Vol. 11, No. 10

MOLECULAR AND CELLULAR BIOLOGY, Oct. 1991, p. 5101-5112 0270-7306/91/105101-12$02.00/0 Copyright © 1991, American Society for Microbiology

GRRI of Saccharomyces cerevisiae Is Required for Glucose Repression and Encodes a Protein with Leucine-Rich Repeats JEFFREY S. FLICK AND MARK JOHNSTON* Department of Genetics, Box 8232, Washington University Medical School, 4566 Scott Avenue, St. Louis, Missouri 63110 Received 13 May 1991/Accepted 15 July 1991 Growth of the yeast Saccharomyces cerevisiae on glucose leads to repression of transcription of many genes required for alternative carbohydrate metabolism. The GRRI gene appears to be of central importance to the glucose repression mechanism, because mutations in GRRI result in a pleiotropic loss of glucose repression (R. Bailey and A. Woodword, Mol. Gen. Genet. 193:507-512, 1984). We have isolated the GRRI gene and determined that null mutants are viable and display a number of growth defects in addition to the loss of glucose repression. Surprisingly, grrl mutations convert SUC2, normally a glucose-repressed gene, into a glucoseinduced gene. GRRI encodes a protein of 1,151 amino acids that is expressed constitutively at low levels in yeast cells. GRR1 protein contains 12 tandem repeats of a sequence similar to leucine-rich motifs found in other proteins that may mediate protein-protein interactions. Indeed, cell fractionation studies are consistent with this view, suggesting that GRR1 protein is tightly associated with a particulate protein fraction in yeast extracts. The combined genetic and molecular data are consistent with the idea that GRR1 protein is a primary response element in the glucose repression pathway and is required for the generation or interpretation of the signal that induces glucose repression.

galactose in the presence of 2-deoxyglucose, a gratuitous inducer of glucose repression (2). Because grrl mutations substantially relieve repression of several glucose-repressed genes, GRRI appears to be of central importance to the glucose repression mechanism. To analyze the role of the GRRI gene in glucose repression, we have isolated new grrl mutants and used these to isolate the gene by complementation. We report here the results of our analysis of GRRI.

In the yeast Saccharomyces cerevisiae, growth on glucose results in the repression of transcription of a large number of genes necessary for alternative carbohydrate metabolism and respiration (for recent reviews, see references 8, 18, 22, and 31). This global regulatory pathway, termed glucose repression, adjusts the physiology of the cell to allow it to utilize selectively glucose, the most efficiently metabolized energy source, while growing in the presence of multiple carbon sources. The mechanisms by which growth on glucose leads to repression of gene expression are poorly understood. Genetic analysis has suggested that glucose repression operates through a branched pathway that involves several genes. For example, repression of the GAL] gene is mediated by at least two mechanisms that can function independently (21). First, glucose repression acts through negative regulatory sites (URSgal) found upstream of the GAL] TATA box. Second, growth on glucose reduces the synthesis of the GAL4 activator protein required for expression of GAL] (23a). Both pathways of GAL] repression, as well as the mechanism of repression acting on the SUC2, MAL, and CYCI genes, are inactivated by mutations in either the GRRI or REGI (HEX2) gene (2, 21, 21a, 42, 45). By contrast, mutations in URRI relieve only the repression operating through the GAL] URS; ga182 mutations appear to relieve only the repression of GAL4 expression (21). These findings suggest that GRRI and REG] act early in the pathway and may be involved in producing the signal for glucose repression whereas URRI and GAL82 may act more directly upon these genes to confer repression. Analysis of the genes that are required for repression of many glucose-repressed genes seems essential for understanding the mechanism(s) by which growth on glucose signals repression. grrl mutants pleiotropically defective in glucose repression were identified by the ability to grow on *

MATERIALS AND METHODS Yeast strains, media, and growth conditions. The yeast strains used are listed in Table 1. Strain construction followed standard methods for genetic crosses, sporulation, and tetrad dissection (52). Standard yeast growth medium was used (52); 5-fluoro-orotic acid (5-FOA) plates for selection of Ura- segregants were prepared as previously described (5). Carbon source utilization was determined on YP medium that contained 2% glucose (YPGlu), 2% galactose (YPGal), 2% raffinose (YPRaf), or 5% glycerol (YPGly). YPGlu medium containing 2.0 M ethylene glycol was used for the testing of osmotic sensitivity (60). Sensitivity to nitrogen starvation was assayed as described previously (64) by replica plating yeast cells from a fresh YPGal master plate (incubated overnight at 30°C) to SD medium lacking (NH4)2 S04 and containing 2% glucose and the appropriate amino acid supplements. This plate was incubated at 30°C for 7 days and then printed to a YPGlu plate to determine cell viability. The YPGlu plate was incubated for 48 h and photographed. Plates used to score expression of a GALIlacZ fusion gene contained 2% glucose, 2% galactose, and 40 jig of 5-bromo-4-chloro-3-indolyl-f3-D-galactoside (X-Gal) per ml in a buffered medium as described previously (50). Yeast RNA and cell extracts were prepared from cells grown on SD medium containing either 2% glucose, 2% galactose, or 5% glycerol plus 0.1% glucose as previously described (21). Plasmids were transformed into yeast cells treated with lithium acetate (29).

Corresponding author. 5101

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FLICK AND JOHNSTON

TABLE 1. Yeast strains Strain

Genotype'

YM923 YM1871 YM2061 YM2920 YM2953 YM2996 YM3135 YM3454 YM3459

YM2915

MATa met LEU2::pRY181 [pBM261] MATa grrl-1121 LEU2::pRY181 MATa met LEU2::pRY181 MATa URA3::pRY171 leu2-3,2-112 grrl: :LEU2 MATa leu2-3,2-112 grrl: :LEU2 MATa leu2-3,2-112 sup4-o grrl::LEU2 MATTa leu2-3,2-112 cdcll-6 MATa met LEU2::pRY181 [pBM2081] MATa ADE2 LYS2 HIS3::GALI/FLP trpl leu2-3,112 prbl-1122 prcl407 pep4-3 [pBM2083] [cir°] MATa met grrl:: URA3 LEU2::pR Y181 MATa ADE2 LYS2 HIS3::GALJ/FLP trpl leu2-3,2-112 prbl -1122 prcl-407 pep4-3 [pBM2081] [cir+] MATa/MATa URA3::pRY171/URA3::pRY171 leu2-

YM3774

MA Ta/MA Ta metlmet GRRJ/grrl::URA3

YM3775

MA Ta/MA Ta trpl/TRPI 1eu2-3,2-11211eu2-3,2-112 gaI80-5341gaI80-534 snfl-31SNFJ GRR1/grrl::LEU2 MA Ta/MA Ta TRPI/trpl-901 grrl:: URA31GRRI LEU2::pRY181/LEU2: :pRY181

YM3492 YM3773

3,2-1121leu2-3,2-112 LEU2::pRY181/LEU2::pRY181 YM3843

a All strains also carry ura3-52 his3-200 ade2-101Je lys2-801 unless indicated otherwise. Brackets denote plasmid carrier state. Plasmids pRY171 and pRY181 (GALI-lacZ) are described in reference 67; plasmid pBM261 (GALIHIS3) is described in reference 32; plasmids pBM2081 and pBM2083 (GRRI) are described in Materials and Methods.

Isolation of grrl mutants. Spontaneous mutations conferring resistance to glucose repression were isolated in YM923 (his3-200), which contains plasmids pBM261 (GALJ-HIS3 fusion) (32) and pRY181 (GALJ-lacZ fusion) (67). YM923 cells are His- and do not synthesize ,-galactosidase when growing on 2% glucose as a result of repression of the GAL] promoter. Liquid cultures (0.1 ml) of YM923 were spread on glucose- and galactose-containing (Glu + Gal) X-Gal plates lacking histidine. After 5 days of incubation at 30°C, a single His+ blue colony from each plate was purified and its phenotype was retested. Seven of fifteen recessive mutants that expressed GAL] at high levels during growth on 2% galactose-2% glucose medium comprised one complementation group (GALl expression was determined by an assay of GALI-lacZ activity as described previously) (67). All mutants of this group displayed an aberrant sausage shape similar to that previously described for grrl mutants (2). Allelism to grrl was confirmed by the failure of a grrl mutant (DGX-3A, provided by R. Bailey) to complement the glucose repression defect in YM1871, a representative strain of this group. Isolation of GRRI clone. YM1871 (grrl-1121) was transformed to Ura+ with a YCp5O-based plasmid library (51) and then replica plated to Glu + Gal X-Gal medium lacking uracil. After 3 days of incubation at 30°C, white colonies were selected as possible GRRJ transformants. GALI-lacZ expression in the transformants was assayed after growth in 2% galactose-2% glucose medium. The dependence of glucose repression on the plasmid was determined by scoring the color on Glu + Gal X-Gal plates of colonies selected on 5-FOA medium (5). Plasmids were recovered from yeast cells into bacteria as described previously (27). Subcloning and plasmid construction. Standard procedures for the manipulation of plasmid DNA and transformation into bacteria were followed (55). Restriction enzyme fragments of pBM1624 were purified after gel electrophoresis and ligated into a YCpSO plasmid (51) that had been cut with

MOL. CELL. BIOL.

