The suppressor gene scl1+ of Saccharomyces cerevisiae is essential for growth

271

Gene, 83 (1989) 271-279 Elsevier GENE 03210

The suppressor gene sell + of Saccharomyces cerevisiae is essential for growth (Recombinant DNA; temperature lethality; cycloheximide resistance; secretory signal peptide; transcription regulation)

Elisabetta BalziP, Weining Cben”, Etienne Capieaux”, John H. McCusker b*, James E. Haber b and AndrC Goffeau p a Unite de Biochimie Physiologique, Universitt!Catholique de Louvain, 1348 Louvain-la-Neuve (Belgium) and b Rosenstiel Center and Department of Biology, Brandeis University, Waltham, MA 02254 (U.S.A.) Tel. (617) 7362462 Received by J. Davison: 19 April 1989 Revised: 15 May 1989 Accepted: 16 May 1989

SUMMARY

In Succharomyces cerevisiue, the SCLI - 1 mutation is a dominant suppressor of the cycloheximide-resistant, temperature-sensitive (ts) lethal mutation, cr13 [McCusker and Haber, Genetics 119 (1988a) 303-3151. The wild-type sell + gene was isolated by screening subclones of the 35-kb region between TRPS and LEUl for restoration of the ts phenotype in an SCLl-1 cr13-2 strain. The sell + mRNA is about 900 nt long and encodes an open reading frame of 810 bp. The polypeptide deduced from sell + possesses a putative secretory signal peptide. The 5’noncoding region may be under multiple controls, since it contains significant homology to the consensus sequences for the DNA-binding proteins, GCN4, GFI and, possibly, TUF. Gene disruption of sell + demonstrates that it is an essential gene.

INTRODUCTION

A large class of pleiotropic mutations conferring resistance to low levels of Cx and inability to grow at 37’ C (ts), were recently isolated in S. cerevisiue by McCusker and Haber (1988a). Twenty-two unlinked Correspondenceto: Dr. A. Goffeau, Place Croix du Sud, 1,.1348 Louvain-la-Neuve (Belgium) Tel. 32/10/47 36 14; Fax 32/10/474745. * Present address: Department of Biochemistry, Stanford University School of Medicine, Stanford University Medical Center, Stanford, CA 94305-5307 (U.S.A.) Tel. (415)7236161. Abbreviations: aa, amino acid(s); bp, base pair(s); crl, mutation encoding Cx resistance and ts lethality; Cx, cycloheximide; 0378-l 119/89/$03.50

0 1989 Elsevier

Science Publishers

B.V. (Biomedical

Cx-resistant ts-lethal (crl) loci were mapped. The crl mutants exhibit pleiotropic phenotypes, including failure to arrest at the Gl stage in response to aa starvation and hypersensitivity to aa analogs and to 3-aminotriazole (McCusker and Haber, 1988b). These features are also characteristic for gcn GCN4,regulator ofgeneral control ofaa biosynthesis; H6,5.8-kb Hind111 insert of plasmid pA-H6; kb, kilobase or 1000 bp; nt, nucleotide(s); ORF, open reading frame; RPG, ribosomal protein gene; sell + , wt allele of x11, lacking suppressor activity; SCLI-1, mutant allele of sell dominant suppressor of cr13 ts lethality; Sell, polypeptide encoded by sell + ; ts, temperature sensitive; wt, wild type; UAS, upstream activating sequence; YNB, yeast nitrogen base; ::, novel joint (fusion, insertion).

Division)

272

mutants, which are unable to derepress the aa biosynthetic pathways in response to aa deprivation. The crl mutants also share some properties with the omnipotent suppressor mutants, which relax protein translation fidelity. Both groups of mutations suppress nonsense mutations, are highly sensitive to aminoglycoside antibiotics, which stimulate translational misreading, and are unable to grow at high osmotic pressure or temperatures. Based on the similarities with omnipotent suppressors and gcn mutations, McCusker and Haber (1988a,b) suggested that the crl mutations increase translational misreading, which becomes more severe at non-permissive temperatures. One extragenic dominant suppressor of crl ts lethality, SCLl-1, was isolated and mapped. SCLl-1 specifically acts on the cr13 alleles by suppressing ts lethality, slightly decreasing Cx resistance, and reducing the suppression, by cr13, of the nonsense mutation metl3-2. To gain further insight into the nature and mode of action of the SCLI-I suppressor, we have undertaken the molecular cloning and characterization of the wt xl1 + gene.

