Role of GCR2 in Transcriptional Activation of Yeast Glycolytic Genes

MOLECULAR AND CELLULAR BIOLOGY, Sept. 1992, p. 3834-3842 0270-7306/92/093834-09$02.00/0 Copyright © 1992, American Society for Microbiology

Vol. 12, No. 9

Role of GCR2 in Transcriptional Activation of Yeast Glycolytic Genes HIROSHI UEMURA* AND YOSHIFUMI JIGAMI Division ofBiological Chemistry, National Chemical Laboratory for Industry, Tsukuba Research Center

(MITI), Tsukuba, Ibarak 305, Japan Received 11 December 1991/Returned for modification 19 February 1992/Accepted 8 June 1992

The Saccharomyces cerevisiae GCR2 gene affects expression of most of the glycolytic genes. We report the nucleotide sequence of GCR2, which can potentially encode a 58,061-Da protein. There is a small cluster of asparagines near the center and a C-terminal region that would be highly charged but overall neutral. Fairly homologous regions were found between Gcr2 and Gcrl proteins. To test potential interactions, the genetic method of S. Fields and 0. Song (Nature [London] 340:245-246, 1989), which uses protein fusions of candidate gene products with, respectively, the N-terminal DNA-binding domain of Gal4 and the C-terminal activation domain II, assessing restoration of Gal4 function, was used. In a Agal4 Agal80 strain, double transformation by plasmids containing, respectively, a Gal4 (transcription-activating region)/Gcrl fusion and a Gal4 (DNA-binding domain)/Gcr2 fusion activated acZ expression from an integrated GAL1/lacZ fusion, indicating reconstitution of functional Gal4 through the interaction of Gcrl and Gcr2 proteins. The Gal4 (transcriptionactivating region)/Gcrl fusion protein alone complemented the defects of both gcrl and gcr2 strains. Furthermore, a Rapl/Gcr2 fusion protein partially complemented the defects of gcrl strains. These results suggest that Gcr2 has transcriptional activation activity and that the GCRI and GCR2 gene products function together.

The glycolytic pathway is a major metabolic route in Saccharomyces cerevisiae, and most of the genes are highly expressed (16, 19). Their 5' regions often have a Rapl (37)-binding site (Rapl is also known as GRF1 [6] or TUF [231), and in some cases mutational analysis shows Rapl binding to be essential for expression (7, 8, 10, 32). CrTCC motifs have also been implicated in expression (7, 9), and some of the genes also have ABF1-binding sites (5, 8). However, neither Rapl-binding sites (23, 29, 37) nor ABF1binding sites (6) are restricted to glycolytic genes, and any special mechanism for glycolytic gene expression likely involves other elements. One such element is Gcrl. gcrl mutants (1, 12, 13, 27) are severely reduced in expression of most glycolytic enzymes at the transcriptional level. The Gcrl protein has been proposed as a transcriptional activator (1, 22), and very recently Baker indicated that it binds to the sequence containing a ClTCC motif (2). Recently we reported a new regulatory locus, GCR2 (38). The levels of reduction of most glycolytic enzymes in gcr2 mutants are very similar to those in gcrl mutants, but the growth defect is less. In this report, we present the GCR2 sequence and also present genetic evidence indicating that there is physical interaction of Gcr2 and Gcrl and that normal Gcr2 action likely contributes an activation domain to a Gcrl/Gcr2 complex.

