Vol. 10, No. 6

MOLECULAR AND CELLULAR BIOLOGY, June 1990, p. 2544-2553

0270-7306/90/062544-10$02.00/0 Copyright © 1990, American Society for Microbiology

SNF6 Encodes a Nuclear Protein That Is Required for Expression of Many Genes in Saccharomyces cerevisiae FRANCISCO ESTRUCHt AND MARIAN CARLSON* Department of Genetics and Development and Institute of Cancer Research, Columbia University College of Physicians and Surgeons, New York, New York 10032 Received

5

December 1989/Accepted 15 February 1990

The SNF6 gene appears to affect transcription from a variety of promoters in Saccharomyces cerevisiae. The cloned, and sequence analysis revealed two completely overlapping open reading frames of 996 and 1092 nucleotides on opposite strands. The SNF6 coding sequence was identified by selective mutagenesis. The predicted 37,604-dalton SNF6 protein is highly charged but overall neutral. A bifunctional SNF6-0igalactosidase fusion protein was localized in the nucleus, as judged by immunofluorescence microscopy. The N terminus of SNF6 contains a sequence homologous to nuclear localization signals and was sufficient to direct (galactosidase to the nucleus. The 5' ends of the SNF6 RNA were heterogeneous and included ends mapping downstream from the first ATG codon. Construction of a frameshift mutation provided evidence that translational initiation at the second ATG yields a partially functional SNF6 product. Null mutations in SNF6 caused a wider range of pleiotropic defects than the previously isolated point mutation, including slow growth. Genetic and molecular evidence suggested that SNF6 is functionally related to the SNF2 and SNFS genes. These genes may function together to affect transcription. gene was

The SNF6 (sucrose nonfermenting) gene of Saccharomycerevisiae was originally identified as a gene required for derepression of the SUC2 (invertase) gene in response to glucose limitation (28). The defect was in derepression of SUC2 mRNA (unpublished data). SNF6 was found also to be required for derepression of acid phosphatase in response to phosphate starvation (1). This finding indicated that the function of SNF6 is not specific to expression of genes controlled by a single signal transduction pathway. Further support for this idea comes from recent studies showing that SNF6 is required for transcription of Ty elements (A. M. Happel, M. S. Swanson, and F. Winston, personal communication). This evidence suggests that SNF6 affects expression of a variety of promoters. Genetic evidence also implicates SNF6 in transcriptional processes. The snf6 defect in invertase derepression is suppressed by mutations in the SPT6ISSN20 gene (30), which is an essential gene that affects transcription (13, 29). The spt6 mutations suppress 8 insertions in the 5' region of HIS4 or LYS2 by altering transcription (13, 42). The ssn2O alleles suppress mutations in cis- and trans-acting elements that are required for SUC2 transcription (29, 30). The cre2-1 mutation, which affects ADH2 expression, is also an spt6 allele (15; C. L. Denis and T. Malvar, Genetics, in press). Similarities in phenotype and genetic behavior suggest that SNF6 is functionally related to SNF2 and SNF5, two other genes identified in the same mutant search (28). Null mutations in these genes cause defects in derepression of SUC2 mRNA, derepression of acid phosphatase (1), and Ty transcription (Happel et al., personal communication). The invertase derepression defects are suppressed by spt6lssn2O mutations (29, 30). In these respects, snJ2, snf3, and snf6 are similar to one another and distinct from other snfmutations. One difference is that snJ2 and snf5 also affect utilization of

galactose and glycerol, for which expression of various glucose-repressible genes is required. In addition, snJ2 and snf5 interact differently with the ssn6 suppressor than does snf6 (28). However, only one snf6 allele (snf6-719) was isolated, and it may represent a leaky mutation. Thus, both genetic and molecular evidence suggests that SNF6 has a general role in transcription and that SNF2, SNF5, and SNF6 constitute a group of genes with related functions. To elucidate the function of SNF6, we undertook a molecular characterization of the gene and its product. The gene was cloned, and sequence analysis revealed overlapping open reading frames (ORFs) on both strands. The SNF6 coding sequence was identified by selective mutagenesis of each ORF. The chromosomal SNF6 locus was disrupted to ascertain the null phenotype, which proved to be more pleiotropic than the snf6-719 mutant phenotype. To facilitate analysis of SNF6 gene products, we constructed a bifunctional SNF6-lacZ fusion. The fusion protein was shown to be localized in the nucleus, and the nuclear localization signal was identified. Finally, the relationship of SNF2, SNF5, and SNF6 was further examined.