the appropriate enzyme. Frameshift mutations (4-bp insertions or deletions) were constructed in plasmid pBM1624 by partial digestion with either HindlIl, Apal, EcoRI, or BglII and treatment with either Escherichia coli DNA polymerase (Klenow enzyme) or T4 DNA polymerase. These bluntended molecules were self-ligated at a low plasmid concentration for 18 h and transformed into bacteria. The frameshift mutations were confirmed first by the loss of a single restriction enzyme cleavage site within the plasmid and then by DNA sequencing. High-copy-number plasmids pBM2081 and pBM2083 contained the 8-kb BamHI fragment of pBM1624 cloned into the BamHI site of YEp24 (7) and pIAl (obtained from P. Hieter), respectively. The copy number of plasmid pIAl is inducible to high levels by galactose (26a). Null mutations in GRRl. Deletions of GRRI were generated by the one-step gene disruption method of Rothstein (53). The LEU2 gene was substituted for amino acids 601 to 1151 of a plasmid copy of GRRI by replacing a 1.8-kb SnaBI fragment with the 2.2-kb SalI-to-XhoI fragment from YEp13 containing LEU2 (7). The grrl::LEU2 disruption is contained on the 4-kb BglII fragment cloned into the BamHI site of pT7-3 (obtained from S. Tabor) and was designated pBM1829. Plasmid pBM1829 was digested with NdeI and SphI prior to transformation of the diploid yeast strain YM2915. The DNA from Leu+ diploid transformants was digested with BglII and SacI and analyzed by Southern blotting. The 32P-labeled 4-kb BgIII fragment from pBM1624 hybridized to a 4.5-kb fragment derived from the grrl: :LEU2 allele in addition to the 2.1- and 1.9-kb fragments of GRRI. A grrl:: URA3 disruption (removing GRRI amino acids 24 to 1151) was constructed by inserting the 3.8-kb BglII-toBamHI fragment (containing URA3) from pNK51 (1) between the Apal and SnaBI sites of the 8-kb BamHI fragment of pBM1624, cloned into the BamHI site of pUC18 to create pBM2101. Plasmid pBM2101 cut with BamHI transformed both haploid and diploid yeast to uracil prototrophy, resulting in the replacement of GRRJ with grrl:: URA3 as determined by Southern blot analysis. As described previously (1), the URA3 gene cloned from pNK51 can be subsequently removed from the disrupted strain. Sequencing. The nucleotide sequence of the 4-kb Bglll fragment partially encoding GRRI was determined with a set of nested deletions by dideoxy sequencing of doublestranded plasmid DNA (12). The BglII fragment was cloned in both orientations into the yeast shuttle vector pUN70 (17), and unidirectional deletions were constructed by the method of Henikoff (26), using a kit obtained from Pharmacia. DNA sequence outside of the BgIII fragment was determined by using oligonucleotide primers. All DNA sequence was determined on both strands except for nucleotides -310 to -315. RNA preparation and Northern (RNA) blot analysis. Total RNA was isolated from yeast cells as described by Elder et al. (16), and mRNA was purified by selection with oligo(dT)cellulose as described previously (35). Poly(A)+ RNA used in primer extension analysis was purified twice with oligo(dT)-cellulose. RNA was detected by Northern blotting as described previously (21). 32P-labeled strand-specific RNA probes (riboprobes) used in Northern blot analysis were synthesized with T7 RNA polymerase by an in vitro runoff transcription reaction as previously described (59) except that [a-32P]CTP (800 Ci/mmol; Amersham) was substituted for radioactive UTP. The template for the GRRJ probe was the 576-bp SnaBI to Avall fragment (GRRI nucleotides +1802 to +2378) cloned in both orientations into the SmaI site of pT7-3. The derived plasmids, pBM1923 (synthesizes

VOL . 1 l, 1991

antisense-strand transcript with the sequence complementary to the mRNA) and pBM1924 (synthesizes sense-strand transcript with the sequence of the mRNA) were cleaved with BamHI prior to RNA synthesis. The template for the SUC2 probe was the 776-bp HindIII-to-BamHI fragment (SUC2 nucleotides +11 to +787) from pSL27 (56) cloned into pT7-3 vector. The derived plasmid, pBM1254, was cleaved with Hindlll prior to RNA synthesis. The template for the LEU2 riboprobe, pBM1117, was described previ-

ously (21).

Primer extension. The transcription initiation sites were mapped by modification of a previously described method (37). A gel-purified oligonucleotide, 5'-TGGTTGTTGTT ATCCTGATCC-3', complementary to nucleotides +3 to +23 of GRRI, was labeled by treatment with T4 kinase in the presence of [-y-32P]ATP (Amersham). Labeled oligonucleotide (0.05 pmol) was combined with 2 p.g of poly(A)+ yeast

RNA in 15 ,u of 100 mM KCI-5 mM Tris (pH 8.3)-2 mM EDTA, denatured at 95°C for 3 min, and then annealed for 3 h at 48°C. Then 15 RI of a reverse transcriptase cocktail (100 mM Tris [pH 8.3], 10 mM MgCl2, 10 mM dithiothreitol, 1 mM each dATP, dTTP, dGTP, and dCTP, 15 U of avian myeloblastosis virus reverse transcriptase [Boehringer Mannheim], 2.5 U of RNasin [Boehringer Mannheim]) was added, and the mixture was incubated at 44°C for 1 h. The extension reaction was then treated with RNase A (50 ,ug/ml) for 10 min at 37°C, extracted with phenol-chloroformisoamyl alcohol (25:24:1), and precipitated with 3 volumes of ethanol. After centrifugation (14,000 x g for 15 min), the pellet was resuspended in 5 ,ul of formamide loading dye and a 2.5-RI sample was subjected to electrophoresis on a 6% polyacrylamide gel containing 7 M urea. The gel was dried and exposed to film with an intensifying screen for 6 days at -700C. Generation of anti-GRRI antibodies. TrpE-GRR1 fusion proteins were produced and purified from E. coli. Plasmid pBM1869 contains the 1,630-bp BclI fragment (encoding GRRJ amino acids 389 to 933) from pBM1624 cloned into the BamHI site of the trpE fusion vector pATH1 (15). E. coli RR1 containing pBM1869 synthesized a fusion protein of 95 kDa upon induction of trpE expression that was purified electrophoretically from an insoluble fraction as described previously (36). Two rabbits with minimal crossreactive preimmune sera to yeast proteins were injected with 200 ,g of the 95-kDa TrpE-GRR1 fusion protein emulsified in Freund's complete adjuvant followed by booster injections of 100 p.g of fusion protein in Freund's incomplete adjuvant at 21, 42, and 63 days after the primary injections (24). Serum was collected 2 weeks after the final injection. The rabbit antisera were provided by East Acres Biologicals (Southbridge, Mass.). Anti-TrpE-GRR1 antibody was titered against a second TrpE-GRR1 fusion protein of 115 kDa (containing GRRJ amino acids 446 to 1151). The 115-kDa fusion protein was expressed from plasmid pBM1876 (2,324bp EcoRV fragment of pBM1624 cloned into pATH2 cut with SmaI) in E. coli and purified electrophoretically from an insoluble fraction. Antibodies were affinity purified from the crude serum of a single rabbit, using the 115-kDa TrpEGRR1 fusion protein fixed to an Immobilon membrane (Millipore) as described previously (40). Yeast extracts. Yeast extracts were prepared essentially as described previously (23, 66) from either YM3459 or YM3773 containing GRRI on a high-copy-number plasmid. The parent yeast strains for both strains are proteasedeficient mutants. Extracts were prepared from YM3459 after growth on uracil-deficient SD medium containing

MOLECULAR ANALYSIS OF GRRI

5103

2% galactose. YM3459 carries GRRJ on a 2pm plasmid (pBM2083) that amplifies to high copy number during growth on galactose as a result of the [cir°] host background and overexpression of the 2,um flip recombinase from a GALIFLP fusion gene integrated at the HIS3 locus (26a). YM3773 carries GRRI on YEp24 (pBM2081) (in a host strain that is [cir+]) and was used to assay the level of GRR1 protein in cells grown on either 2% glucose or 5% glycerol plus 0.1% glucose. Cultures of 100 ml were harvested during late log phase (optical density at 600 nm of 3 to 4), washed once in 10 mM NaN3, resuspended in 2 ml of 1.2 M sorbitol-10 mM MgCI2-2 mM dithiothreitol-50 mM Tris (pH 7.5), and digested with 200 U of zymolyase for 90 min at 30°C. The resulting spheroplasts were washed three times with 2 ml of 1 M sorbitol-50 mM Tris (pH 7.5)-10 mM EDTA-0.5 ,uM phenylmethanesulfonyl fluoride-4 p.M leupeptin-2 ,uM pepstatin-1.5 mM benzamidine, resuspended in 0.4 ml of TEA buffer (0.8 M sorbitol, 1 mM EDTA, 10 mM triethylamine [pH 7.2]) containing protease inhibitors, and then lysed with 20 strokes in a Wheaton glass tissue grinder. The lysed cells were centrifuged at 450 x g for 3 min, and the supernatant, designated S(450g), was recovered. This supernatant was centrifuged at 10,000 x g for 10 min to produce a pellet fraction, designated P(104g), and a supernatant fraction, designated S(104g). A sample of the S(104g) fraction was centrifuged at 100,000 x g for 2 h. The resulting pellet [P(105g)] and supernatant [S(105g)] fractions were recovered. Protein concentrations were determined by using Bradford reagent with bovine serum albumin as the standard (6). Solubilization of GRR1 protein from the P(104g) fraction was performed essentially as described previously (54). The pellet was homogenized in TEA buffer, divided into six aliquots, and centrifuged at 10,000 x g for 10 min. The pellet from each sample was resuspended in TEA buffer (control) or TEA buffer containing either 1 M NaCl, 50 mM Tris (pH 7.5)-10 mM EDTA, 1% Triton X-100, 6 M urea, or 0.2 M Na,C03 (pH 11.0). The mixtures were incubated on ice for 10 min and then centrifuged at 10,000 x g for 10 min. The supernatant was removed, and the pellet was resuspended in an equal volume of TEA buffer. Equivalent samples of the supernatant and pellet fractions were analyzed by immunoblotting. Immunoblotting. For sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, samples were boiled in sample buffer containing 1% SDS and run on 6.5% gels according to Laemmli (39). Protein was electroeluted to an Immobilon membrane (Millipore) for 2 h at 22°C at 10 Volts as described previously (65). Membranes were blocked by incubation at room temperature for 1 h in TTN buffer (50 mM Tris [pH 7.5], 0.1% Tween 20, 150 mM NaCI) containing 5% nonfat powdered milk (3). GRR1 protein was stained in TTN plus 5% nonfat dry milk with affinity-purified anti-GRR1 antibody (0.25 pug/ml) for 1 h at room temperature. The membrane was then washed four times for 5 min in TTN. The membrane was incubated in secondary antibody (goat anti-rabbit immunoglobulin G conjugated to alkaline phosphatase [Tago]) at a dilution of 1:5,000 in TTN for 1 h at room temperature and then washed four times for 10 min in TTN. Color development was performed as described previously (19). Nucleotide sequence accession number. The GenBank accession number of the reported GRRI sequence is M59247. RESULTS Isolation of a GRRI clone. A genomic clone of GRRI was isolated from a plasmid library by complementation of the