transformed by the method of Ito et al. (24). Growth on different carbon sources was scored by measuring average colony size under a microscope. Media. E. coli cells were grown in LB (14). Yeast cells were grown in rich medium (41) and synthetic complete medium (SC) (36) or SC dropout medium, depending on the selective pressure for plasmids; 2% glucose or 2% glycerol plus 2% lactate was added as indicated. When necessary, the respiration inhibitor antimycin A was added to a final concentration of 1 ,ug/ml. DNA manipulation. Standard techniques for DNA manipulation used in this study are described by Sambrook et al. (35). S. cerevisiae chromosomal DNA was prepared by the method of Sherman et al. (36). Isolation of plasmid DNA from yeast cells was done by the method of Hoffman and Winston (21). Plasmid constructions. Cloned GCR2 DNA was derived from pGCR2 (38), a derivative of YCp5O. pML16-2, pML17-1, pML18-1, and pML19-1 are dropout plasmids of pGCR2 with deletions of EcoRI, HindIII, BamHI, and SalI-SacI fragments, respectively (Fig. lb) (38). YEp351 (20), a multicopy plasmid with a LEU2 selection marker, was used to construct a yeast genomic DNA library by cloning partially Sau3A-digested chromosomal DNA (average length of 10 to 20 kb) to its BamHI site. PD206-1 and PD210-1 (Fig. lb) were obtained from this DNA library. pL133-2 contains GCR2 on YEp351, constructed by cloning a SalI-XhoI fragment of pGCR2 into the SailI site of YEp351. pCL1 is a YCp5O derivative containing the ADHI promoter (PADl)-GAL4(1-881) gene (15). pCTC13 is a multicopy plasmid with a LEU2 marker, kindly provided by Stanley Fields (State University of New York at Stony Brook). It encodes a fusion of a nuclear localization signal and Gal4(768-881) plus additional residues encoded by a BamHI linker sequence under the control of the ADHI promoter and terminator. To construct pML77-8, the SacI-

MATERIALS AND METHODS Strains and genetic methods. The S. cerevisiae strains used are listed in Table 1. Escherichia coli DH5a [F- endAl4 hsdR17 supE44 thi-1 recAl gyrA96 relAl A(lacU169 +80 dlacAAM15)] was used to propagate all plasmids (18). Matings, diploid selection, sporulation, and dissection were carried out by the usual methods (31). Yeast cells were *

Corresponding author. 3834

YEAST GCR2 PROTEIN AS A TRANSCRIPTIONAL ACTIVATOR

VOL. 12, 1992

3835

TABLE 1. S. cerevisiae strains Strain

Genotype

Comment or reference

2845 YHU2012 MWGL29 NW9-19-1 DFY643 YHU3002-8A YHU3002-8B YHU3002-8C YHU3002-8D GGY1::171 DFY644

a leu2-3 leu2-112 ura3-52 his6 a leu2-3 leu2-112 ura3-52 a gcrl-6 leu2-3 leu2-112 ura3-52 his6 a gcr2-1 leu2-3 leu2-112 ura3-52 his6 a Agcr2::URA3 leu2-3 leu2-112 ura3-52 his6 a Agcr2::URA3-big leu2-3 leu2-112 ura3-52 his6 a leu2-3 leu2-112 ura3-52 GCR2 a Agcr2::URA3-big leu2-3 leu2-112 ura3-52 his6 a leu2-3 leu2-112 ura3-52 GCR2 a Agal4 Agal80 leu2 his3 a Agcrl::LEU2 leu2-3 leu2-112 ura3-52 his3

38 Isogenic with 2845,a (38) gcrl mutant of 2845 (38) gcr2 mutant of 2845 (38) Agcr2 mutant of 2845 (38) See text See text See text See text GALI-lacZ fusion gene integrated at the URA3 locus (17) 38

a YHU2012 was constructed from a His' revertant of 2845 by converting the mating type through transformation with a multicopy plasmid carrying the HO gene, curing of the plasmid from the diploid, and segregation.