ces

MATERIALS AND METHODS

Strains and genetic methods. The strains of S. cerevisiae used in this study are listed in Table 1. Crossing, sporulation, and tetrad analysis were done by standard genetic methods (39). Yeast cells were transformed by the method of Ito et al. (19). Growth on different carbon sources was scored by spotting cell suspensions on medium containing the indicated carbon source. Cloning of SNF6 gene. A library prepared in the vector YEp24 from genomic DNA of an S288C-derived strain (7) was used to transform MCY953 to uracil prototrophy. Transformants (-90,000) were replica plated onto supplemented synthetic medium (39) lacking uracil and containing 2% raffinose as the carbon source, and the plates were incubated anaerobically in GasPaks (BBL Microbiology Systems, Cockeysville, Md.). Fifteen raffinose-fermenting transformants were identified and purified by single-colony isolation.

* Corresponding author. t Permanent address: Departamento de Bioquimica y Biologia Molecular, Facultades de Ciencias, Universitat de Valencia, Valen-

cia, Spain. 2544

YEAST SNF6 GENE ENCODES A NUCLEAR PROTEIN

VOL. 10, 1990

TABLE 1. S. cerevisiae strains used Straina Genotype MCY939 ..... MATa ssn2O-l his4-539 ura3-52 SUC2 MCY953 ..... MATa snf6-719 his4-539 ura3-52 ade2-101 SUC2 MCY1093 ..... MATa his4-539 ura3-52 lys2-801 SUC2 MCY1094 ..... MATot ura3-52 ade2-101 SUC2 MCY1208 ..... MATa snjf5-18 his4-539 lys2-801 ura3-52 SUC2 MCY1250 ..... MATa snj2-AJ::HIS3 lys2-801 ura3-52 his3-A200 SUC2 MCY1389 ..... MATa ura3-52 leu2::HIS3 SUC2 MCY1802 ..... MATa ssn6-A9 his4-539 lys2-801 ura3-52 SUC2 MCY1811 ..... MATo his3-A200 lys2-801 SUC2 a Strains are congenic to S288C.

Plasmid DNA was recovered from these transformants by passage through Escherichia coli HB101 (18). To demonstrate linkage of the cloned DNA to the SNF6 locus, we constructed the integrating plasmid pEY12 by subcloning the 6.5-kilobase (kb) SmaI-EcoRI fragment from pEL3.10 into YIp5 (4). pEY12 was used to transform MCY953 (snf6 ura3). Four stable SnfI Ura+ transformants were crossed to MCY1811 (SNF6 URA3), and the resulting diploids were sporulated and subjected to tetrad analysis. All plasm id

Sm

pEL3.1 0

}

B

the 22 tetrads examined showed 4+ :0- segregations for the Snf phenotype, indicating that pEY12 was integrated at a site tightly linked to snf6. To confirm that the plasmid had integrated at only one chromosomal location, we crossed two of the transformants to MCY956 (snf6 ura3). Tetrad analysis of the diploids showed 2+:2- segregations for the Snf and Ura phenotypes and cosegregation of the SNF6 and URA3 markers in 13 tetrads examined. Construction of subclones and lacZ fusions. Subclones were prepared from DNA of pEL3.10 (Fig. 1) by standard methods (24). pEY81, pEY83, pEY84, pEY88, pEY90, pEY91, and pEY111 are subclones in the centromere-containing vector YCp5O (35). pEM1 and pEM3 are derivatives of M13mpl8, and pEM2 is a derivative of M13mp19 (31). pEB81 and pEB89 are subclones in pUC19 (44). pEB89 contains the 1.1-kb HindlIl fragment carrying the URA3 gene inserted into the HinclI site at position + 122 in SNF6. pEY103 and pEY110 contain the indicated restriction fragments in YIp5 (4); the EcoRV fragment is deleted from pEY103, and the HincIl fragment is deleted from pEYllO. Plasmid pSNF6(254)-lacZ, carrying a gene fusion between codon 254 of SNF6 (at the PstI site) and lacZ, was constructed in the vector YEp354 (26). pSNF6(23)-lacZ and pSNF6(41)-lacZ carry fusions to lacZ at the indicated K K P