Moleculaire, Gif-sur-Yvette, France), containing the URA3 gene. The resulting plasmid, pA-H6 : : URA, contains thus a sell + gene disrupted by URA3 (xl1 :: URA3). The 7-kb Hind111 insert of pAH6 : : URA was used for one-step gene replacement (Rothstein, 1983)inthediploidUS86(ura3-l/ura3-1) and in the haploid 22295-C (a, ura3-1). (b) DNA and RNA preparations and analysis Genomic DNA for Southern blotting was prepared according to Davis et al. (1980). Total RNA for Northern blotting was extracted as previously described (Balzi et al., 1987). Poly(A) + RNA was selected on mAP paper (Amersham Corp.). For analysis of sell + mRNA in conditions of aa starvation, total RNA was extracted from the strain Y55-1163 (HO, adel-1, ura3-1; McCusker and Haber, 1988a), after overnight growth in duly supplemented minimal medium (0.7% YNB/2% glucose) to a density of about 35 x 106/ml, followed by 6 h incubation in the presence of 10 mM 3-amino-l, 2,4-triazole. Radioactive DNA probes were prepared by nick-translation with [ a-32P]dCTP.

MATERIALS AND METHODS

RESULTS AND DISCUSSION

(a) Strains, plasmids and transformations

(a) Cloning and disruption of the sell + gene: sell + is essential for cell growth

Saccharomyces cerevisiae strains Y55-282 (HO, cr13-2, trp5-1, SCLI-l,leul-1, ura3-1) and Y55-1161 (HO, cr13-2, SCLI-1, adel-1, ura3-1) (McCusker and Haber, 1988a) were used as SCLI-1, cr13 hosts

for transformation, as previously described (Balzi et al., 1987). The plasmids used to clone xl1 + , cosA and its subclones, pA-X30, pA-X28, pA-B6.5 and pA-B3, were described (Balzi et al., 1987). To subclone sell + , one additional plasmid, pA-H6, was constructed by insertion of the 5.8-kb Hind111 fragment (H6) from pA-X28, into the Hind111 site of the vector, pHCG3. For gene disruption, H6 was introduced into the Hind111 site of a pBR322 derivative, in which the BamHI site was removed by cutting with EcoRV + BamHI, filling of the protruding ends and religation. This plasmid carries a single BamHI site located within the sell + gene. Into this site was introduced a 1.17-kb BgjII fragment from pFL44 (a kind gift of Dr. F. Lacroute, Institut de Genetique

The genetic location of SCLI-1, between LEUl and PDRl on chromosome VII (McCusker and Haber, 1988a), predicts the presence of sell + on the cosA plasmid, which contains the 35-kb region of chromosome VII comprised between LEUl and TRP.5 (Fig. 1). CosA, as well as its subclones depicted in Fig. 1, were used to transform a SCLI - 1, cr13 double mutant. Functional complementation of the suppressor SCLI-I was scored by appearance of cr13 ts lethality at 37°C. As partly shown in Fig. 2, the transformants containing the entire cosmid or one of the subclones, pA-X28, pA-H6 and (not shown) pA-X30, complemented SCLl-1. In contrast, the transformants obtained with plasmids pA-B6, pA-B6.5 (not shown), or pA-B3 did not complement SCLI - 1. This indicates that sell + overlaps the first BamHI site distal to LEUI (Fig. 1). In this position a major transcript of about 900 nt

213

1 kb Y

PMAl

L---d SS H

CO6 A

SmHSS

LEUl

sell r,

I HS

H

B

v SmXSt

TRPS 4

PDRl

HXH

B

s

HHH

6

HH

SHHXH

BHB

SB

H

B

1

-Y

pA-X30 +

+

PA-X26 +

+

pA-H6

PA-B6

PA-B6.5

~A-63

Fig. 1. Plasmids used to clone sell + . The cosA plasmid and its subclones were previously described (Balzi et al., 1987). Subclones pA-X28 and pA-X30 are deleted of the fragment bounded by ( + ). The fragments complementing SCLI-1 are presented as blackened bars. The vector (pHCG3), common to all the plasmids shown, is depicted in cosA, as an open bar. The positions and orientations of the genes sell (this paper), PMAI, LEUZ, PDRl and TRP5 (Balzi et al., 1987) are indicated by arrows.

