nth1::URAS constructions in the yeast genome: the disrupted mutants (lanes 1 and 2) show a 1.1-kb bandshift as compared to the wild type (lane 3).

Function of the Neutral Trehalase in Conditions with Tem- perature Stress-dependent Changes of Trehalose Concentra- tion-Transfer of exponentially growing yeast cells at 30 "C to the heat stress temperature of 39 "C for 40 min causes a rapid and drastic increase of the trehalose concentration (14, 15, 19). The trehalose accumulation is completely reversible when the cells are brought back subsequently to 30 "C for further 40 min (14, 15, 19). The role of neutral trehalase in this temperature-dependent change of trehalose concentration was studied with the neutral trehalase-disrupted mutant YMN5 (nth1::URAS) and the corresponding isogenic wild- type DF5a (NTH). As can be seen in Table I11 the steady state concentration of trehalose during exponential growth is 3-fold higher in the mutant than in the wild type. After transfer from 30 to 39 "C (second line in Table 111) the concentration of trehalose is again 3-fold higher in the mutant as compared to the wild type. At subsequent shift down from 39 to 30 "C the decrease of the trehalose concentration is small in the mutant and almost total in the wild type. This is a consequence of the missing trehalose-degrading activity of neutral trehalase in the mutant. Taken as a whole, these experiments demonstrate an important role of neutral trehalase in trehalose degradation in yeast and no or only a minor function of acid trehalase. This latter conclusion is further supported by the observation that, in a pral mutant, which shows no acid trehalase activity, the rate of trehalose degradation at shift from 39 to 30 "C is not different from the wild type with active proteinase A and acid trehalase (data not shown).

DISCUSSION

The structural gene for neutral trehalase (NTHl ) from the yeast S. cereuisiae was cloned by complementation of a neutral trehalase-deficient yeast mutant obtained by EMS mutagenesis. Mutants, isogenic to the wild-type yeast strains YS18 and YHH65, were constructed by gene disruption and homologous recombination. These mutants, YMN3 and YMN4, respectively, have a single defect in the structural NTHl gene for neutral trehalase without other effects due to the EMS treatment. Nucleotide sequence analysis indicated a location of the structural gene of neutral trehalase near the centromeric elements CDEI, CDEII, and CDEIII on chromosome 4, found by Mann and Davis (40). This implies a localization of neutral trehalase on chromosome 4. The consensus boxes CDEI, CDEII, and CDEIII consist of extremely A-T-rich sequences, encoding four putative consensus sequences MA- TAAA) for polyadenylation in the 3"noncoding region of the neutral trehalase gene. Whether these hexanucleotide stretches function as polyadenylation signals for termination of specific mRNA transcription in addition to the centromere mitotic stabilization effects by the centromere sequences can- not be decided. However, only a small number of sequenced yeast genes contain the AATAAA polyadenylation consensus sequence, whereas a large number of genes, in spite of coding for a poly(A) tail do not have this sequence. For example, CYC7, TRPl, histone HzA2, Matal, M a t 4 HIS4 URA3 (411, and FBPl (42) do not have the AATAAA sequence. Zaret and Sherman (41) postulated a tripartite structure, on the basis of computer analysis of 3"nontranslated regions of these


FIG. 5, Nucleotide- and nucleotide derived amino acid sequence of NTHl. The MIGl binding sequence from -652 to -641 is doubled underlined. Sequences in bold letters in the 5'-noncoding regions indicate the TATA elements, whereas they indicate three putative N-glycosylation sites in the coding region. A four-amino acid spanning sequence in the N-terminal region indicates the CAMP-dependent phosphorylation site. The phosphorylatable serine residue in position 25, is marked with an asterisk. In the 3'-noncoding region several possible polyadenylation signals are underlined. An open reading frame for the NTH1 gene starts at nucleotide 1 and extends 2079 bp (693 amino acids) to a termination codon at position 2080. From position 2091 to position 2112 the underlined (AT),, stretch, indicates an alternative signal sequence for t.he mRNA 3'-end (57).

4772 Neutral Trehalase Gene from S. cerevisiae

FIG. 6. Alignments for maximal amino acid similarities of neutral trehalase from S. cerevisiae with periplasmic trehalase from E. coli and small intestinal trehalase from rabbit. Identical residues among all trehalase enzymes are marked with an asterisk (*). Conservative substitutions are marked with a point (.). Amino acid sequences were aligned to give the best fit. When sequence alignment suggests an addition or deletion, the gap is represented by a dash. Y, yeast s. cereuisiae; E, E. coli; R , rabbit.