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MOL. CELL. BIOL.

FLICK AND JOHNSTON TABLE 2. Complementation of grrl

Relevant Relevant

GALI expressionb

~Plasmid

genotypea

Gal

Gal+Glu

2 511 GRRI YCp5O 506 333 grrl YCp5O 595 4 grrl pBM1624 678 394 grrl::LEU2 none 521 421 grrl:: URA3 none a Yeast strains assayed were GRRI, YM2061; grrl, YM1871; grrl::LEU2, YM2920; and grrl:: URA3, YM3492.

b Units of lacZ expression from a GALJ-lacZ fusion plasmid, pRY181, assayed as described previously (67) in yeast cells grown on SD medium containing either 2% galactose (Gal) or 2% galactose plus 2% glucose (Gal+Glu).

glucose repression defect in a grrl mutant (see Materials and Methods). In one transformant, repression of GAL] gene expression was completely restored during growth on Glu + Gal medium (Table 2). In addition, the aberrant cell morphology of the parent grrl yeast strain (see below) was restored to wild type in this same transformant. The plasmid (designated pBM1624) was rescued from yeast cells into bacteria and upon retransformation into yeast cells was able to complement the grrl mutation. Plasmid pBM1624 was confirmed to carry GRRI by demonstrating that the cloned DNA is able to direct integration of the URA3 gene to the GRRI locus. A 7-kb HindIII fragment from pBM1624, subcloned in the integrating plasmid YIp5 (62), directed integration of this plasmid (pBM1865) (cleaved at the unique SacI site within the insert) into the genome at a site that was tightly linked to the GRRJ locus: no recombinants between grrl and URA3 were detected in 37 tetrads examined from a cross between a strain containing the integrated pBM1865 and a grrl strain.

Localization of GRRI in the cloned DNA. Analysis of pBM1624 showed that it contains approximately 10 kb of inserted yeast DNA (Fig. 1). The GRRJ gene was localized to the 4-kb BglII fragment (pBM1723) by testing various subclones for complementation of grrl yeast cells (Fig. 1). GRRJ was further mapped to the left-hand 3 kb of the BglII fragment by analysis of a series of unidirectional deletions of the BglII fragment in pBM1723. Deletion of as little as 144 bp of DNA from the left-hand end of the BglII fragment (as diagramed in Fig. 1) destroyed complementation activity, whereas deletion of approximately 1 kb from the right-hand end did not affect complementation ability (data not shown). DNA sequence analysis. The DNA sequence of the essential region of the BglII fragment in pBM1723 was determined on both strands (see Materials and Methods). The sequence of this region contains a single long open reading frame (ORF) that terminates with a TAA codon but extends in the 5' direction beyond the left-hand end of the BglII fragment. Continued sequencing upstream of the BglII fragment revealed an extended ORF of 3,453 bp that would encode a protein of 1,151 amino acids (Fig. 2). Confirmation that this ORF encodes GRRI was obtained by a series of frameshift mutations generated at restriction sites in pBM1624 (Fig. 1). Frameshift mutations occurring within the ORF abolished the ability of pBM1624 to complement grrl (ApaI, codon 24; EcoRI, codon 157; BglII, codon 283; EcoRI, codon 548); mutations occurring either 5' or 3' to the ORF retained GRRI activity (Hindlll at -111; EcoRI at approximately +4500). The longest alternative ORF (initiated with a methionine codon) found on either strand extends for only 53 amino acids. It is somewhat surprising that although the 4-kb BglII fragment is missing a considerable portion of the complete ORF, it nevertheless complements the grrl phenotypes when cloned in either orientation in a low-copy-number vector. We believe that this is a result of transcription initiating from within vector sequences

cO)MPLEMENTATION

PLASMID B

pBM 1624 -

+

B

pBM 1679

RV

II

H Sm

H A

IIIA AUG

I

RI

H

I

I

I

B

+

Bg N RV RI Sn

RI

B

I

GRR 1

Sc

Sn RV RI Sp Bg B II I IAIA UAA 1 kb

il

pBM 1720

--I

pBM 1723

+

pBM1715 pBM 1675 pBM 1676 pBM 1829

pBM2 101

URA3

FIG. 1. Restriction map of GRRI plasmid clones. Shown are yeast DNA (thin line) contained in YCp5O vector (filled box) in plasmid pBM1624 and a detailed restriction map for the 8-kb BamHI fragment (pBM1679). The bold line indicates the position of a 3,453-bp ORF. + and symbols above restriction sites indicate the ability of a plasmid with a 4-bp frameshift mutation at that position to complement grrl::LEU2. Indicated on the right is the ability of fragments subcloned in YCp5O to complement grrl, which was scored in transformants of YM1871 (grrl-1121) by assay of GALI-lacZ expression. Restriction sites: A, ApaI; B, BamHI; Bg, BglII; H, Hindlll; N, NdeI; RI, EcoRI; RV, EcoRV; Sc, Sacl; Sm, SmaI; Sn, SnaBI; Sp, SphI. -

-479 -360

-240

-120 HindIII -1

ApaI MND

NN

N

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N

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240 80

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600 200

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720 240

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840 280

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960 320

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BglIII. R Q

S

L

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N

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F L

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TNM

V

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S N

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D IVEK

K Y

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SEK

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W A

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N Y

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1080 360 1200

D

400

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1320 440

TTTCCCAAGTGGGATTCACGGATAAGGACGTTCGATGCGTTTTGTACCTAGGACAATTGCCCGAGTCAGGCTTTATGTCCCAGGAAGGATGAA Q S V D I T G I RD V S D D V F D T L A T Y C P R V Q G F Y V P Q A R N V T

14404 480

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K

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D

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V

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TTCGATTCACTGCGGAATTTCATAGTCCATTCCCCGATGTTGAAAAGAATAAAAATCACAGCAAAC)AATAACATGAATGACGAATTAGTAGAACTATTAGCCAACAAATGCCCTTTGCTT F D S L RHN F I V H S PHN L K R I K IT A N NH NHM DE L VE L L A NK C P L L

1560 520

EcoRI. 'AGGTTAGTTCAG,;CTGAGGGAATTCAGAATAACTCATAATACGAATATTACGGATAAT R L V Q L R EF R I TNH N T N I T D N

1680 560

CTTTTCCAGGAGCTTTCTAAAGTAGTTGACGATATGCCCTCTTTAAGATTGATTGATCTTTCTI'GGATGTGAAAAT,rATTACAGATAAAACTATAGAAAGTATAGTCAATTTAGCCCCTAAA

1800

V

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600

P

TTACGTrAAATGTTrTriTTCTAGGCAAGTGTAGCC~GA vATTACAGATGCAR7TCGTTG'T[TCCAATTATC~GJAAGCTGGGCAAAkARACTTCAAACACLGGTG4CATrTTTTGGGCACCTTGTTTCAATATAACTGAT L

R

N

V

F

L

G

KC

S

R

I

T

D

A

S

L

F

Q

L

K

S

L G

K

N

LQ T

V

H

FG

C

H

FN

ITOD

AACGGGxGTAAGA kGICACTCTTTCATTCATGTACAUvAkGAATACAGTATTGGTGGACCTTTTGCGTGCTGTJrp'ACGAATTTAACC:APiATAGAACTCTTI.11TATGAARCITAGCAGACCTTTACCAAAATTAAAGAGA H G

V

R

A

L

F

H

S

C

T

R

I

Q

Y

V

D

F

A

C

T N

C

L

T

R

T

L

Y

E

LAOD

L

PEKLEKR

1920 640

2040 680

ATTGGCr. cCTTGTC::ARAATGTAC2GCAAATGACTGAOxGAGGGTTTGTTC GAAATATGXGTTTCCTTGCZGA(kc,,GGCCGAAATGAT,rARhCTTTAGAAAGGC ;GGTACATrTTTATCTTACCTTGwTCrAATTTAACAATA I G L V K C T QNX T D E G L L NHX V S L R G R N D T L E R V H L SY C S N LT I

2160 720

TATCCCG3ARTATATIrGrIAGCTTCTAATGTCTTGCCCAU0AGGCTCTCACATMTTGTCTrTTTGACTGCTGTT4rc'CCGTCATTTTTA,NCCGCCCCGATATAJLAACGATGr. 7TATTGCAGGrpcCCTGC.ACCCTCAGACTTT