(Fig. 3) and an 810-bp ORF, described below, were identified. Complementation of the SCLl- 1 suppressor, despite its dominant character, might be explained by the multiple copy number of the plasmidborne sell + . Disruption of scfl was performed by inserting URA3 into the BumHI site of sell + , and by replacement of the homologous region in the chromosome of haploid and diploid hosts (Fig. 4B). Ura+ transformants were obtained only from the diploid strain. Tetrad analysis of nine asci from one diploid transformant, showed 2: 2 segregation for viability (Fig. 4A). All viable spores were uracil auxotrophs, revealing a tight linkage of the lethal function to the URA3 marker inserted into xl1 + . The nonviable spores arrested growth after two to three generations and no uniform point of arrest was observed. The sell + gene product is thus probably not involved in cell cycle control. Southern-blot analysis (Fig. 4) showed that in the parental diploid, one disrupted sell : : URA copy and one normal sell + copy are both present, while in the two surviving spores only the wt copy is present. The genetic linkage of spore viability to uracil auxotrophy and the Southern-blot hybridization results imply that disruption of sell + is lethal and that the sell + gene product is essential for cell growth.

(b) The 5’- and 3’-flanking regions of sell + : potential for multiple transcriptional control The 5’ region flanking sell + contains elements typically observed in yeast promoters. About 115 bp upstream from the translation start, a pyrimidinerich block (19 CT out of 20 nt from nt position -112 to -131, or 37 out of 46nt from -112 to -157) is observed. Similar structures, reported for several yeast promoters (Dobson et al., 1982; Oberto and Davison, 1985) are believed to prevent the accumulation of erroneous transcripts (McNeil, 1988). A TATATA-box is present 189 bp upstream from the translation start. Such a sequence (T,) was recently shown to be involved in regulated transcription, probably by binding a specific TATA-protein which interacts with transcription-activator proteins (Chen and Struhl, 1988). In particular, in the HZS3 promoter, T,, together with the GCN4 binding site, regulates the expression of HZS3, in response to aa starvation (Struhl, 1986). Similarly, in the sell + promoter, we observe the presence not only of a T, element, but also of two putative GCN4-binding sites, TGACTC, located 381 and 627 (not shown) bp upstream from the translation start. The nt flanking TGACTC were also shown to be important for GCN4 binding and the expanded consensuses

274

ICG3

t-m -X28

ABFl (TATCATTNNNNACGA, Buchman et al., 1988). Factors GFI and ABFl, as well as SBF-B (Shore et al., 1987; Brand et al., 1987), are related DNA-binding factors capable of binding, though not exclusively, autonomously replicating sequences.

t-B3

A kb

SI

Fig. 2. Functional complementation of SCL1-1 by pA-H6. The mutant Y55-282, containing both the SCLZ-1 and cr13-2 mutations, was transformed with cosA and its subclones. Ura’ transformants containing the vector pHCG3 (1st row) or the plasmids pA-H6 (2nd and 3rd rows), pA-X28 (4th row) or pA-B3 (5th row) were grown two days at 30°C and 37°C on minimal medium omitting uracil(O.7% YNB/2% glucose/40 pg L-leucine per ml, 20 pg L-tryptophan per ml/40 pg adenine per ml). Strains Y55-1161 and Y55-1162 (HO, crZ3-2,adel-1, ura3-1; McCusker and Haber, 1988a) grown in minimal medium, as described above and with the addition of 4Opg uracil/ml, are shown as references for the crl3 + SCL1-1 and 013 phenotypes, respectively.