N

I

II

A A NcRs K III

N

I

Hc

2545

E E

RRH

H

X

III

BNcX KBP I I I.. Is

Hc

H

w.www.

complementation

pEYl 11 pEY81, pEB81 pEY83 pEY90 pEY91 pEY88 pEY84 pEM1, pEM2 pEM3

++ ++ ++

|4

allele pEB89 pEY1 03 pEY110

snf6- 1 ::URA3 snf6-A 1 snf6-A2

0.5 kb I FIG. 1. Restriction maps of SNF6 locus and plasmids. Plasmids are described in the text. Only the yeast DNA segment is shown, except that part of the vector sequence (hatched bar) is shown for pLE3.10. Symbols: thin line, yeast DNA; solid bar, SNF6 coding region; wavy arrow, RNA. An arrow indicates the 5'-to-3' direction of the bacteriophage DNA strand of pEM1 that was available for hybridization to SNF6 RNA in the experiment shown in Fig. 2; the opposite strand of pEM2 was available. A scale is shown for the expanded map. For episomal plasmids, complementation of a snfj mutation was judged by testing several transformants of MCY953 for anaerobic growth on supplemented synthetic medium lacking uracil and containing raffinose. The allele designations of chromosomal snfb mutations constructed with plasmids are indicated. Restriction sites are as follows: A, AccI; B, BglII; E, EcoRV; H, HindIII; Hc, HincII; K, KpnI; N, NheI; Nc, NcoI; P, PstI; R, EcoRI; Rs, RsaI; X, XhoI. Not all AccI, EcoRV, HincII, or RsaI sites are shown.

2546

ESTRUCH AND CARLSON

codons (at the RsaI and HinclI sites, respectively) in the vector YEp356R (26). Nucleotide sequence analysis. Restriction fragments were cloned into M13mpl8 and M13mpl9 (31), and the sequence was determined by the method of Sanger et al. (36) with the 17-nucleotide sequencing primer (Amersham Corp., Arlington Heights, Ill.). The sequence of both strands was determined for the region -196 to + 1158. Disruption of chromosomal SNF6 locus. To construct the chromosomal snf6-1:: URA3 allele, we digested plasmid pEB89 with KpnI, which cleaves in the gene and in the polylinker at the edge of the yeast DNA segment, to release a fragment of yeast DNA carrying the URA3 insertion. The digested DNA was used to transform strain MCY1094 to uracil prototrophy. Gel transfer hybridization analysis (40) of the transformants confirmed that the SNF6 locus carried the insertion. To introduce the deletions snf6-AJ and snf6-A2 into the genome, we used the integrative plasmids pEY103 and pEY110, respectively, to transform a diploid from the cross of MCY1094 x MCY1389 to uracil prototrophy. Urasegregants were selected on medium containing 5-fluoroorotic acid (3). Those segregants in which loss of the integrated plasmid resulted in replacement of the wild-type sequence on one homolog with the deletion were identified by Southern blot analysis. For each deletion, two such heterozygous diploids were subjected to tetrad analysis. Oligonucleotide-directed mutagenesis of each ORF. The 2.5-kb NheI-XhoI fragment containing the gene was cloned in M13mpl8. Oligonucleotide-directed in vitro mutagenesis was done with the Bio-Rad Muta-Gene M13 in vitro mutagenesis kit and the oligonucleotides 5'-GAACGTGTATGAC TTAG-3' and 5'-CACAAAACTAATAGCTGCG-3' (purchased from Research Genetics). The nucleotide representing the base substitution is underlined. The first oligonucleotide changes codon 84 of the SNF6 ORF from CGA (Arg) to TGA (stop) and changes one serine codon to another in ORF2 on the other strand. The second oligonucleotide replaces codon 172 of the SNF6 ORF with another leucine codon (CTA) and changes codon 184 of ORF2 from AAG (Lys) to TAG (stop). For each mutation, the 2.5-kb yeast DNA fragment from a recombinant bacteriophage was subcloned into pUC19 and then transferred to YCp50 for analysis in yeast cells. The resulting plasmids have the same structure as pEY111. The region containing each mutation was sequenced by subcloning a small fragment from the YCp50 plasmid (KpnI-HindIII for the snf6 mutation and KpnI-EcoRV for the orJ2 allele) into M13mpl9 and using the 17-nucleotide sequencing primer (Amersham). Construction of frameshift mutation. Plasmid pEB81 was digested with the NcoI endonuclease, which cleaves at a single site after codon 10. The ends were filled with Klenow fragment and ligated with T4 ligase. A fragment containing the resulting +2 frameshift mutation was used to reconstruct the SNF6 gene in the vectors YCp5O and YEp354, thereby generating pEYlllfs and pSNF6(254)fs-lacZ, respectively. Preparation and analysis of RNA. Poly(A)-containing RNAs were isolated from glucose-repressed or derepressed cultures as described previously (37). 32P-labeled singlestranded probes were prepared from pEM1, pEM2, and pEM3 by the M13 chain extension method (36), omitting chain terminators, as described previously (38). Enzyme assays. Glucose-repressed cells were prepared by growing cultures to the exponential phase in medium containing 2% glucose, and cells were derepressed by a shift to medium containing 0.05% glucose. Secreted invertase activ-