2280

Y

P

I

Y

E

L

L

S

C

P

R

L

SNH

L

S

L

T

A

P S

V

F L

R

P0D1

T

AGTGAAkAAATCAA,kcOGTCAAATATTCTGCGTATTT,ri'TCAGGGAAAGGTTGGTTCATrAAAACTTCGCCAT,riTATTTAGTAAATrT!TAACGCTGCCGCx S E NHQ R Q I F CV F S G K G V H L R H Y L V N L T SP A

N

Y

C

P

R

AP

SOD

F

760

rGGGACCACATTGGTCGATGTAAATGATGTT V DV NODV G P H

2400 800

TTGACAKA~AA~AT,rAPLTTAGATCCAAGAATTTGATA'ki6TTTAACGGTGAAPLAA~CACTTrGIAAGATGCTCTTJrp'AGGAGAATCATAkARCTGATTTAAATN ICCAAGATrTTCCGCTGCi KAATTATAGCTGCTACAGGA

2520 840

L

T

Y

I

R

S

EN

L

I

F

NHG

L

F

H

Q

G

I

L

NHN

D

F

T

L

E

D

A

R

L

R I

I

TOD

L

N

QOD

S

A

AATTA ATCAATrT TTTGAAC2GAATA(kc, Q

I

N

F

E

R

I

I

A

~GATGAAGTATTC::AFiGTTGGTATCTCJ:AAATACTrT

TTAAATrcCAAATC::ARACGGTCTAAATAACGATT rc L

E

F

I

A

A

T

G

CAATTAGGATGAGCTCGGAG

FTTGATGGC

D

E

V

F

SHWY

L

N

T

FODG

I

R

N

S

S

2640

880

E

GAAGTTrAAACTCARNCCTATTATTGCAAGTAAACAAGJU;ACGTTTTGTGAAikGGATCCAKTTTTAGTGAT'GTG4xGACGATGATCAA kr.rIATTATGTCGTA(6cGCACCTrGGGTGTAAACccCGGGAAATTAACAGTGAA E V L L L V S Q NEK T F C E D P F SOD V D D D Q D Y V V A P G V N RE IN S E

2760 920

ATGTGTrcIATAT'I rGGTTAGAAAATTCCATGAGTTA uLAATGATCATATTTGGATGATMTTCGAGGTGAAT4rc'GTTGCTAGTTTG 3GI.TAAGAGTTCAGII1TTTCAGr.7TTTACTGGTrT-TTTTAC?-CATGAAATG

2880 960

N C

H

I

V

REKF

H

E

L

I

NON

DOD

F

E

V

N

V A

S

L

V

RV

Q

F

Q

F

TG

F

L

L

HE

N

ACTCAP ALAACCTATrAATGCAAATGATTGAATTAAACN:AGACAAATTTGTT17TTAGTAPicCAAAAAACGGTT4rcC.AGGAATCGGGC::ARACATAGATTACG IC RGGGGCTTTTAKAATATGGCGACTTTTATTC T Q T Y NI E L N R I C K T V L V Q Q HI D Q Y Q E S G QEK G L L I WHRL L F

3000 1000

ATTGACCAAAATTCCAATTATGGTGGTTCAGAAGTACN:AAGCTCTCCACCccITTGTTr'lTTGAGACTATArmITTAAAAGATAACnkTAACATTGTTAh LAACCAGARCCAAAGAGAP RCCTATTAATAGCCCACCAA V V I D K F IN K Y L S T V V L R L Y Q LEKD N I T L L T R QR E L L I ANH Q

3120 1040

AGATCAPicGCATGGSAAATAACAATAATGACAATGAO1(.GCCAACCGGAACccGCCAACCAAACATAGTGAAT ri,ATTGTATCGGATrGGCTGGGGCAAACG xGATACi hAAGTAACAATTGGAAACTAACAATGGTAAT

3240 1080

R S

A

D

N ET

W

NHN

NHN

D

N

D

A

N

RHN

A

N

N

I

V

N

I V

S D

A

G

A

N

L

Q

D

T

SN"

N

E

T

R N

Q

NHN

N

HG

N

3360 D

E N

PHN

F

H R

Q

F

G

H R

N

Q

I SP

D

Q

N

R N

H G

L

V

R N

1120

3480 H FIG.

of

a

2.

N

N

H

T

I

D

E

S

N

DNA sequence of the GRRJ

line is numbered

on

the

P

D

T

A

gene and

right (+ 1 is assigned

I

D

S

predicted to the

Q

N

D

E

A

S

G

T

P

D

amino acid sequence of the

E

D

N

L

1151

protein product. The last nucleotide

first base of the initiation codon). An asterisk above

start site. The restriction sites that were used to construct frameshift mutations are labeled.

5105

a

or

amino acid

nucleotide indicates

an

RNA

1 2

409 Y 435 V 461 L 487 F 513 L 549 L 568 V 594 I 620 L 646 671 700 i

MOL. CELL. BIOL.

FLICK AND JOHNSTON

5106

F

L A

I A

L V

UVS F

EZLL R

L hf

3

4

5 6

V G C R G C T Y C V S NKR T R L D D M N L A K L G H S C A D L G R N -

-

K K P P P V

7

8

L

Q S K

K N T R K D T

C P

-

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

T D Y K D R D F H D G H

E R Q S Q G K R V E R E R L R N Q T Q Y KR E R

N F R N

L

-

-

V

-

V F T G I P Q A T A N T L S T H N SGC GK C G H C A C C VK C S Y C L

-

-

C

PD

ITIDKNT

i

-

N I T D

-

-

L

-

-

serse sarand

aarti-sense strand

probe,

434 S A 460 D T 486 R N 512 E L 548 LK Q E L S K 567 593 E S F Q 619 R A 645 Y 670 L N M V S 699 Y E 726

H TS V P V R D I S F D S R N I N N iNDE L P N FTID S S T N T DN L EN S R ITIDIA S Fi ITIDIN G TNR T TX T E G T Q TIYP S

IS ->.Ie ~~~~~F777~~~~~I') 7

r,,>

>>

,,, 1

._.

4 _2

4

consensus

FIG. 3. Sequence of leucine-rich repeats of GRR1 protein. The 12 tandem repeats of 26 amino acids are shown. The residue numbers within the GRR1 protein of the first and last amino acids of a repeat are indicated on the left and right, respectively. Conserved positions are boxed. Amino acids present in at least five of the repeats were assigned to the consensus sequence shown below. hf, hydrophobic residue.

allowing translation of GRRI beginning at the methionine codon at residue 319. Predicted protein sequence of GRRI. The most striking feature of the predicted GRR1 protein is the presence of 12 tandem repeats of a 26-amino-acid sequence that is similar in composition to leucine-rich repeats found in several other proteins (Fig. 3; see Discussion). The encoded GRRI protein is rich in asparagine and leucine residues (11 and 12%, respectively) and contains 23 potential sites for N-linked glycosylation (Asn-X-Ser/Thr) found throughout the protein. A hydrophobic signal sequence that might suggest transport into the secretory pathway is not apparent at the amino terminus (33). Hydrophilic domains rich in asparagine and glutamine residues are found at both ends of the protein: the amino-terminal 146 amino acids contain 46 residues (32%) that are either asparagine or glutamine (including a run of 12 consecutive asparagine residues from amino acids 38 to 49); the carboxyl-terminal domain (amino acids 1040 to 1124) contains 34 asparagine or glutamine residues (40%) followed by a short, very negatively charged (-9) tail of the carboxylterminal 24 amino acids. GRRI is expressed constitutively at low levels. Northern blot analysis identified three transcripts expressed at low levels from the GRRI locus. A strand-specific riboprobe (GRRI nucleotides + 1802 to + 2378) hybridized to sizefractionated poly(A)+ RNA from GRRI yeast cells detected a 3.8-kb and a 2.7-kb RNA with the sequence of the coding (sense) strand of the GRRJ gene (Fig. 4). In addition, a riboprobe of the opposite strand detected a 2.7-kb RNA with the sequence of the noncoding (antisense) strand of GRRI. All three RNA species are absent from yeast cells containing the grrl::LEU2 deletion, demonstrating that these signals are encoded by GRR1 and are not the result of crosshybridization of the probes to related sequences. The steady-state level of the GRRI RNAs was considerably less than that of the moderately abundant LEU2 RNA and is similar in yeast cells grown on repressing (2% glucose) and nonrepressing (5% glycerol) carbon sources. Because only the 3.8-kb species is of sufficient size to encode the predicted GRR1 protein and hybridizes only to the proper strandspecific probe, it is likely that this is the GRRI mRNA. The absence of the splice site sequence TACTAAC (30) in the DNA sequence suggests that differential splicing is not the source of the two sense-strand transcripts. Primer extension analysis determined that RNA synthesis