1 1 1

43 56

52

117 79 90

176 122 114

236 161 145

296 219 203

346 264 255

403 317 308

462 371 359

422 521

4 10

577 472 457

633 524 506

684 57 7 554

Y R E

Y R E

Y R E

Y R E

Y R E

Y R E

Y R E

R Y

E

Y R E

R Y

E

Y R E

Y R E

Y R E

genes, as polyadenylation signal and established the following consensus sequence.

Termination codon. . .l-140. . .(T-rich). These sequences are also present in part in the NTHl and in the FBPl genes as follows.

~VFDNVSPFKKT-----GFGK~QTRRGSEDDTYSSSQE

MKSPAPSRPQKMALIPACIFLCFMLSVQAEETPVTPQPPD--IL-----" M--PGSTWELHLLLL-------- LGLGLGSEQALPPPCESQ--IYC--"" HGELLHQV

LGPLFNDV * . .*.. . . . . . *. . DTDKNYQITIEDTGPKVLKVGTANSYGYKHINIRGTYMLSNL~ELTIAKSFGRHQIFLD QPW(LYP------DDKQFVDMPLST----AP~VLQSF--AEL------MTYN-----" QNAKLFP------DQKTFADAVPNS---DPL"QQN------QSGFD------- . . . . . . * . . . . . . . . . . . . EARINENPVNRLSRLINTQFWNSLTRRVDLNNVGEIAKDTKIDTPGA-I(NPR1YVPYDCP "" NTVPREQLEKFVQEHF-------------QAVGQELESWTPGD~ESPQF~KISD -----------LRHFVNVNF-------------TLp------------ KEGEKYVPPEGQ

.... .* *. ... EQYEFYVQASQMHPSLKLEVEYLPKKITAEYVKSVNIYPPGLLALAnEEHFNPSTGEKTLI PKLRAW--AEQLHLLWKK----LGKKIKPEVLSQPERF-SLIYSQH-------------- S-LR-----EHIDGLWPV----LTRST-----ENTEKWDSLLPLPE-------------- .... * ... . . .*. . GYPYAVPGGRFNELYGWDSYMMALGLLEANKTDVARGMVEHFIFEINHYGKILNANRSYY --PFIVPGGRFVEFYYWDSYWVMEGLLLSEMAETVKGMLQNFLDLVTAYGHIPNGGRVYY --PYWPGGRFREVYYWDSYFTMLGLAESGHWDKVADMVANFAHEIDTYGHIPNGNRSYY *. ****** **** ** . . . . .*".* .. **.* *..* ** LCRSQPPFLTEMALWFKKLGGRSNPDAVDLLKRAFQASIKAS--- - - - - - - - ~RSQPPLLT~DRYVAHTGD------LAFLRENIETLALEL-D~AE-------- LSRSQPPFFA~ELLAQHEGD------M-LKQYLPQMQKEY-AYWMDGVENLQAGQQE

NRT

* ****** . . . *. . *. . * . * . ---PRLDPETGLSRYHPNGLIPPETESDHFDTVLLPYASKHGVTLDEFKQLYNDGKIKE

KAWKLQDGTLLNRYWDDRDTPRPESWVEDIATA-------KSNPNRPATEIYRDLRSM ISVSSGGNSHTLNRYHVPYGGPRPESYSKDTELA------- HTLPEGSWETLWAELKAGA

. . *.** . .**. . . . . . . . . . . . . . PKLDEFFLHDRGVRESGHDTTYRFEGV-CAYLATIDLNSLLYKYEIDIADFIKEFCDDKY ESGWDF----SSRWLVGSPNPDSLGSIRTSKLVPVDWAFLCQAEELLSGFYSR~NE-- A S G W D F - - - - S S R W " - - D N ~ Q L N T ~ T T S I V P V D W S ~ F ~ E K I ~ R A S ~ G D N - - . .* . . . . . . . . . . ....***... . . . . . . EDPLDHSITTSAMWKEMAKIRQEKITKYMWDDESGFFF-DYNTKIKHRTSYESATTFWAL

SQATKYRNLRAQRIAALTALLWDEDKGAWF-DYDLENQKKNHEFYPSNLTPL

. . . . . . .. .*... .. **. . . . .... .* WAG----LATKEQAQKI.NEKALPKLEMLGGLAACTERSRGPISISRPIRQWDYPFGWAPH WAGCFSDPAIADKALQYLQDSQI-LNHRHGI---------PTSLQNTGQQWDFPNAWAPL "" YVNAAAKDRANKMATATKTHLLQPGGL---------NTTSVKSGQQWDAPNGWAPL