MOL. CELL. BIOL.

ity was assayed in whole cells (16). 3-Galactosidase activity was assayed (25) in permeabilized cells (17). Immunofluorescence microscopy. Cells were prepared as described previously (12) and stained with monoclonal antibody to 3-galactosidase (Promega Biotec, Madison, Wis.) and fluorescein isothiocyanate-conjugated F(ab')2 fragment of sheep antibody to mouse immunoglobulin G (Sigma Chemical Co., St. Louis, Mo.). Both incubations with antibody were done overnight at 4°C. Cells were stained with 4',6-diamidino-2-phenylindole and were observed and photographed as before. RESULTS Cloning the SNF6 gene. Plasmids carrying the SNF6 gene were isolated from a genomic library (7) by complementation. The defect in derepression of invertase caused by snf6 leads to an inability to utilize raffinose, which is hydrolyzed by invertase. The library was used to transform MCY953 (snfj-719 ura3) to uracil independence, and transformants were screened for ability to grow anaerobically on medium containing raffinose, as described in Materials and Methods. Four different plasmids carrying overlapping segments of cloned yeast DNA were recovered from 15 Raf+ transformants. The restriction map of one of these plasmids, pEL3.10, is shown (Fig. 1). The cloned DNA was shown by genetic analysis to direct integration of a plasmid to the SNF6 locus, as described in Materials and Methods. To delimit the SNF6 gene on the cloned DNA, we constructed subclones in the centromere-containing vector YCp5O (35) and tested their ability to complement the raffinose-nonfermenting phenotype of snf6 by transforming MCY953. The results suggest that the gene lies in the 1.5-kb region between the AccI and XhoI sites (Fig. 1). Expression of RNA from SNF6 locus. To identify RNAs encoded by the SNF6 locus, we examined poly(A)-containing RNA isolated from glucose-repressed and derepressed cultures of a wild-type (SNF6) strain by gel transfer hybridization analysis. Three RNA species (3.3, 1.8, and 1.2 kb) were detected by hybridization with probe prepared from pEB81 DNA or the 1-kb NheI-HincIl fragment from pEB81 (data not shown). Only the 1.2-kb RNA was detected with probe prepared from either the 0.6-kb HincII-PstI fragment or the 0.6-kb NcoI-BglII fragment, each of which lies within the DNA region required for complementation of snf6 (Fig. 2a). These findings suggest that the 1.2-kb RNA is encoded by SNF6. The direction of transcription was determined by using single-stranded probes prepared from pEMl and pEM2, subclones of the HincII-PstI fragment in M13mpl8 and M13mpl9, respectively. The pEMl probe hybridized to the 1.2-kb RNA (Fig. 2c), and the pEM2 probe did not, indicating that the direction of transcription of the SNF6 locus is as shown in Fig. 1. The expression of the putative SNF6 RNA was not regulated in response to glucose availability. The relative abundance of the SNF6 and TUB2 (tubulin) RNAs was approximately the same in glucose-repressed and derepressed cells (Fig. 2b). Sequence of SNF6: large ORFs on both strands. The nucleotide sequence of the cloned SNF6 locus was determined. The region that complements the raffinose utilization defect of snf6 includes two large ORFs, one on each strand. The ORF of 996 nucleotides could be encoded by the 1.2-kb RNA and was tentatively designated the SNF6 ORF. A definitive genetic experiment identifying it as SNF6 is de-