Agr_ -; P

A blot analysis of GRRJ expression. RNA was FIG. 4. Northern prepared from GRRJ strain YM2061 and grrl::LEU2 strain YM2953 2% glucose grown on leucine-deficient SD medium containing either (Glu) or 5% glycerol plus 0. 1% glucose (Gly). Poly(A) + RNA (20 p.g per lane) was fractionated by electrophoresis and blotted to a membrane along with lambda DNA cut with EcoRI and HindIll (labeled with 32p) . As indicated, the left and right halves of the blot were hybridized with a radioactive antisense-strand riboprobe and a sense-strand riboprobe, respectively. The riboprobes contained GRRI sequences + 1802 to +2378 (deleted in grrl::LEU2). The 3.8-kb RNA (thick filled arrow) and the 2.7-kb RNAs (thin filled arrows) encoded by GRRI are indicated. The 1.3-kb RNA (open arrow) in each lane is LEU2 RNA probed as an internal control for RNA loading. Strand-specific riboprobes containing GRRI sequences + 1633 to + 1807 (174-bp PvuII-to-SnaBI fragment) and +2641 to +3614 (Sacl-to-SnaBI fragment) detected identical patters of GRRJ RNA (data not shown). initiates at a cluster of sites upstream of the GRRJ ORF (Fig. 5). The major sites of initiation were found at -293 and -276 relative to the ATG codon of GRRJ. No sequence motifs upstream of the RNA start sites match the consensus TATA box element (TATAAA) (13). While the 5' nontranslated leader RNA of GRRJ is unusually long (238 to 306 bases) compared with most yeast genes (20 to 60 bases in length [14]), no intervening ATG codon is found upstream of the initiator ATG codon (+1 in Fig. 2). GRR1 is located on chromosome 10. A 32P-labeled DNA fragment containing GRRJ was hybridized to an ordered array of lambda clones containing yeast DNAs that have been mapped in the yeast genome (47). This analysis revealed that GRRJ lies on the right arm of chromosome 10. GRR1 was then mapped more precisely by genetic recombination with chromosome 10 markers SUP4-o and cdcll-6

(Fig. 6). GRRJ null mutants are viable and possess pleiotropic growth defects. To examine the phenotype of a null mutant, GRRJ was disrupted by replacement of the coding region with a restriction fragment containing either the LEU2 (removes GRRJ amino acids 602 to 1151) or URA3 (removes GRRI amino acids 24 to 1151) gene (Fig. 1). Tetrads of a diploid disrupted for GRRJ (see Materials and Methods) yielded four viable spores, indicating that GRRJ is not an essential gene. The grrl mutation causes a severe change in cell morphology characterized by elongated sausage-shaped cells and buds (Fig. 7) (2). This cell morphology defect is most severe during growth on glucose. The null mutation (Leu phenotype) cosegregated with the loss of glucose repression of GAL] expression and the sausage-shaped cell

MOLECULAR ANALYSIS OF GRRI

VOL . 1 l, 1991

Agrr 1

x

GRP I G3A

T C

5107

e

4C

*M

4" .

-

-306

-

-293

-

-286

-

-2 6/

- -2

.,

-

YPGlu

I

60

-2 3 8

..

YPG1y

U-

FIG. 5. Primer extension analysis of GRRI RNA. A 5'-endlabeled oligonucleotide primer (GRRI +3 to +23) was annealed to either 2 ,ug of poly(A)+ RNA from YM3454 (Yep24/GRRI) grown in 2% glucose medium or 10 ,ug of yeast tRNA and extended by avian myeloblastosis virus reverse transcriptase. Products were resolved on a 6% polyacrylamide gel containing 7 M urea next to dideoxysequencing reactions of pBM1624 by using the same primer and are labeled by their positions from the ATG codon (+1) of the GRRI sequence. The same pattern of initiation sites was obtained with an oligonucleotide primer complementary to GRRI sequences + 152 to + 169 and also with RNA from glycerol-grown cells (data not shown).

morphology in 22 complete tetrads examined. The defect in glucose repression of GALI expression in yeast cells containing the grrl::LEU2 or grrl::URA3 null mutation was similar to effects of the grrl-1121 point mutation (Table 2). Two spore clones of each tetrad that contained the grrl disruption formed significantly smaller colonies on glucosecontaining medium. To examine the effects of a grrl null

Chr.