XhoI fragment of pGCR8, which contains residues 68 to 844 of Gcrl, was blunted with T4 DNA polymerase and ligated to pCTC13 that had been digested with BamHI and treated with Klenow fragment. This construction yielded an inframe fusion between codon 881 of Gal4 and codon 68 of Gcrl separated by five codons contributed by linker sequence. pMA424 (30) is a multicopy plasmid with HIS3 and encodes Gal4(1-147), plus additional residues encoded by a polylinker sequence, under the control of the ADH1 promoter and terminator. pMA424E was constructed by digesting pMA424 with EcoRI, filling in the 5' protruding end with Klenow fragment, and religation. Gal4(1-147)/Gcrl fusion plasmid pML78-2 was constructed as follows. The same SacI-XhoI fragment of pGCR8 used to construct pML77-8 was blunted with T4 DNA polymerase and ligated to pMA424E that had been digested with BamHI and treated with Klenow fragment. This construction yielded an inframe fusion between codon 147 of Gal4 and codon 68 of Gcrl separated by seven codons contributed by the polylinker sequence. pL83-11 is a LEU2 version of pML78-2, made by digestion of pML78-2 with BglII to delete a part of HIS3 and replacement with the LEU2-containing BglII fragment of YEpl3. Two kinds of Gal4(1-147)/Gcr2 fusion plasmids were constructed. For pL41-14, the HpaI fragment of pGCR2, which contains residues 48 to 534 of Gcr2, was ligated to pMA424 that had been digested with BamHI and treated with Klenow fragment. This construction yields an in-frame fusion between codon 147 of Gal4 and codon 48 of Gcr2 separated by six codons contributed by the polylinker sequence. To construct pL46-1, the SalI-XhoI fragment of PD206-1 (the Sall site being derived from the polylinker sequence of YEp351), which contains residues 223 to 534 of Gcr2, was ligated to pMA424E that had been digested with SalI. This construction yielded an in-frame fusion between codon 147 of Gal4 and codon 223 of Gcr2 separated by 12 codons contributed by the linker sequence (Fig. ld). For GCRI and GCR2 under PENOz control, first a multicopy plasmid with the ENOI promoter plus initiation codon, pML53-1, was constructed as follows. YEp352 (20), a multicopy plasmid with a URA3 selection marker, was digested with EcoRI plus BamHI, and the EcoRI-BamHI fragment of pMC1403-ENO-C (39), which contains 724 bp of ENO1 5' noncoding region and a BamHI restriction site just downstream of ATG, was ligated. To express GCRI, pL58-11 was constructed. The same SacI-XhoI fragment of pGCR8 was blunted by T4 DNA polymerase and ligated into pML53-1 that had been digested with BamHI and treated with Klenow

fragment. This construction made the 68th codon of Gcrl the 6th codon of truncated Gcrl as a result of the linker sequence (Fig. ld). GCR2 was expressed from pL45-3. The SacI-XhoI fragment of PD206-1 was ligated into pML53-1 which had been digested with SalI. This construction made 724 bp of 5' ENOI noncoding region plus ATG fused in frame to codon 223 of Gcr2 separated by 10 codons contributed by the polylinker sequence (Fig. lb). pL88-1 contains RAPI on YEp352, constructed by cloning an EcoRI fragment of D56 into the EcoRI site of YEp352. D56 is a pUC19 derivative containing RA4PJ, and it was kindly provided by David Shore (Columbia University). Rapl/Gcr2 fusion plasmid pL87-6 was constructed as follows. First, a BglII fragment of D56, which contains the 5' noncoding region and residues of 1 to 701 of Rapl, was ligated into the BamHI site of YEp352. Then it was partially digested with SacI, and the Sacl fragment of pML41-1, a plasmid containing GCR2 on YEp352 (38), which contains residues 118 to 534 of Gcr2, was ligated. This construction yielded an in-frame fusion between codon 701 of Rapl and codon 118 of Gcr2 separated by four codons contributed by linker sequence. Nucleotide sequence analysis. Restriction fragments were cloned into Ml3mpl8 and M13mpl9 (33), and the sequence was determined by the dideoxy-chain termination method, using an Applied Biosystems model 373A DNA sequencer (Applied Biosystems, Foster City, Calif.) with Sequenase (U.S. Biochemical Corp., Cleveland, Ohio) and the 18nucleotide sequencing primer -21M13 primer (Applied Biosystems). Enzyme assays. For ,B-galactosidase assays, transformants were grown to mid-log phase in synthetic dropout medium (36) containing 2% each galactose, ethanol, and glycerol, and ,-galactosidase activity was assayed as described previously (40). For glycolytic enzyme assays, transformants were grown to mid-log phase in synthetic dropout medium containing 2% each glycerol and lactate. Cells extracts were prepared by vortexing cells with glass beads for 2 min (four times for 30 s each time) at 4°C and assayed as described previously (38). Nucleotide sequence accession number. The sequence shown in Fig. 2 has been given GenBank/EMBLVDDBJ accession number D10104. RESULTS Sequence of the GCR2 gene. Dropout plasmid pML16-2 complemented gcr2, but pML17-1, pML18-1, and pML19-1

3836

UEMURA AND JIGAMI (a)