YEAST SNF6 GENE ENCODES A NUCLEAR PROTEIN

VOL. 10, 1990

R

D

R

D

R

D

M

1

42,645-dalton protein encoded by ORF2 is shown in Fig. 4.

2

_ * *X a j5-

W **

a

*

b

w

c

- -

2547

-

d

FIG. 2. Analysis of SNF6 RNA. Poly(A)-containing RNAs were prepared from glucose-repressed (R) and derepressed (D) cultures of wild-type strain MCY1093. (a to c) Northern blot analysis. RNAs were separated by electrophoresis in a 1.8% agarose gel containing formaldehyde (24) and were transferred to nitrocellulose (41). DNA fragments were used as size standards. RNA was detected by hybridization with labeled probes and autoradiography. The following probes were used: (a) 32P-labeled NcoI-BglIl fragment from pEB81 prepared by nick translation (34); (b) 32P-labeled TUB2 DNA (27). The same filter shown in panel a was reprobed. (c) 32P-labeled single-stranded probe prepared from pEM1. No RNA was detected by using single-stranded pEM2 probe (not shown), and the presence of SNF6 RNA on the blot was confirmed subsequently. (d) S1 mapping of the 5' ends of the SNF6 RNAs. Hybridization of RNA ( glucose-repressed cells to 32P.aee P-labeled sige-tane single-stranded romguoerpesdclst p.g)g from probe prepared from pEM3 was done at 500C in 80% formamide for 14 h as described previously (14). Hybrids were treated with 200 U (lane 1) or 600 U (lane 2) of S1 nuclease (Boehringer Mannheim Biochemicals, Indianapolis, Ind.) per ml. Samples were analyzed by electrophoresis on a 5% polyacrylamide gel containing urea. Similar results were obtained by using RNA from glucose-repressed cells (not shown). No protected fragments were detected in control samples in which tRNA (10 ,ug) was hybridized to the probe (not shown). M, Marker size ladder generated from a SNF6 DNA fragment. Arrowheads indicate prominent clusters of protected fragments.

scribed below. The nucleotide sequence and the amino acid sequence of the predicted 37,604-dalton protein are shown in Fig. 3. The second large ORF, designated ORF2, is present on the opposite strand from the putative SNF6 gene and includes 1,092 nucleotides. ORF2 completely overlaps the SNF6 ORF; the first ATG of ORF2 lies at position 1065 of the SNF6 sequence and the termination codon is at position -30 (Fig. 3). The amino acid sequence of the predicted

Although we did not detect any stable RNA transcribed from this strand, this negative result does not exclude the possibility that ORF2 is expressed. The amino acid sequence of the predicted SNF6 protein was compared with the sequences in GenBank (release 58.0) translated in all six reading frames by using the program TFASTA (32). No significant homology was found. The protein is highly charged but has nearly equal numbers of acidic and basic residues. Overall, 28% of the amino acids are charged. The middle one-third of the protein (residues 129 to 251) has 40% charged amino acids. The N terminus is basic (8 of the first 16 residues), and the C terminus is acidic (10 of the last 32 residues). Additional noteworthy features are a largely uncharged region near the N terminus (residues 17 to 74; three charges) and a glutamine-rich stretch near the C terminus (positions 264 to 276). Comparison of the predicted protein sequence for ORF2 with the sequences in the Protein Identification Resource (release V20.0) of the National Biomedical Research Foundation by using the computer program FASTP (23) revealed