10

*

~~~~SUP4 CDC I /

GRR1

H I-

|

l-1

cM

23

cM

31 4 CM

FIG. 6. Genetic map of the right arm of chromosome 10. The genetic linkage of GRRI to SUP4 and CDCII is indicated in centimorgans (cM). Linkage was determined by tetrad analysis of a cross between YM2996 and YM3135 of the following gene pairs: grrl: :LEU2 x sup4-o (parental ditype [PD] = 39, nonparental ditype [NPD] = 2, tetratype [T] = 37); grrl::LEU2 x cdcll-6 (PD = 104, NPD = 0, T = 14); and cdcll-6 x sup4-o (PD = 45, NPD = 1, T = 32). Genetic map distance was calculated by using the formula: map distance (in centimorgans) = 100 [(T + 6NPD)/2(PD + NPD + T)] (52). The gene order was deduced in a three-factor cross: 29 of 30 SUP4-CDCJH recombinants are also recombinant for SUP4-GRRI; only 30 of 46 of the SUP4-GRRI recombinants are also recombinant for SUP4-CDCII.

FIG. 7. Cell morphology of a grrl null mutant. Spore cultures A (GRRI) and D (grrl:: URA3) from Fig. 8 were grown to mid-log phase in the media indicated and photographed by using Normarski optics and the 63x objective lens of a Ziess Axioskop microscope.

mutation on growth, strains derived from a tetrad of a GRRl/grrl:: URA3 diploid (YM3774) were tested for growth on plates containing various carbon sources. As shown in Fig. 8, growth of the grrl mutants (spores C and D) was severely reduced on glucose and raffinose plates compared with wild-type growth (spores A and B). In contrast, growth of GRRI and grrl:: URA3 yeast cells was nearly the same on either galactose or glycerol plates. The doubling times in liquid cultures of the strains derived from this tetrad confirmed the plate results (Figure 8): the grrl:: URA3 mutation increases the doubling times of yeast cells on glucose and raffinose media by nearly 100% but resulted in only a 25% increase in their doubling time in galactose or glycerol medium. We discovered two additional growth defects conferred by the grrl null mutation. First, grrl mutants are sensitive to the osmotic stress caused by ethylene glycol (Fig. 8B). Second, grrl mutants are sensitive to starvation for a nitrogen source. After 7 days on nitrogen starvation plates, grrl cells had a much greater loss of viability than did GRRI cells (Fig. 8B). SUC2 regulation is reversed in grrl mutants. It was surprising that the grrl:: URA3 mutation reduced the growth rate on raffinose, since grrl mutants produce high levels of invertase (encoded by SUC2) during growth on glucose (2). To examine the possibility that grrl cells grow poorly on raffinose as a result of decreased expression of invertase, the level of SUC2 RNA was analyzed in both glucose-repressed and nonrepressed yeast. We observed that grrl cells failed to express significant levels of SUC2 RNA when grown on galactose but contained high levels when grown on galactose plus glucose (Fig. 9). This pattern of SUC2 regulation (induction by glucose) is opposite that in GRRJ cells and

MOL. CELL. BIOL.

FLICK AND JOHNSTON

5108

A.

YPGlu

YPGal

GRR I Agrr I R C I)

A

I

GRP I

Agrr i

A

C

R

11I

g (hrs)

4.7

3 .2

2.

YPRaf A

B

C

3

YPGly

D

B

A

D

C

L

g

(hrs)

B

2.5

4 7

5.' P

'12 +

_"

2

MDN

1

6.C

ethylene

vYyoi

FIG. 8. Growth defects of grrl null mutants. (A) Cultures from four spores (labeled A to D) of a tetrad derived from a ura3-521 ura3-52 GRRllgrrl::URA3 diploid were spotted on YP plates containing the carbon sources indicated (YPGlu, 2% glucose, YPGal, 2% galactose; YPRaf, 2% raffinose; YPGly, 5% glycerol). A multiprong inoculating grid was used to transfer approximately 2 x 104 cells from each spore culture to the top row of each panel and approximately 4 x 103 cells to the bottom row. Plates were incubated at 30°C for 24 h (YPGlu) or 48 h (YPGal, YPRaf, and YPGly) and then photographed. Indicated below each panel is the growth rate of spore cultures in YP broth (same carbon source as plate assay) determined in an independent experiment. The doubling time (g) of mid-log-phase cultures was calculated by measuring culture turbidity over time, using a Klett-Summerson colorimeter. (B) The same tetrad as in panel A was tested for osmotic sensitivity (top), viability after nitrogen starvation (middle) or the presence of the grrl::URA3 allele (bottom) by replica plating from a YPGal master plate to the media indicated. Nitrogen starvation conditions are described in Materials and Methods.

indicates that the grrl mutation converts SUC2 from a glucose-repressed gene into a glucose-induced gene. The defect in SUC2 derepression during growth on galactose in the grrl mutant is in contrast to the apparently normal derepression of the GAL] gene (Table 2). The snfl grrl double mutant is not viable. The SNFJ gene encodes a protein kinase that is required for the derepression of glucose-repressed genes in yeast cells (11). We wished to determine the epistatic relationship of SNFJ and GRRI, since these genes are positive and negative regulators, respectively, of glucose-repressed genes. Sporulation and tetrad dissection of a diploid heterozygous for both grrl: :LEU2 and snfl-A3 null mutations (YM3775) resulted in a failure to recover a viable segregant containing both mutations: all of four complete tetrads were parental ditype; in 16 tetrads with only three viable spores, the genotype of the dead spore was inferred to be grrl snff for 10 of the tetrads and grrl SNFJ for the remaining 6 tetrads. These data strongly suggest that the combined effects of the grrl and sntfi mutations are lethal in S. cerevisiae. However, because it is possible that germination is specifically sensi-

FIG. 9. Northern blot analysis of SUC2 expression in a grrl mutant. Each lane was loaded with total RNA (20 ,ug) from GRRJ (YM2061) or grrl-1121 (YM1871) cells grown in leucine-deficient SD medium containing either 2% galactose (Gal) or 2% galactose plus 2% glucose (Gal + Glu). The RNA was fractionated by gel electrophoresis, transferred to a membrane, and probed for SUC2 and LEU2 RNAs. The 1.9-kb SUC2 RNA and the 1.3-kb LEU2 RNA are indicated.

tive to the double mutation, the diploid strain YM3775 was transformed to Ura+ with a YEp24 plasmid containing SNFJ (pCE9) (10). This diploid was sporulated, and tetrads were dissected on YPGlu medium. Four spores from a tetratype ascus (regarding chromosomal GRRI and SNFI alleles) that all contained the SNFI plasmid were tested for their ability to grow in the absence of the SNFJ plasmid by plating on 5-FOA medium (Fig. 10A). We observed that one spore clone (deduced genotype grrl ::LEU2 snfl-A3) did not grow on 5-FOA medium, whereas strains that contain a chromosomal copy of either GRRJ or SNFJ grew in the absence of the SNFI plasmid. These results clearly demonstrate that the grrl snfl double mutant is not viable. grrl mutants are dependent on aromatic amino acid prototrophy. An unusual phenotype of grrl mutants was observed in crosses with trpl or tyri strains. We observed that recombinant yeast cells containing both grrl and trpl have a severe growth defect on either YPGlu or YPGal that is much more severe than that of grrl TRPI strains (Fig. 10B). In addition, we were unable to construct a grrl tyri double mutant from a cross of grrl with tyri mutants. From this cross only two complete tetrads were obtained, and both were parental ditype with respect to grrl and tyri. Ten of the eleven three-spored tetrads recovered were inferred to be tetratypes, with the genotype of the dead spore being grrl tyri. Similar results were obtained in crosses of grrl mutants with arolC and aro7 mutants. In control crosses of grrl with other nutritional auxotrophs (ura3, his3, lys2, ade2, met, and leu2), recombinants that did not exacerbate the grrl growth defect were recovered at the expected frequency (data not shown). GRRI encodes a 135-kDa protein found in the particulate fraction of yeast extracts. To detect GRR1 protein, antibodies were produced in rabbits immunized with a TrpE-GRR1 fusion protein (containing GRRI amino acids 389 to 933) and affinity purified from crude rabbit sera. The anti-GRR1 antibodies were used to probe immunoblots of fractionated extracts prepared from yeast cells. The anti-GRR1 antibodies recognized a protein of approximately 135 kDa in the low-speed supernatant [S(450g)] of extracts from yeast cells containing a high-copy-number GRRI plasmid that is not found in extracts from grrl:: URA3 yeast cells (Fig. 11). The molecular size of this protein agrees well with the predicted size of the GRRJ primary translation product (132.7 kDa) and suggests that GRR1 protein is not extensively modified in yeast cells. GRR1 protein was found in the P(104g) fraction. A small amount of the 135-kDa protein was detected in the P(105g) but not in the S(105g) fraction. These results suggest that GRR1 protein is not a soluble cytoplasmic protein in S. cerevisiae.

VOL. 11, 1991

MOLECULAR ANALYSIS OF GRRI

A.

High Copy

,dgrrl

IAorr ASnR1

r

5109

GRR1

--

k-

t88-

a..w

-AIdz"

I: 6-

11

-

49

-GRR1

-

*Sbl411s1

.

.'

-URA

5-FOA

5848.5-

B. 8 4

YPGlu

YPGal

FIG. 10. Genetic interactions of the grrl mutation. (A) Loss of SNFJ plasmid is lethal to the grrl::LEU2 snfl double mutant. Four spore clones of a tetratype ascus from the diploid YM3775 (grrl::LEU2/GRRJ SNFllsnfl-3, Yep24/SNFI [pCE9]) were replica plated from a YPGlu master plate to uracil-deficient SD and 5-FOA plates and incubated for 3 and 5 days, respectively, at 30°C. The deduced genotypes of the spore cultures are labeled. (B) Reduced growth rate of the grrl trpl double mutant. Four spore clones of a tetratype ascus from the diploid YM3843 (TRP11trpl-901 grrl:: URA31GRRI) were restreaked on YPGlu and YPGal plates and incubated for 3 days at 30°C. The deduced genotypes of the spore cultures are labeled.

FIG. 11. Immunoblot identification of GRR1 protein in yeast extracts. Yeast extracts were prepared from a deletion mutant, YM3492 (grrl::URA3), or from a strain (YM3459) containing a high-copy-number GRRI plasmid (pIAl/GRRl) (both grown on uracil-deficient SD medium containing 2% galactose). Spheroplasts from each strain were lysed and centrifuged for 5 min at 450 x g to produce a cleared supernatant, S(450g). The S(450g) fraction was centrifuged at 10,000 x g for 10 min to produce the P(104g) and S(104g) and S(104g) fractions. The S(104g) fraction was centrifuged at 100,000 x g for 1 h to produce the P(10'g) and S(105g) fractions [the S(450g) fraction of YM3492 was centrifuged at 100,000 x g, omitting the 10,000 x g step]. Samples from each fraction proportional to the original cell volume were boiled in SDS loading buffer and subjected to electrophoresis in a 6.5% polyacrylamide-1% SDS gel as described by Laemmli (39). Proteins were electroblotted to an Immobilon membrane, and GRR1 protein was detected by using affinitypurified rabbit anti-GRR1 antibodies as described in Materials and Methods.

the leucine-rich repeats, which in other proteins are thought to mediate protein-protein interactions, makes this suggestion tenable.