1986) or ETGACTC (Arndt and Fink, ETGACTCattt (Hill et al., 1986) were proposed. The flanking nt of the xl1 + TGACTC sequences, tTGACTCgt and tTGACTCtg, do not match well with the expanded consensuses mentioned above. However, several naturally occurring GNC4-binding sites do not show the optimal consensus. In conditions of GCN4 derepression, that are caused by histidine starvation induced by 10 mM aminotriazole, no marked increase in the level of sell + mRNA could be detected in Northern-blot analysis (data not shown), as compared to normal growth conditions. However, this observation does not rule out the possibility that GCN4 interacts with the sell + promoter, since frequently GCN4 has little effect on the transcription of genes that it controls. Another UAS motif is also present in the sell + promoter. Between nt -81 and -66, we observed a sequence, TATCACAATAGACGAA, corresponding to the binding site of factor GFI (RTCRNNNNNNACGNR; Dorsman et al., 1988). This sequence matches, with 13 out of 15 nt, the more defined consensus sequence binding factor

B SC/l 4

HS

H

B

SmX St

HXH

B

SI Sn Fig. 3. The sell transcript. (A) Northern blot of 10 pg poly(A)+ RNA hybridized to the probes SI and SII shown in (B). The two probes, one distal and one proximal from the BarnHI site cutting sell + , hybridize to a common major RNA molecule of about 900 nt. In addition, SI hybridizes to the 3-kb LEUl transcript and to a mRNA of about 600 bp, while SII reveals a 1.5-kb transcript. The latter two mRNAs correspond to genes flanking sell. Gel was 1.5% agarose/6% formaldehyde, transferred to nylon membranes and hybridized according to the supplier’s (Du Pont-New England Nuclear) instructions.

215

In addition, we found in the sell + promoter two sequences homologous to the UAS, (review by Mager, 1988) binding site for the closely related factors, TUF (Vignais et al., 1987), RAP1 (Shore and Nasmyth, 1987) and GRFI (Buchman et al., 1988), believed to control the transcription of the ribosomal protein genes (Leer et al., 1985), and other essential genes (Capieaux et al., 1989 and references therein). One RPG-like box, gCCCATAttTaT, starting from nt -248, is preceded, with an interval of 17 bp and in the opposite orientation, by an HOMOLl-like sequence, AAgATCTcaGCT. In conclusion, transcriptional regulation of sell + is potenti~y mediated by multiple tr~sc~ption~ activation systems. In the 3’ flanking region of xl1 +, a TATGT.. .lTT sequence, consensus for messenger RNA termination and polyadenylation proposed by Zaret and Sherman (1986), is present 52 bp downstream from the translation stop site. Another known eukaryotic polyadenylation signal, AATMA (Proudfoot and Brownlee, 1976), is also found, 228 bp from the end of the coding region.

(c) The s&+-encoded polypeptide: presence of a secretory signal sequence The 1564-bp nt sequence containing the 8 lo-bp xl1 + ORF is presented in Fig. 5. The low codon

bias index (Bennetzen and Hall, 1982) of 0.08, the codon adaption index (Sharp and Li, 1987) of 0.13, and the low degree of third nt pyrimidine bias (total and third-position G + C contents of 0.4 and 0.35, respectively) of sell + characterize poorly expressed yeast genes (Sharp et al., 1986). The putative Sell polypeptide has a calculated M, of 30339 and a theoretical isoelectric pH of 7.22. As shown in Fig. K, the N-terminal region of Sell displays a structure co~esponding to a tripartite secretory signal, which specifies translocation across the endoplasmic reticulum (review by von Heijne, 1988). The likelihood of this sequence to be a signal peptide is indicated by the score (von Heijne, 1986) of 8.6, which is well above the threshold score of 3.5, The Sell putative signal is likely to be cleaved off, a potential cleavage site, conforming to the ‘(-3, -1) rule’ (von Heijne, 1988), is predicted between aa 37 and 38. Sell exhibits other features generally attri-