homology. Disruption of chromosomal SNF6 locus. Previously, we isolated only one snf6 allele, snf6-719, which is probably a point mutation. To determine the phenotype of a null mutation at the SNF6 locus, we used the cloned DNA to construct an insertion and two deletion mutations, as described in Materials and Methods. First, the insertion in pEB89 (Fig. 1) was introduced into the genome of a haploid strain to replace the wild-type SNF6 allele. The resulting allele was designated snf6-J:: URA3. The insertion mutant, like the snf6-719 mutant, was defective in anaerobic growth on raffinose but capable of anaerobic growth on galactose and aerobic growth on glycerol. Diploids homozygous for snf6-1:: URA3 failed to sporulate, as was true for diploids homozygous for snf6-719. The two deletions in pEY103 and pEYllO were each introduced into a wild-type (SNF6ISNF6) diploid strain. a diploi e iploduceto Diploids heterozygous for each(SNF6) mutation (snf6-AJISNF6 and and snf6-A2ISNF6) were sporulated and subjected to tetrad no

analysis. Tetrads showed 2+ :2- segregations

not

only for

anaerobic growth on raffinose but also for anaerobic growth on galactose and for aerobic growth on glycerol. In addition, small spore clone size cosegregated with these phenotypes, and the snf6 segregants subsequently produced smaller c colonies than wild type when streaked on rich medium containing glucose. Segregants from each cross were defectiende sosSereted eachrcrossawererded of secreted invertase and produced tive in derepression lower activity than snf6-7J9 strains (Table 2). Homozygous mutant

diploids failed to sporulate.

The three new mutations were recessive and failed to complement snf6-719 for growth on raffinose or sporulation. This result confirms that the cloned locus is SNF6. Since the snf6-A2 allele removes most of the coding region, we condude that the phenotype of the two deletion mutants corresponds to the null phenotype and that the snf6-719 and

snf6-J::

URA3 alleles provide partial function. In addition, the finding that snf6-A2 strains are viable shows that neither SNF6 nor ORF2 is an essential gene. Selective mutagenesis of each ORF. To identify definitively the ORF corresponding to the SNF6 gene, we used oligonucleotide-directed mutagenesis to introduce two mutations that selectively disrupted each of the two ORFs, as described in Materials and Methods. One of the mutations, designated snf6-84, was a base substitution that changed codon 84 of the putative SNF6 ORF to the termination

2548

ESTRUCH AND CARLSON

MOL. CELL. BIOL.

-240

GAGGCACATTCGTTTGTAGCCGACGTGCTTCATTTATCAGGTT AGTTGGCAGCGGCTGGACCATACAGGTTCTCACAATGTGTTGGTCTACGTTCGGTCATGCACCACGGTTGTTCCGTCAAA

-160

CGGTCAGAAAACACACGCGGAAAATCCAAAGGAAAAGATGGGAGAGAAAAAACATGAAGTTGAGAAGCAACGAAACAAGG

-283

-80

1

TTTGATCCAGAAAGGGL8AMCTAAAGGCGAAAAAGGCGAAAGAAAAGCGT

GAGAATTTTACAAACGTAGTA8AMTA _0 0RF2

NcoI ATG GGT GTC ATC AAG AAG AAA AGA-.CG CAC CAT GGA AAG GCT TCG CGC CAA CAA TAC TAC Met Gly Val Ile Lys Lys Lys Arg Ser His His Gly Lys Ala Ser Arg Gln Gln Tyr Tyr

20

TCT GGG GTA CAG GTT GGG GGA GTA GGC AGC ATG GGC GCC ATA AAC AAT AAC ATC CCG TCG Ser Gly Val Gln Val Gly Gly Val Gly Ser MHt Gly Ala Ile Asn Asn Asn Ile Pro Ser

40

121

TTG ACG AGC TTC GCG GAG GAA AAC AAC TAT CAG TAC GGA TAC AGC GGC TCC AGT GCC GGC Leu Thr Ser Phe Ala Glu Glu Asn Asn Tyr Gln Tyr Gly Tyr Ser Gly Ser Ser Ala Gly

60

181

ATG AAT GGC AGA TCG CTT ACG TAC GCG CAG CAA CAG CTT AAT AAG CAA AGA CAG GAC TTC Met Asn Gly Arg Ser Leu Thr Tyr Ala Gln Gln Gln Leu Asn Lys Gln Arg Gln Asp Phe