DISCUSSION In addition to the 135-kDa protein, several proteins of lower molecular weight were recognized by anti-GRR1 antibodies. These proteins were absent from extracts from the grrl:: URA3 deletion strain, demonstrating that they are encoded by the GRRI gene. Although we cannot rule out that these faster-migrating proteins are potential translation products of the smaller 2.7-kb sense-strand transcript- from GRRI, the increased abundance of these bands in stored extracts suggest that they are degradation products of the 135-kDa protein (data not shown). We have been unable to reliably detect GRR1 protein in yeast cells containing a single copy of the GRRI gene, a result that is consistent with the low level of expression of the GRRI RNA. To analyze the insoluble nature of GRR1, we attempted to release GRR1 protein from the P(104g) fraction by using a variety of agents that solubilize membranes and disrupt protein-protein interactions. GRR1 protein was not significantly solubiized by 1 M NaCl, 10 mM EDTA, 0.2 M Na2CO3, or 1% Triton X-100 (Fig. 12). The only agent that significantly solubilized GRR1 protein was urea, a protein denaturant. These results suggest that the insolubility of GRR1 protein is due to its association with a particulate protein complex rather than with a membrane component of this particulate fraction. The presence in GRR1 protein of

Previous genetic analysis has demonstrated that the GRRI gene is required for glucose repression in S. cerevisiae yeast

ps

AN_

FIG. 12. Solubilization of GRR1 protein from the P(104g) fraction A P(104g) fraction containing GRR1 protein was prepared from YM3459 as for Fig. 10. The pellet was extracted with homogenation buffer (control) or homogenation buffer containing 1.0 M NaCl, 50 mM Tris (pH 7.5)-10 mM EDTA, 1% Triton X-100, 0.2 M Na2CO3 (pH 11.0), or 6 M urea as described in Materials and Methods. The mixtures were incubated on ice for 10 min and then centrifuged at 10,000 x g for 10 min to produce a pellet fraction (P) and a supematant fraction (S). Equivalent samples of the pellet and supernatant were brought to 1% SDS in loading buffer and boiled for 3 min prior to electrophoresis and electroblotting to an Immobilon membrane. GRR1 protein was detected with affinity-purified rabbit anti-GRR1 antibodies.

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(2). GRRI is essential for repression, operating on both the UASgal and URSgal systems of the GAL] promoter (21), as well as repression of other genes involved in carbon source metabolism (SUC2 and MAL [2]) and respiration (CYCI [21a]). The requirement of GRRJ for glucose repression of many genes suggests that GRRI functions early in the pathway, perhaps in the generation of the signal of glucose repression.

We observed that grrl mutants display a number of phenotypes in addition to the defect in glucose repression. First, the growth rate of grrl mutants is significantly reduced on media containing glucose and raffinose but is relatively unaffected on galactose or glycerol medium (Fig. 8). The slow growth rate on glucose may indicate that grrl mutants are defective in some aspect of glucose metabolism that is not shared with galactose, such as transport. Mutations in two other genes required for glucose repression, regi and ssn6, also result in reduced growth rates, but these growth defects are not limited to glucose or raffinose media (9, 28). Second, we discovered a dependence of grrl mutants on aromatic amino acid prototrophy: yeast cells that are grrl trpl have a much more severe growth defect on all carbon sources than do grrl TRP+ strains (Fig. 10); grrl tyri, grrl aroiC, or grri aro7 strains appear inviable. This finding suggests that the grrl mutation either prevents utilization of exogenous aromatic amino acids (possibly by blocking transport) or causes an accumulation of a toxic intermediate of aromatic amino acid biosynthesis in an aro mutant background. Third, grrl mutants also display enhanced sensitivity to hypertonic media and nitrogen starvation (Fig. 8B), the bases of which are unknown. Finally, grrl mutations cause an abnormal cell morphology (Fig. 7). The numerous growth defects of grrl mutants may reflect a general defect in an environmental sensing mechanism(s). The poor growth on raffinose medium can probably be attributed to an unexpected consequence of the grrl mutation on SUC2 expression. We observed that grrl mutants are defective in derepression of SUC2 RNA during growth on nonrepressing carbon sources (Fig. 9) (21a). Remarkably, SUC2 expression is induced by glucose in grrl mutants, thereby converting SUC2 from a glucose-repressed gene into a glucose-induced gene. This result has been verified by Vallier and Carlson (65a). The inverted glucose regulation of SUC2 suggests that either the regulatory signal conferred to SUC2 is reversed in grrl mutants or the elimination of glucose repression reveals a cryptic dependence on glucose metabolism for high-level SUC2 expression. Since derepression of both GAL] and CYCI genes is normal in grrl mutants (Table 2) (21a), we favor the latter possibility. The idea that glucose, the product of invertase activity on sucrose, is required for high-level SUC2 expression is analogous to the situation in E. coli, in which lac operon expression is induced by allolactose, a product of ,3-galactosidase activity on lactose (4). Consistent with this proposal is our observation that low levels of glucose are required for the continued high-level expression of SUC2 RNA during growth of wildtype yeast cells on glycerol (21a). Derepression of glucose-repressed genes is under coordinate control and requires the SNFI gene, which encodes a protein kinase (11). Previous genetic analysis of glucose repression mutants demonstrated that the snfl defect in derepression of SUC2 is epistatic to the defects in repression of regi and hxk2 (44) but is suppressed by ssn6 mutations (9). We found that the grrl snfl double mutant is inviable. This is puzzling given that yeast cells containing snfl in combination with the other glucose repression mutations are viable

MOL. CELL. BIOL.

and suggests an as yet undetermined regulatory defect specific to grrl mutants. The predicted sequence of GRR1 protein does not bear significant similarity to any protein currently in the NBRF or GenBank data base (48). Analysis for internal homology revealed the presence of 12 tandem copies of a 26-aminoacid repeat (Fig. 3). The GRRI repeat is similar in amino acid composition and length to previously described leucine-rich repeats found in several diverse proteins from a variety of species. Tandemly repeated motifs of 22 to 26 amino acids that are rich in leucine residues have been found in adenylate cyclase (34) and the sds22+ protein phosphatase regulatory subunit (46) from yeasts, the serum at2-glycoprotein (63), extracellular matrix glycoprotein PG40 (38), and platelet transmembrane glycoprotein Iba (41) from humans, the Toll transmembrane protein (25) and cell surface chaoptin protein from Drosophila melanogaster (49), and the leucine-rich repeat gene from trypanosomes (61). The repeats in GRR1 contain a characteristic periodicity of leucine or conserved hydrophobic residues that is different from the previously described repeat motifs. In addition, the GRR1 repeats contain two conserved cysteine residues at positions 5 and 16, suggesting the possibility of disulfide bridges within this domain. The repeat domain of GRRI is found in the central core of the protein and comprises 28% of its sequence. The presence of a leucine-rich repeat domain in GRRJ suggests that it may function in mediating protein-protein interactions as has been postulated for the related domains in other proteins. The repeats in the platelet transmembrane glycoprotein lb alpha chain are known to bind von Willebrand factor (41), and those in adenylate cyclase appear to mediate activation by RAS protein (20). The Sds22+ protein of Schizosaccharomyces pombe, which consists almost entirely of leucine-rich repeats, appears to activate the dis2+encoded protein phosphatase (46). Results consistent with the possibility that the GRRJ repeats mediate protein-protein interactions were obtained by immunoblot analysis of GRR1 protein in yeast extracts. Cell fractionation revealed that GRR1 protein is in a particulate fraction in yeast extracts. This could be due to the nonspecific aggregation of GRR1 protein or its specific association with other cellular components. We believe that GRR1 protein is not associated with a membrane or membrane-bound compartment in S. cerevisiae, because it is not released from the particulate fraction by a nonionic detergent (Fig. 12). Rather, it appears to be a component of particulate protein complex because it is released by the protein denaturant urea. Additional studies will be required to determine whether the leucine-rich repeats are essential for the association of GRR1 protein with this complex. GRRI RNA and protein are both expressed at low levels in S. cerevisiae and are not regulated by the carbon source (Fig. 4 and data not shown). Furthermore, mutations in the GAL82, GAL83, REGI, HXK2, SSN6, or SNF1 regulatory gene have no effect on the expression of GRRJ RNA (21a). The low level of expression observed is consistent with the role of GRR1 protein as a regulatory factor in cells rather than a structural element or enzyme of glucose metabolism. Although the defect in transcriptional repression in grrl mutants implies that GRRJ is formally a repressor, we believe it unlikely that GRRI encodes a DNA-binding repressor protein for three reasons. First, the protein sequence of GRRJ does not contain any known DNA-binding motifs. Second, GRR1 protein does not appear to be in the nucleus, since lysis of nuclei in the P(104g) fraction with Triton X-100 does not solubilize GRR1 protein. Furthermore, staining of

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MOLECULAR ANALYSIS OF GRRI

GRR1 protein in yeast cells by indirect immunofluorescense reveals a faint but reproducible general cytoplasmic staining (21a). Third, overexpression of GRR1 protein had no effect on GALI gene expression as might be expected for a direct negative regulator of gene expression (21a). Such an effect on SUC2 expression has been shown for the SSN6 gene, which encodes a nuclear-localized regulatory protein (57, 58) and on SUC2 and GALI expression by the MIGJ gene, which encodes a protein containing a zinc finger DNAbinding domain (43). The aberrant cellular morphology of grrl mutants and the cell fractionation results raise the possibility that GRR1 protein is associated with the subcellular matrix in vivo. ACKNOWLEDGMENTS This work was supported by Public Health Service grant GM32540 from the National Institutes of Health. J.S.F. was supported by Public Health Service training grants GM07067 and GM08036 from the National Institutes of Health, by the Division of Biology and Biomedical Sciences of Washington University, and by the Lucille P. Markey Charitable Trust. M.J. was an Established Investigator of the American Heart Association during most of this analysis. We thank Jim Dover and Jim Murphy for excellent technical assistance; J. Dutchik and L. Riles for the physical mapping of GRRI; M. Goebl for first pointing out the internal homology in GRRI and M. Boguski for further analysis of the leucine-rich repeat; R. Bailey, J. Pringle, M. Carlson, and P. Hieter for sending plasmids and yeast strains; and L. Vallier and M. Carlson for communicating results prior to publication. REFERENCES

1. Alani, E., L. Cao, and N. Kleckner. 1987. A method for gene disruption that allows repeated use of URA3 selection in the construction of multiply disrupted yeast strains. Genetics 116: 541-545.