buted to signal peptides. Pro residues are absent from aa -3 through + 1, relative to the cut point; a her-bre~g Gly is present at aa position -5, and a large Leu residue is observed at aa position -2. Also, as generally observed, a second potential cleavage site is predicted at aa positions 35-36 with a score of 7.27. One membr~e-sprung fragment is predicted by the method of Klein et al. (1985) between aa 22 and 38 of Sell, corresponding to the h- and c-regions of the putative signal peptide. Even though one transmembrane helix may be predicted at aa positions 85 to 101 by the method of Rao and Argos (1986), which sets the ~nimum helix length to 16 aa, no membrane-association structure is predicted by other methods (Eisenberg et al., 1982; Klein et al., 1985; von Heijne, 1988), out of the cleavable signal peptide region of Sell. Therefore, anchoring of Sell to the endoplasmic reticulum membr~e seems unlikely. However, it has recently been reported that a viral glycoprotein, VP7 (Stirzaker and Both, 1989), possessing no membrane-anchor domain out of the cleaved signal peptide, is retained in the endoplasmic reticulum membrane through the signal peptide itself, despite its rapid cleavage. If this was also the case for Sell, this polypeptide could interact with the translocation and processing of nascent secretory proteins. Alternatively, the Sell protein could be translocated into the endoplasmic reticulum lumen. The HDEL signal, specifying retention of soluble proteins in the endoplasmic reticulum of yeast (Pehhnan et al., 1988), is absent in Sell. Sell is thus likely to enter the secretory pathway as a vacuolar protein or as a polypeptide to be directed to the periplasm, the cell wall or the ex~ace~~~ medium. All known proteins entering the secretory pathway are glycosylated. The Sell putative polypeptide possesses two potential sites for N-glycosylation at aa positions 54 and 141. Among the well characterized glycoproteins of S. cercvisiae, one could possibly correspond to the predicted Sell polypeptide. It is a major component of the cell wall, a 29-kDa mannoprotein composed of a 26-kDa peptide moiety, N-glycosylated with one core oligosaccharide (Pastor et al., 1984). It is also interesting to note that, like many secreted proteins, the putative Sell deprived of its signal peptide would have an N-terminal aa, Phe, that confers a short half life to the protein in the cytoplasm (Bachmair et al., 1986).

276

CEN

H”

8

il

+----_(

C

kb 23+

Fig. 4. The lethal sell gene disruption. (A) A tetrad analysis showing the 2 (viable): 2 (nonviable) segregation of spores dissected from nine asci, obtained from the diploid disruptant D scL2. Tetrad analysis of asci derived from the nondisrupted diploid host, US%, is shown on the IeR. (B) A scheme for the recombination event which took place between the H6:: URA fragment, containing xi1 + disrupted

211

A

6 -477 -387 -297 -207 -I

17 -27 +b4

+I54 +244 +334 l4 24 +5 14 +bO4 +694 t784 +a74

TTAAATGGCTMCTCATTATAATCTTCATGCTAAATCATATMGGGCAGAGACGMGCAMGCGAAA~CATATTACMTCATGTCGG MLNHIRAETKOSEKN ILQSCR Gn;Cn;CTGCTGCATCTGCTGCTGGTTATGACAGGCACATCACTATC~T~CCCCGA~GGTCGTTTATCMGTAGMT~TGCC~TMA VLLLHLLLVMTGTSLSFPPRVVYQVEYAFK GCGACTAATCMACTMCATAAACTCACTCACTA~~TCAGAGGTAAAGA~GTACAGTGGTGATAAGTCAG AAAAAAGTCCCTGATAAACTG ATNQTNINSLAVRGKDCTVV ISQKKVPDKL TIY;GATCCMCTACTGmCGTATATTTTTTGTATTTCAAGTGCG LDPTTVSY I S R T IGMVVNGPIPDARNA I F C GCCCTMGAGCCAAGGCTGAGGCTGCAGM~CCGTTATAC ALRAKAEAAEFRYKYGYDMPCDVLAKRMAN CmCCCAAATCTATACTCAAAGAGCATATATGAGACCATTAC LSQIYTQRAYMRPLGVILTFVSVDEELGPS AmACAAAACTGACCCTGCAGGTTATTACGTTGGCTAC~~TACTGCGACAGGACC~C~CA~AGA~ACMC~C~AGAA IYKTDPAGYYVGYKATATGPKQQEI T T N L MCCATTTCAAAAAGAGTAAAATCGACCATATTAATGAAGGACGCA NHFKKSKIDHINEESWEKVVEFAITHMIDA CTGGGTACCGMTTTTCAAAGM~A~~MGTCGGTGTCGCTAC~GGACAMTTCT~ACC~GAGT~~AGMCA~GMGM LGTEFSKNDLEVGVATKDKFFTLSAENIEE AGGCTAGTAGCMTTGCTGMCMGATTAACATACATAC~CCTGCCACMCATAATGTG~~ACAC~GTCMCACTCMTA~ATGG RLVAIAEQD ACACGTPTCATTTAGGATTTAmTATAGGAAAAAGTAGGAMAAGTAGATCMGTAATAGAAAATCGMTC~CMGCC~GTCGCGT~TAC