80

241

GAA CGT GTA CGA CTT AGA CCA GAA CAG CTC AGC AAT ATC ATA CAT GAC GAG AGC GAC ACG Glu Arg Val Arg Leu Arg Pro Glu Gln Leu Ser Asn Ile Ile His Asp Glu Ser Asp Thr

100

301

ATA TCG TTC CGA TCC AAC CTT TTG AAG AAC TTT ATA AGC TCG AAC GAC GCA TTT AAC ATG Ile Ser Phe Arg Ser Asn Leu Leu Lys Asn Phe Ile Ser Ser Asn Asp Ala Phe Asn Met

120

361

CTG AGT TTG ACC ACG GTA CCG TGC GAC AGA ATT GAG AAG TCC AGA TTG TTC AGT GAA AAA Leu Ser Leu Thr Thr Val Pro Cys Asp Arg Ile Glu Lys Ser Arg Leu Phe Ser Glu Lys

140

421

ACA ATA AGA TAT CTC ATG CAA AAA CAA CAC GAA ATG AAA ACA CAA GCG GCA GAG TTG CAA Thr Ile Arg Tyr Leu Met Gln Lys Gln His Glu Met Lys Thr Gln Ala Ala Glu Leu Gln

160

481

GAA AAG CCT CTG ACG CCA CTT AAA TAC ACA AAA CTT ATA GCT GCG GCA GAG GAC GGA AGC Glu Lys Pro Leu Thr Pro Leu Lys Tyr Thr Lys Leu Ile Ala Ala Ala Glu Asp Gly Ser

180

541

CGT AGC ACA AAG GAT ATG ATA GAT GCT GTC TTC GAG CAA GAT AGT CAT TTG AGG TAC CAG Arg Ser Thr Lys Asp Met Ile Asp Ala Val Phe Glu Gln Asp Ser His Leu Arg Tyr Gln

200

601

CCG GAC GGC GTG GTC GTA CAT CGT GAC GAT CCT GCG CTG GTG GGT AAA CTC CGC GGA GAT Pro Asp Gly Val Val Val His Arg Asp Asp Pro Ala Leu Val Gly Lys Leu Arg Gly Asp

220

661

CTC CGC GAA GCG CCG GCG GAC TAC TGG ACG CAT GCT TAT AGG GAT GTT TTG GCG CAA TAC Leu Arg Glu Ala Pro Ala Asp Tyr Trp Thr His Ala Tyr Arg Asp Val Leu Ala Gln Tyr

240

721

CAC GAG GCC AAG GAG CGT ATC AGG CAG AAG GAA GTA ACT GCA GGT GAA GCA CAG GAC GAA His Glu Ala Lys Glu Arg Ile Arg Gln Lys Glu Val Thr Ala Gly Glu Ala Gln Asp Glu

260

781

GCC AGC TTG CAG CAG CAA CAG CAG CAA GAT TTG CAG CAA CAG CAA CAA GTA GTG ACT ACA Ala Ser Leu Gln Gln Gln Gln Gln Gln Asp Leu Gln Gln Gln Gln Gln Val Val Thr Thr

280

841

GTT GCC TCG CAA AGT CCT CAT GCA ACT GCA ACG GAA AAG GAG CCA GTA CCC GCC GTG GTT Val Ala Ser Gln Ser Pro His Ala Thr Ala Thr Glu Lys Glu Pro Val Pro Ala Val Val

300

901

GAC GAC CCA CTG GAG AAC ATG TTC GGA GAT TAT TCC AAT GAG CCG TTC AAC ACC AAT TTC Asp Asp Pro Leu Glu Asn Met Phe Gly Asp Tyr Ser Asn Glu Pro Phe Asn Thr Asn Phe

320

961

GAC GAT GAA TTT GGA GAT CTT GAT GCT GTA TTT TTT TAA TAGCCATGGGCGGCTACTTCTTGGAAA Asp Asp Glu Phe Gly Asp Leu Asp Ala Val Phe Phe *