2. Bailey, R. B., and A. Woodword. 1984. Isolation and characterization of a pleiotropic glucose repression resistant mutant of Saccharomyces cerevisiae. Mol. Gen. Genet. 193:507-512. 3. Batteiger, B., W. J. Newhall, and R. B. Jones. 1982. The use of Tween 20 as a blocking agent in the immunological detection of proteins transferred to nitrocellulose membranes. J. Immunol. Methods 55:297-307. 4. Beckwith, J. 1987. The lactose operon, p. 1444 1452. In F. C. Neidhardt, J. L. Ingraham, B. Magasanik, K. B. Low, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella typhimurium: cellular and molecular biology. American Society for Microbiology, Washington, D.C. 5. Boeke, J. D., F. LaCroute, and G. R. Fink. 1984. A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol. Gen. Genet. 197:345-346. 6. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254. 7. Broach, J. R. 1983. Construction of high copy yeast vectors using 2-micron circle sequences. Methods Enzymol. 101:307325. 8. Carlson, M. 1987. Regulation of sugar utilization in Saccharo-

myces species. J. Bacteriol. 169:4873-4877. 9. Carlson, M., B. C. Osmond, L. Neigeborn, and D. Botstein. 1984. A suppressor of SNFJ mutations causes constitutive high-level invertase synthesis in yeast. Genetics 107:19-32. 10. Celenza, J. L., and M. Carlson. 1984. Structure and expression of the SNFJ gene of Saccharomyces cerevisiae. Mol. Cell. Biol.

4:54-60. 11. Celenza, J. L., and M. Carlson. 1986. A yeast gene that is essential for release from glucose repression encodes a protein kinase. Science 233:1175-1180.

5111

12. Chen, E. Y., and P. H. Seeburg. 1985. Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA 4:165-170. 13. Chen, W., and K. Struhl. 1988. Saturation mutagenesis of a yeast his3 "TATA element": genetic evidence for a specific TATA-binding protein. Proc. Natl. Acad. Sci. USA 85:26912695. 14. Cigan, A. M., and T. F. Donahue. 1987. Sequence and structural features associated with translational initiator regions in yeast-a review. Gene 59:1-18. 15. Dieckmann, C. L., and A. Tzagoloff. 1985. Assembly of the mitochondrial membrane system. J. Biol. Chem. 260:1513-1520. 16. Elder, R. T., E. Y. Loh, and R. W. Davis. 1983. RNA from the yeast transposable element Tyl has both ends in the direct repeats, a structure similar to retrovirus RNA. Proc. Natl. Acad. Sci. USA 80:2432-2436. 17. Elledge, S. J., and R. W. Davis. 1988. A family of versatile centromeric vectors designed for use in the sectoring shuffle mutagenesis assay in Saccharomyces cerevisiae. Gene 70:303312. 18. Entian, K.-D. 1986. Glucose repression: a complex regulatory system in yeast. Microbiol. Sci. 3:366-371. 19. Ey, P. L., and L. K. Ashman. 1986. The use of alkaline phosphatase-conjugated anti-immunoglobulin with immunoblots for determining the specificity of monoclonal antibodies to protein mixtures. Methods Enzymol. 121:497-509. 20. Field, J. H.-P. Xu, T. Michaeli, R. Ballester, P. Sass, M. Wigler, and J. Colicelli. 1990. Mutations of the adenylyl cyclase gene that block RAS function in Saccharomyces cerevisiae. Science 247:464-467. 21. Flick, J. S., and M. Johnston. 1990. Two systems of glucose repression of the GAL] promoter in Saccharomyces cerevisiae. Mol. Cell. Biol. 10:4757-4769. 21a.Flick, J. S., and M. Johnston. Unpublished data. 22. Gancedo, J. M., and C. Gancedo. 1986. Catabolite repression mutants of yeast. FEMS Microbiol. Rev. 32:179-187. 23. Goud, B., A. Salminen, N. C. Walworth, and P. J. Novick. 1988. A GTP-binding protein required for secretion rapidly associates with secretory vesicles and the plasma membrane in yeast. Cell 53:753-768. 23a.Griggs, D., and M. Johnston. Proc. Natl. Acad. Sci., in press. 24. Harlow, E., and D. Lane. 1988. Antibodies: a laboratory manual, p. 93-120. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 25. Hashimoto, C., K. L. Hudson, and K. V. Anderson. 1988. The Toll gene of Drosophila, required for dorsal-ventral embryonic polarity, appears to encode a transmembrane protein. Cell 52:269-279. 26. Henikoff, S. 1984. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28:351359. 26a.Hieter, P. Personal communication. 27. Hoffman, C. S., and F. Winston. 1987. A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57:267-272. 28. Hovland, P., J. Flick, M. Johnston, and R. A. Sclafani. 1989. Galactose as a gratuitous inducer of GAL gene expression in

yeast growing on glucose. Gene 83:57-64. 29. Ito, H., Y. Fukuda, K. Murata, and A. Kimura. 1983. Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153:163-168. 30. Jacquier, A., J. R. Rodriguez, and M. Rosbash. 1985. A quantitative analysis of the effects of 5' junction and TACTAAC box mutants and mutant combinations on yeast mRNA splicing. Cell 43:423-30. 31. Johnston, M. 1987. A model fungal gene regulatory mechanism: the GAL genes of Saccharomyces cerevisiae. Microbiol. Rev. 51:458-476. 32. Johnston, M., and R. W. Davis. 1984. Sequences that regulate the divergent GALI-GALIO promoter in Saccharomyces cereiisiae. Mol. Cell. Biol. 4:1440-1448. 33. Kaiser, C. A., D. Preuss, P. Grisafi, and D. Botstein. 1987. Many random sequences functionally replace the secretion signal

5112

FLICK AND JOHNSTON

sequence of yeast invertase. Science 235:312-317. 34. Kataoka, T., D. Broek, and M. Wigler. 1985. DNA sequence and characterization of the S. cerevisiae gene encoding adenylate cyclase. Cell 43:493-505. 35. Kingston, R. E. 1989. Preparation of poly(A)+ RNA, p. 4.5.1. In F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (ed.), Current protocols in molecular biology, vol. 1. Greene Publishing Associates and Wiley-Interscience, New York. 36. Kleid, D. G., D. Yansura, B. Small, D. Dowbenko, D. Moore, M. Grubman, P. McKercher, D. Morgan, B. Robertson, and H. Bachrach. 1981. Cloned viral protein vaccine for foot-andmouth disease: responses in cattle and swine. Science 214:11251129. 37. Krainer, A. R., T. Maniatis, B. Ruskin, and M. R. Green. 1984. Normal and mutant human ,-globin pre-mRNAs are faithfully and efficiently spliced in vitro. Cell 36:993-1005. 38. Krusius, T., and E. Ruoslahti. 1986. Primary structure of an extracellular matrix proteoglycan care protein deduced from cloned cDNA. Proc. Natl. Acad. Sci. USA 83:7683-7687. 39. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London)

227:680-685. 40. Lillie, S. H., and S. S. Brown. 1987. Artifactual immunofluorescent labelling in yeast, demonstrated by affinity purification of antibody. Yeast 3:63-70. 41. Lopez, J. A., D. W. Chung, K. Fujikawa, F. S. Hagen, T. Papayannopoulou, and G. R. Roth. 1987. Cloning of the alpha chain of human platelet glycoprotein Ib: a transmembrane protein with homology to leucine-rich alpha2-glycoprotein. Proc. Natl. Acad. Sci. USA 84:5615-5619. 42. Matsumoto, K., T. Yoshimatsu, and Y. Oshima. 1983. Recessive mutations conferring resistance to carbon catabolite repression of galactokinase synthesis in Saccharomyces cerevisiae. J. Bacteriol. 153:1405-1414. 43. Nehlin, J. O., and H. Ronne. 1990. Yeast MIG1 repressor is related to the mammalian early growth response and Wilm's tumour finger proteins. EMBO J. 9:2891-2898. 44. Neigeborn, L., and M. Carlson. 1987. Mutations causing constitutive invertase synthesis in yeast: genetic interactions with snf mutations. Genetics 115:247-253. 45. Niederacher, D., and K.-D. Entian. 1987. Isolation and characterization of the regulatory HEX2 gene necessary for glucose repression in yeast. Mol. Gen. Genet. 206:505-509. 46. Ohkura, H., and M. Yanagida. 1991. S. pombe gene sds22+ essential for a midmitotic transition encodes a leucine-rich repeat protein that positively modulates protein phosphatase-1. Cell 64:149-157. 47. Olson, M. V., J. E. Dutchik, M. Y. Graham, G. M., C. Helms, M. Franc, M. MacCollin, R. Scheiman, and T. Frank. 1986. Random-clone strategy for genomic restriction mapping in yeast. Proc. Natl. Acad. Sci. USA 83:7826-7830. 48. Pearson, W. R., and D. J. Lipman. 1988. Improved tools for biological sequence comparison. Proc. Natl. Acad. Sci. USA

85:2444-2448. 49. Reinke, R., D. E. Krantz, D. Yen, and S. L. Zipursky. 1988. Chaoptin, a cell surface glycoprotein required for Drosophila photoreceptor cell morphogenesis, contains a repeat motif found in yeast and human. Cell 52:291-301. 50. Rose, M. D., and D. Botstein. 1983. Construction and use of gene fusions to lacZ (P-galactosidase) that are expressed in yeast. Methods Enzymol. 101:167-180.

MOL. CELL. BIOL. 51. Rose, M. D., P. Novick, J. H. Thomas, D. Botstein, and G. R. Fink. 1987. A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. Gene 60:237243. 52. Rose, M. D., F. Winston, and P. Hieter. 1990. Methods in yeast genetics: a laboratory course manual. Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 53. Rothstein, R. J. 1983. One-step gene disruption in yeast. Methods Enzymol. 101:202-211. 54. Ruohola, H., and S. Ferro-Novick. 1987. SecS3, a protein required for an early step in secretory protein processing and transport in yeast, interacts with the cytoplasmic surface of the endoplasmic reticulum. Proc. Natl. Acad. Sci. USA 84:84688472. 55. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 56. Sarokin, L., and M. Carlson. 1984. Upstream region required for regulated expression of the glucose-repressible SUC2 gene of Saccharomyces cerevisiae. Mol. Cell. Biol. 4:2750-2757. 57. Schultz, J., and M. Carlson. 1987. Molecular analysis of SSN6, a gene functionally related to the SNFI protein kinase of Saccharomyces cerevisiae. Mol. Cell. Biol. 7:3637-3645. 58. Schultz, J., L. Marshall-Carlson, and M. Carlson. 1990. The N-terminal TPR region is the functional domain of SSN6, a nuclear phosphoprotein of Saccharomyces cerevisiae. Mol. Cell. Biol. 10:4744-4756. 59. Sellect, S. B., and J. Majors. 1987. Photofootprinting in vivo detects transcription-dependent changes in yeast TATA boxes. Nature (London) 325:173-177. 60. Singh, A., and F. Sherman. 1978. Deletions of the iso-1-cytochrome C and adjacent genes of yeast: discovery of the OSMI gene controlling osmotic sensitivity. Genetics 89:653-665. 61. Smiley, B. L., A. W. Stadnyk, P. J. Myler, and K. Stuart. 1990. The trypanosome leucine repeat gene in the variant surface glycoprotein expression site encodes a putative metal-binding domain and a region resembling protein-binding domains of yeast, Drosophila, and mammalian proteins. Mol. Cell. Biol. 10:6436-6444. 62. Struhl, K., D. T. Stinchcomb, S. Scherer, and R. W. Davis. 1979. High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules. Proc. Natl. Acad. Sci. USA 76:1035-1039. 63. Takahashi, N., Y. Takahashi, and F. W. Putnam. 1985. Periodicity of leucine and tandem repetition of a 24-amino acid segment in the primary structure of leucine-rich a2-glycoprotein of human serum. Proc. Natl. Acad. Sci. USA 82:1906-1910. 64. Tanaka, K., K. Matsumoto, and A. Toh-e. 1989. IRAI, an inhibitory regulator of the RAS-cyclic AMP pathway in Saccharomyces cerevisiae. Mol. Cell. Biol. 9:757-768. 65. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350-4354. 65a.Vallier, L., and M. Carlson. Genetics, in press. 66. Walworth, N. C., B. Goud, H. Ruohola, and P. J. Novick. 1989. Fractionation of yeast organelles. Methods Cell Biol. 31:335356. 67. Yocum, R. R., S. Hanley, R. West, Jr., and M. Ptashne. 1984. Use of lacZ fusions to delimit regulatory elements of the inducible divergent GALI-GALIO promoter in Saccharomyces cerevisiae. Mol. Cell. Biol. 4:1985-1998.