-"""- " """ AMANQYETLANARQKGIEKYLWNDQQG-WYADYDLKSHKVRNQLTMALFPL

* . .* . . * *. . . . . .*** .*** QILAWEGLRSYGYL---TVTNRLAYRWLFMMTKAFVDYNGIWEKYDVTRGTDPHRVEAE QDLVIRGLAKSPSARTQEVAFQLAQNWIRTNFDVYSQ-RSAMYEKYDISNAQP-----" QWVATEGLQNYG---QKEVAMDISWHFLTNVQHTYDR-EKKLVEKYDVSTTGT-------- .. ** . .*. . . . . . . . . . ****.. . YGNQGADFKGAATEGFGWVNARYILGLKYMNSYERREIGA--------- -GGGG-EY--EVQEGFGWTNG---VA~LLDRYGDRLSSGTQLALLEPHCLAAALLLLSFL

CIPPISFFSSL

-GGGGGEY--PLQDGFGWTNG---VTLKMLDLICPKEQPCDNVPATR~VKSAT-"--- *. .. .****.*. . .*. . . . ..

-RPQERNLYGL------- TRPQGGLPAPLFIKPLTG TQPSTKEAQP------TP

sequence, whereas Mann and Davis found "AATA." In mul- tiple sequencing reactions on both DNA strands we could

.TAG. . . . . /TAT,,. . . (A-T-rich) . .TTT. . . . TAGT

FBPl +47 * TAGTACGCGAAAMAMMTCTGTATATGTCCTTATATATATATATATTT . .

NTHl +83..TAGATAGTTCTAAGTCATTGAGGTTCATCAACAATTGGATTT..

SEQUENCES 1-3

Further different classes of polyadenylation sites were postulated by Irninger et al. (43) and by Henikoff et al. (44, 45). But none of these sequences are found, neither in NTHl nor in FBPl. Mann and Davis (40) found two open reading frames within the nucleotide sequences around the three centromere boxes. One of them can now be ascribed to the NTHl gene from yeast. However, a minor difference in the DNA sequence exists in positions 1366-1369 of the NTHl gene (positions 2086-2089 in the CEN4 sequence). We found a "TGCG"

confirm the "TGCG" sequence. This difference may be due to different strains of S. cereuisiae or to sequencing artefacts.

Because of the localization of the NTHl gene close to the centromere, this gene should be a useful tool to determine the distance of unknown genes relative to their centromere by crossing a yeast strain of interest with the yeast strain YMN3, carrying centromere linked markers, and subsequent tetrad analysis (37).

Transformation of both the EMS and the disruption mutants with pNTH (cf. Fig. 1) did not lead to overexpression


2.6 kb-

1.5 kb-

FIG. 7. Southern blot analysis. Genomic DNA was isolated from yeast strains YMN3 (nthl::URA3, lane 1 ) and YMN4 (nth1::URAJ pralAEN::HISS, lane 2) and wild-type yeast YHH65 (NTH1 pralAEN::HIS3, lane 3 ) (see Materials and Methods) and digested with EcoRI. The DNA was separated on 1% agarose gel and blotted to a Hybond N' membrane. The 1.5-kb EcoRI fragment from the NTHl gene was used as radioactively labeled probe.

TABLE 111 Change of trehalose concentration due to temperature stress in wild-

type strain DF5a (NTHl) and the isogenic disruption mutant YMN5 (nthl::URA3)

DF5a (NTHI) YMN5 (nth2::URAS) mM trehalose

Exponential growth (OD578 0.4 1.1

40 min at 39 "C 8.0 29 Followed by 40 min at 30 "C 0.1 23

-1 at 30 "C)

milliunitslmg Activity of neutral trehalase 36 (3

(milliunits/mg)

of neutral trehalase (cf. Table 11). The centromere linkage of the NTHl gene very likely is responsible for that. The centromere elements are assumed to have a depressing influence on the number of plasmid copies per cell. I t is likely that, due to the CEN4 sequence within the insert fragment, the plasmid is recognized as a centromeric vector by the yeast cell. Even if a wild-type cell was transformed with the complementing plasmid pNTH, no overproduction was observed by measuring the enzyme activity or by Western blot experiments (data not shown).

Analyses of the 5"nontranslated region of the NTHl gene revealed besides several putative TATA boxes (see Fig. 5) a possible binding element for the MIGl protein. MIGl was cloned in yeast (46) as multicopy inhibitor of the GAL1 promotor. The authors found that MIGl is a DNA binding protein with a zinc finger structure, that also binds to two sites in the SUC2 upstream region. Recently, Mercado et al. (48) discovered a MIGl binding consensus sequence (47, 48) in the promotor of the yeast FBPl gene. We found the MIGl consensus sequence in the upstream region of NTHl.