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FIG. 1. DNA sequencing strategy and plasmids. Plasmids are described in the text. (a) DNA sequencing strategy. Open box and thin line indicate cloned yeast DNA and YCp5O DNA, respectively. The open arrow above the map denotes the long open reading frame proposed to encode the GCR2 gene product. Dotted and hatched areas indicate the asparagine-rich region and the region containing 38% of charged residues. The arrows below the map represent the direction and extent of sequences obtained from a given M13 clone. (b) Deletion and complementation. Deleted regions in dropout plasmids are indicated by blanks. Complementation of a gcr2 mutation was judged by the growth of transformants of NW9-19-1 on glucose-plus-antimycin A plates at 37°C. (c) Allele designations of chromosomal gcr2 mutations constructed with plasmids. The URA3 replacement Agcr2::URA3 was described previously (38); Agcr2::URA3-big was constructed analogously but by replacing the SacI-to-BamHI region of GCR2 with the URA3 gene and integrating the HpaI-EcoRI fragment into the genome of diploid strain at the GCR2 locus, followed by dissection. (d) Fused GCR2 genes. (e) Truncated and fused GCR1 genes. Coding regions of Gcrl (residues 68 to 844) are indicated by dotted bars. Restriction sites: B, BamHI; Bg, BglII; C, ClaI; E, EcoRI; H, HindlIl; Hp, HpaI; K, KpnI; N, NcoI; P, PstI; Sc, Sacl; S, Sall; Sp, SphI; Ss, SstI; Xh, XhoI. Restriction sites in parentheses are not conserved.

did not (Fig. lb); thus, GCR2 likely was in the ca. 2.9-kb EcoRI-SalI fragment. The nucleotide sequence of this fragment was determined. The sequencing strategy is shown in Fig. la. A single open reading frame containing 534 codons was identified. The size and location of this open reading frame accorded well with the results of a complementation test. Figure 2 shows the nucleotide sequence and the amino acid sequence of the predicted 58,061-Da protein. The nucleotide sequence was compared with the sequences in GenBank, but no significant homology to other gene sequences was found. Comparison of the deduced amino acid sequence of Gcr2 with other sequences in EMBL protein data base showed no significant homology with any catalogued sequences. The codon usage of GCR2 is not biased (codon bias index [3] of -0.036), which suggests that Gcr2 is a regulatory protein or is expressed at a low level. We cite four features of the amino acid sequence. First, there is a cluster of 15 asparagines (52%) between amino acids 255 and 283. Second, the C-terminal region is highly charged, the segment from amino acids 474 to 534 containing

12 acidic residues and 11 basic residues (38%). According to the rules of Chou and Fasman (11), this sequence might be an ot helix. Third, a possible nuclear localization signal was found at codons 281 to 288. Figure 3 shows a comparison of this sequence with some known nuclear localization sequences. Fourth, the most striking feature of the predicted Gcr2 protein is the considerable stretch of sequence similarity, based on the method of Lipman and Pearson (28), with the Gcrl protein (Fig. 4). Between residues 250 and 483 of Gcr2, 17% identical (40 residues) and 52% conservative replacement (122 residues) were obtained when several gaps were introduced into the sequence. Within this region, residues 329 to 347 and 393 to 422 of Gcr2 had 58 and 37% identity, respectively, with stretches of Gcrl. Neither gene in multicopy suppressed a mutant of the other gene (38), arguing against similar functions. Rather, the protein similarity suggests the possibility that they function together (as discussed below). Deletion of most of the GCR2 coding sequence is not lethal. Previously, the chromosomal GCR2 locus was disrupted by

-300 -240 -160 -80

GATCAATTTTATGAATGTCTTTCAATGGAATCTCAATACGGGTAATAACGTCATATTTCT

TATTTTTCTTGGACAATTTGTCCAAAATAAAATTTTCACGACTTTTCTCCTTCAAGTACCGCGACTTCTGCGATAGTTGT CCGTTAGACATAATAATCATTAACGCATCCATAAGGTGCTTGCGGTCTGTATTTGACTTTCTTCACATTCTTACGCCTAG ACTGAATGGGAAAACAACCAAGAAACCAAAAAGGAACCTGAGAACACAAAGAGTATTTGACGAAAAGTTACACTCACATA ATG CAT CAC CAA ACT AAG TTA GAT GTA TTC ATA ATC AGA GCT TAT AAT TTA CTG TCT AAC N L S N L A Y I F I R V D L T K N Q H H