21 51 81 I I I I4 I I71 E

20 I 231 26 I

+964 +I054

C

GGGCMTGTTTACMTACTCATCCCMTCTTTTC

1.0

KQSE:NILQSCRVLLLHLLLVMTGTSL 0 0

7

1I

38

F

PP

n-c----h--c+

Fig. 5. Nucleotide sequence of&l+ and deduced aa sequence. The 6-kb Hind111 insert of PA-H6 and its 1.4-kb HindHI-BumHI, 2.3-kb BarnHI-XhoI and 1.5-kbHindIII-EcoRI fragments, were cloned into the pEMBL8 vectors and progressively deleted (Barnes et al., 1983). Nucleotide sequencing was performed following the dideoxy chain-termination procedure @anger et al., 1977). (A) The strategy used to sequence the 1564-bp fragment containing sell + (B) The nt sequence of the sell + ORF with its flanking regions, and, under it, the deduced aa sequence of the putative Sell protein. Position + 1 is assigned to the putative start codon. Nucleotides and deduced aa are numbered on the left and on the right margins, respectively. Elements typical of yeast promoters and terminators, as discussed in RESULTS AND DISCUSSION, section b, are underlined. (C)The putative secretory signal peptide of Sell. The predicted cleavage site is represented by a slash. The tripartite structure ofthe signal sequence (von Heijne, 1988), including a positively charged N terminus (n-region), followed by a hydrophobic core (h-region) and by a more polar tract (c-region), is shown. Positively and negatively charged aa of the n-region are indicated.

by URA3, and the chromosomes of a diploid host (chr VII H). In the diploid Ura + disruptants, one chromosome integrated the H6:: URA fragment by replacement of the homologous resident region (chr VII D). After meiosis, the viable spores inherited the chromosome containing the sell + wt copy (chr VII S). (C)The genomic restriction patterns of dierent structures of chromosome VII in a Southern-blot analysis ofHind and BarnHI genomic digests from the diploid nondisrupted host (US86), two diploid disruptants (D,,,2 and D,,,3), and two viable spores (SPZ-1C and SpZ-ID) of a tetrad derived from D,,2. The probe shown as a dashed line in (B) was used. The labeled fragments indicate the presence of a wt (5.8-kb HindIII, 6-kb BamHI) or/and of a disrupted (7-kb HindIII, 13.5-kb BamHI) sell gene. Gel was 0.9% agarose, followed by transfer to nitrocellulose filters and hybridization as previously described (Balzi et al., 1987).

278

{d) The suppressor function of Sell Informational suppression results generally from alteration in either tRNA, ribosomal constituents or their modifiers, or other translation-related factors. Considering its low codon bias, it is unlikely that the scit + gene encodes a ribosomal protein. One could however envisage that sell + encodes some weaklyexpressed cytoplasmic translation-related factor. Such functions have been attributed to other yeast suppressor genes: SUFl2 (Wilson and Culbertson, 1988) and SUP2 (Kushnirov et al., 1988), both essential and weakly expressed genes encoding elongation-factor-related proteins, and SUP45 (Himmelfarb et al., 1985), which was suggested to code for nonribosomal translation functions. However, the presence of an endoplasmic reticulum targeting signal suggests that the scl2 + product is located in a specific compartment. If Sell proceeded through the secretory pathway, it could suppress the ~13 mutations by interactions taking place elsewhere than at the translation level. Alte~atively, if by analogy with the VP7 glycoprotein, Sell was retained in the endoplasmic reticulum membrane, it could interfere with the translation, tr~slocation or processing of nascent secretory proteins occurring on the surface of the endoplasmic reticulum, or with the glycosylation of secretory proteins occurring in the reticulum lumen.

We gratefully acknowledge Eric Van Dyck for having performed the tetrad analysis.

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