NTHl (-652 to -641) TCATCAGATTTT

s u c 2 (consensus) (-501 to -490) TCCCCRGATTNT

SEQUENCES 4 and 5

These two sequences share homologies of more than 83% making it possible that MIGl binds to the upstream region of the NTHl gene as well as to promotor sequences of the SUC2, FBPl, and the GAL genes. MIGl is not only thought to be a glucose-sensitive repressor of GAL1, but also to be an inducer of glucose-regulated metabolic pathways. Neutral trehalase, a glucose-forming enzyme, might therefore belong to the group of glucose-repressed enzymes, the genes of which are under control of MIG1. This supports the idea of a regulatory role of MIGl in trehalose metabolism.

A comparison of the available sequences of the different

F B P genes from E. coli, rabbit, and S.cerevisiae shows excep- tionally high homology in distinct domains. The regions of identity are directly correlated with the catalytic domain. This may apply also to the trehalases from E. coli, rabbit small intestine, and the cytosol of S. cerevisiae. Amino acid sequence comparison of all three trehalases (cf. Fig. 6) shows a highly conserved central core, spanning about 70 amino acid residues between the amino acid positions: 240-310 for yeast, 145-215 for E. coli, and 161-231 for rabbit intestine. An identity of 44% and a homology of 68% are calculated by comparison of these three sequences. This region might therefore participate in the formation of the catalytic domain. A second cluster of homologous amino acid sequences is located near the C-terminal region of the three trehalases corresponding to the sequence from position 565 to 654 in the yeast protein with 25% identity and 52% homology, respectively. I t may be, therefore, that not only the central core but also the C-terminal homologous region contributes to the catalytic center.

There is no homology with respect to the N-terminal region. The N-terminal region of the yeast enzyme extends further than that of the E. coli and the rabbit enzyme by 92 amino acids. Such an N-terminal extension has also been observed for the FBP gene from S. cerevisiae as compared to the FBPases from pig and sheep (42) as well as to the FBPases from Saccharomyces pombe and pig kidney (49). In yeast malate dehydrogenase, an N-terminal extension has been observed in the case of the cytosolic enzyme as compared to the mitochondrial isoenzyme (50). The N-terminal region of neutral trehalase contains the CAMP-dependent protein kinase recognition site Arg-Arg-X-Ser (51) in positions 22-25 (cf. Fig. 5). This is the only phosphorylation site found in the trehalase protein and therefore must be the one that mediates the regulatory increase of trehalase activity by CAMP-dependent phosphorylation (5) of a serine residue (12), which now is localized as serine 25.

Protein sequencing data indicated that the N terminus of the neutral trehalase is blocked? This is in accordance with many other cytosolic proteins which have blocked N termini (51). The immunostainable band of neutral trehalase in SDS- polyacrylamide gel electrophoresis migrates as a polypeptide of approximately 86,000 daltons (cf. Fig. 3). However, from the open reading frame of the NTHl gene, encoding a protein of 693 amino acids, the molecular mass is calculated as 79,569 daltons. Similar differences have been observed for a large number of proteins (52). In our case, the difference could be due to glycosylation of three potential N-glycosylation sites (Asn-X-Thr/Ser), in the open reading frame (see Fig. 5). Treatment with endoglycosidase F, however, did not show a bandshift to lower molecular weight. Several other cytosolic proteins from yeast also have putative N-glycosylation consensus sequences, but are not glycosylated, for example, hex- okinase A (53), glucokinase (54), and fructose bisphosphatase (42).

A codon bias index (55) was calculated from the sequencing data as 0.25. Hence, neutral trehalase has a weak codon usage and may belong therefore to the lower expressed class of yeast genes (55).

Acknowledgments-We are grateful to Dr. D. J. Klionsky for help with the enzymatic overlay test. We want to thank Prof. Dr. Ernst Helmreich, Dr. Hans Rudolph, PD Dr. Matthias Muller, and Prof. Dr. Nikolaus Pfanner for critical reading of the manuscript. We also want to thank Helga Doge and Wolfgang Fritz for help with the

Unpublished Edman degradation experiments carried out by H. App and H. Holzer with F. Lottspeich.

4774 Neutral Trehalase Gene from S. cerevisiae manuscript and with the figures and Markus Burgert for technical des Gens fur Neutrale Trehalose aus Saccharomyces cerevisiae und Un- assistance. thesis, Facultv of Chemie and Pharmazie, Universitv of Freiburtr. Ger- tersuchungen zur Funktion und Regulation uon Trehalose in Hefe. Ph.D.

Note Added in Proof-The mutation nthl-? induced by EMS mutagenesis and the mutation nthl::URA3 introduced by chromo- somal disruption of the NTH1 gene were shown to be allelic: Sporu- lation of diploid cells YMN2/1-YMN3 (nthl-?-nthl::URA3) containing both mutations, resulted in four spores, which were all defective in the activity of neutral trehalase.

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