20

TTG CAG AGT GTT ACA AAC TCG CCA CAG ACG ACA ACG T T T P Q T N V S Q S L

40

GGG GCG GTT GGA ACA GGG ATA GCT AAT CCA ACA GGG G T P A N G I G T V A G

60

181

TTG ATG GGG TCT GAT AGC ACA CCT AAC ATC GAT GAG ATT ATA ACT AGC ACT GGT AGT AAT N T G S T S I I E D P N I T S S G D M L

80

241

GCT CTG ACG AAA ACC AAC TCA GAT AGC GCT AAT GGT ACG CCG AAT GGT AAT TCA AGT G N P N S S G T A N D S K S N T T L A SacI ACC TCA GCC ATT AGC AAT GCA AGC AAT CCT GCC ACT ACT GGT AAT AAT GCG AGC TCT N A G N S T S T P A N S A S N I S A T

TCT S

100

AGT S

120

GCC AAA ATA TCA S K I A

140

ACA TCA CCA AAA K P T S

160

481

TCG GCA ATC GAA CTA TAT CAA AGA TTT CAA CAG ATG ATT AAG GAA CTA GAG CTG AGT TTT F L S L E K E M I Q Q F Q Y R L E I A S

180

541

GAC GCA AGT CCT TAC GCA AAA TAC TTC CGC CGG TTG GAT GGA AGG CTT TGG CAA ATA AAG K I W Q L G L D R R K F R Y Y S P A D A

200

601

ACA GAC TCA GAA TTA GAA AAC GAT GAA TTG TGG CGA TTA GTC TCA ATG AGC ATA TTT ACA T F S I S M V L W L R D E N E L E S D T

220

661

Sau3A GTA TTC GAT CCT CAG ACC GGC CAA ATT CTA ACT CAA GGA CGC AGG AAG GGA AAC TCC TTA L S G N R K G Q R T I L Q G T V Q P D F

240

721

AAT ACA TCA ACT AAA GGC TCC CCA TCA GAT TTA CAG GGA ATA AAC AAC GGG AAC AAT AAT N N N G N Q G I N L D P S S G K T T S N

260

781

GGG AAC AAT GGT AAT ATT GGA AAT GGG AGT AAT ATT AAG AAC TAT GGA AAT AAA AAC ATG M N G N K N Y I K S N G N I G G N N G N

280

841

CCA AAC AAC CGA ACG AAA AAA AGA GGC ACC AGG GTG GCT AAA AAT GCT AAA AAT GGG AAA K G K N K A V A N T R G R T K K N R N P

300

901

AAC AAT AAA AAT AGT AAT AAA GAG AGA AAC GGC ATT ACA GAT K D T N G I R E S N N N K N ACA ACA ATA AGC AAC CCA GGT ACC AAT ATG CTT TTT GAT CCA D P S N L F P T M N G T I T

ACG AGT GCA TTC AGT AAT F S N A T S

320

TCA TTG TCT CAA CAG TTA L Q Q S S L

340

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CAA AAA CGA CTG CAA ACG CTA TCA CAA GAT GTC AAT TCT CGT TCG TTG ACA GGA TAT TAT Y Y G T L S N S R Q D V Q T S L L K R Q

360

1081

ACA CAG CCA ACC AGT CCT GGC TCA GGA GGA TTT GAA TTT GGT TTG AGT CAT GCA GAT D H A S G L E F G G F S S P G P T Q T Ba-HI AAC CCC AAT GCT TCC AGT AAT ACC ATG GGC TAT AAT ACA ATG TCC AAT AAT GGA TCC S N N G S N Y T M M G S T N S P N A N

1 61

121

301 361 421

961

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GCC ACC TCA AAT GGA ATA TAT ACG CAA GCG CAA TAT TCT CAA CTT TTC F Q L Y S Q A Q T Y G I S N T A Sau3A AAA CTA TAT AAC GCT ACA CTA TCA TCT GGG TCA ATT GAC GAT AGA TCA R S D D S I G S S L A T N Y L K