332

61

Y

V

1027

ORF2 _ TATATACTTTCCTTTTACATATTTAGTATAGTTATA

1107

TTTTCTGTGTATTCCTTGTGACATAGTGGATTTGTGCAGCTACTGCTTATAGGAAGCAGTGCCTGCAAAACAATTCATTC

~TGGATGTGTATGGAGGTAAGATCAGATAGACCCTTCACTG

1187 GTTGGTTGATGTGGCGCGCTGACTGAATGTGTAGGGTCTGTTGTTACCCCCTTGCTCCGGGG FIG. 3. Nucleotide sequence of SNF6 gene and predicted amino acid sequence. Nucleotides are numbered on the left, and amino acids are numbered on the right. Positions of major clusters of 5' ends of the SNF6 RNAs are underlined. The second Met codon is in boldface type. Asterisks indicate termination codons. The first termination codon in the +2 reading frame following the marked NcoI site is indicated by a dashed underline; this NcoI site was used to construct the +2 frameshift mutation in pSNF6(254)fs-lacZ. Arrowheads mark the positions of the snJ6-84 and or42-184 mutations. Initiation and termination codons for ORF2 on the opposite strand are boxed.

VOL. 10, 1990

YEAST SNF6 GENE ENCODES A NUCLEAR PROTEIN M Y N Y T K Y V K G K Y I F P R S S R P W L L K K Y S I K I S K F I V E I G V E

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R L I G I I S E H V L Q W V V N H G G Y W L L F R C S C M R T L R G N C S H Y L

80

L L L L Q I L L L L L L Q A G F V L C F T C S Y F L L PD T L LG L V V L R Q N

120

I P I S M R P V V R R R F A E I S A E F T H Q R R I V TM Y D HA V R L V P Q M

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T I L L E D S I Y H I L C A T A S V L C R S Y K F C V FK W R QR L F L Q L C R 200 L C F H F V L F L H E I S Y C F F T E Q S G L L N S V AR Y R GQ T Q H V K C V

240

V R A Y K V L Q K V G S E R Y R V A L V M Y D I A E L FW S K SY T F E V L S L

280

L I K L L L R V R K R S A I H A G T G A A V S V L I V VF L R EA R Q R R D V I

320

V Y G A H A A Y S P N L Y P R V V L L A R S L S M V R SF L L DD T H Y F T T F

360

V K F S

364

FIG. 4. Predicted amino acid sequence of ORF2. Amino acids are indicated by the standard single-letter code.

codon TGA. This mutation was neutral with respect to ORF2 as it simply altered codon 272 of ORF2 from one serine codon to another. The other base substitution, designated orJ2-184, changed codon 184 of ORF2 to the termination codon TAG but did not affect the coding capability of SNF6. Plasmids carrying each mutant allele cloned in a centromere-containing vector were used to transform snf6A2 strains of both mating types. The plasmid-borne snf6-84 allele failed to complement the chromosomal snf6-A2 deletion for growth on raffinose, galactose, or glycerol and also failed to complement for ability to sporulate in a snf6A21snf6-A2 diploid. In contrast, the orj2-184 allele provided wild-type function with respect to all Snf phenotypes tested. Thus, the ORF that was tentatively identified as SNF6 does in fact correspond to the SNF6 gene, and it is the loss of this coding sequence that is responsible for all the mutant phenotypes associated with deletion of the locus. Mutation of ORF2 confers no phenotype that we detected. Nudear localization of SNF&6-galactosidase fusion proteins. If the SNF6 protein affects transcriptional processes by a fairly direct mechanism, then the protein might be expected to reside in the nucleus. To facilitate its subcellular localization, we tagged the SNF6 protein with ,B-galactosidase. We joined codon 254 of SNF6 to the E. coli lacZ gene, generating a gene fusion designated SNF6(254)-lacZ (Fig. 5). This fusion encodes a bifunctional SNF6-p-galactosidase fusion protein that complements a snf6 mutation in yeast TABLE 2. Invertase activity in snf6 mutantsa Invertase activity"

Relevant genotype

Wild type

snj6-719

snJ6-l:: URA3

snj6-Al

snf6-A2

snJ6-A2(pEY81)

snJ6-A2(pEY111) sn.f6-A2(pEYlllfs) snJ6-A2(pSNF6(254)-lacZ) snJ5-A2(pSNF6(254)fs-lacZ)

Repressed

Derepressed