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CAT H

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1201

TCG TGG AAA CGA AGG TCA CTG GGA TCG TTA GAT GTT AAT ACG CTG GAT GAC GAA GCG GTG V D A D E T L S N K L D V W S S L G R R

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460

1381

AAA GTT GAT TTA AAT CCT TCA AAC S K N P N V D L HindIII CCA TTG AAA GAA GCT TAT GAC GCA Y A D L K E A P

AGC ATG GGA CCT ATA GAT ACA GAA GCC GTG ATA CGC V E I A R D T G P I S M

480

ATC ATT TCT GAA AAA GGC CAA AGA ATT GTG CAA TTA V K I L Q E G R S I I Q

500

1501

GAA AGA GAA TTG GAA TTA CAG CGC CAA GAG ACG CAG TGG TTA AGG AAA ATG TTA ATT GAA W M E K L I L R Q T E L E L R Q R E E Q

520

1561

GAC ATG GGT TGT GTT AGA AGT ATG TTA AGG GAT TTA CAA AGA TGA CACGATAATAATGTTTAACA R D L R L S M R Q D G C V M

534

1626 1706 1786 1866 1946 2026 2106 2186 2266 2346

TATATTGCTTTCTCTTTTAATTTTCTGGTGTAGATTGGCGGTATAATTAACTATTTAACAGTGACGTTTACTATCTTACT GTTTCTTGTACTCTTTTATAATGAACAGACGTTATTATATAACTACGAACTAATGATAATTCTTTTTACGTTATTCCTGA TGTAAAGGATACAAAATGGGTTTCTTTATTTTGCAGCAGAACCGTAAAATTTACGCTACTTTTACCGTGGGTCCTCTTAT TTGATTGTTTCCGTCGCTACGAGCGAACAAGGGGTGGAGACAATAGGGATGGTGAATATATACACATACGTTCATATATA

1141

1441

AGAAAAAAACTCAAGAAGAATATCAAACGACCAGGATCCTTGCTTATATTATTGTCTGTTGCTGGGCCAACACATATTTT CATTTCAACACACCAATCTCAGGTATTAAAGAGAAATCATTGTAAGGTAAAAATGTCTACGTTGAAAGTTGTTTCATCAA AGCTTGCAGCTGAAATCGACAAAGAATTGATGGGTCCTCAAATCGGATTCACTCTACAACAGCTAATGGAGTTAGCTGGG TTTAGTGTCGCGCAGGCTGTATGTCGCCAGTTTCCACTGAGAGGCAAGACAGAAACGGAAAAGGGCAAACATGTATTTGT TATTGCTGGGCCAGGTAACAATGGCGGGGATGGTCTCGTGTGCGCAAGACATTTGAAGCTTTTTGGTTACAACCCTGTTG

TTTTCTACCCCAAGAGAAGCGAGCGCACTGAATTC

FIG. 2. Nucleotide sequence of the GCR2 gene and predicted amino acid sequence. Nucleotides are numbered the left, and amino acids are numbered on the right. The first ATG codon of the open reading frame was assigned the +1 position. Asterisks mark the termination codon. HpaI, SacI, Sau3A, BamHI, and HindIll sites relevant to the construction of plasmids are marked. on

3837

3838

UEMURA AND JIGAMI

SV40 large T

SV40

VP1

124

MOL. CELL. BIOL.

Thr ,Pro Pro LyS LYS LyJa A.r

1

Ala

Pro

LYE Val

TABLE 2. Growth of GCR2 disruptants on plates

T

Growth (colony size [mm]) onb:

Polyoma large T

278

Thr Po ,Pro LyS LY8 Ala Arg Glu Asp

Polyoma large T

188

Pro Val Ser Arg

Gcr2

281

FM Asn Asn Arx Thr Lys LyS Arg Gly

Relevant genotypea

Strain

IaYa Arg Pro Arx Pro

2845 Wild typeC NW9-19-1 gcr2-1 DFY643 Agcr2::URA3 YHU3002-8C Agcr2::URA3-bigc

FIG. 3. Homology with known nuclear localization signals. The Gcr2 sequence from amino acids 281 to 289 is compared with sequences that have been identified as nuclear localization signals in simian virus 40 (SV40) large T antigen (25, 26), simian virus 40 VP1 protein (42), and polyomavirus large T antigen (34). Numbers at the left indicate the position of the first residue shown.

YPD ____ 300C 370C

2.3 1.5 1.5 1.2

2.3 0.4 0.4 0.4

YPGL,

SC (Glu) + anti 37°C

0.8 0.7 0.7 0.6

1.5 0.2