Mutational Analysis of the Tup1 General Repressor of Yeast

Corresponding author: Richard S. Zitomer, Department of Biological Sciences, University at Albany/SUNY, Albany, NY 12222, RZ144{at}cnsvax.albany.edu (E-mail).

Communicating editor: M. JOHNSTON

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The Tup1 and Ssn6 proteins of Saccharomyces cerevisiae form a general transcriptional repression complex that regulates the expression of a diverse set of genes including aerobically repressed hypoxic genes, a-mating type genes, glucose repressed genes, and genes controlling cell flocculence. To identify amino acid residues in the Tup1 protein that are required for repression function, we selected for mutations that derepressed the hypoxic genes. Three missense mutations that accumulated stable protein were isolated, and an additional three were generated by site-directed mutagenesis. The mutant protein L62R was unable to complex with Ssn6 or repress expression of reporter genes for the hypoxic and glucose repressed regulons or the flocculence phenotype, however, expression of the a-mating type reporter gene was still repressed. The remaining mutations fell within the WD repeat region of Tup1. These mutations had different effects on the expression of the four Tup1 repressed regulons assayed, indicating that the WD repeats serve different roles for repression of different regulons.

BAKER'S yeast, Saccharomyces cerevisiae, is a facultative aerobe that can derive energy aerobically either through oxidative phosphorylation or a mixture of fermentation and oxidative phosphorylation, or anaerobically through fermentation. In the intermediate hypoxic state, yeast induce the expression genes, which enable them to utilize available oxygen more efficiently. The aerobic repression of hypoxic genes is heme dependent and is mediated through the heme activated repressor Rox1 (LOWRY et al. 1990 ; KENG 1992 ). Under fully aerobic conditions, heme is synthesized, Rox1 is induced and binds to a 12-bp site in the promoter of hypoxic genes to repress their transcription (BALASUBRAMANIAN et al. 1993 ; DECKERT et al. 1995A ). Under hypoxic conditions less heme is synthesized, and ROX1 is no longer expressed. The cessation of ROX1 transcription and the decay of the Rox1 protein (ZITOMER et al. 1997b) result in the derepression of the hypoxic genes.

In addition to Rox1, aerobic repression of the hypoxic genes requires the general repressors Tup1 and Ssn6, (ZHANG et al. 1991 ; BALASUBRAMANIAN et al. 1993 ). The high molecular weight complex formed by these two proteins also regulates the a-mating type genes in an -mating type cell through interactions with the 2 repressor (KELEHER et al. 1992 ; KOMACHI et al. 1994 ), the glucose repressed genes through interactions with Mig1 and Mig2 (TRUMBLY 1992 ; TREITEL and CARLSON 1995 ; JOHNSTON and LUTFIYYA 1996 ), and a number of other less well defined systems including DNA damage inducible genes (ZHOU and ELLEDGE 1992 ), sporulation specific genes (FRIESEN et al. 1997 ), and flocculence genes (TEUNISSEN et al. 1995 ).

Several studies have defined the functional domains of Tup1. The first 72 amino acids of Tup1 are involved in formation of the repression complex that consists of either three (REDD et al. 1997 ) or four Tup1 molecules (VARANASI et al. 1996 ) and one Ssn6 molecule. Amino acids 73–385 associate with histones H3 and H4 (EDMONDSON et al. 1996 ). The carboxy terminus of Tup1 contains six WD repeats (WILLIAMS and TRUMBLY 1990 ; ZHANG et al. 1991 ). These repeats were first identified in Gß transducin and have since been found in over 40 other, for the most part, unrelated eukaryotic proteins. Each of the four to eight repeats found in these proteins includes a conserved core of 27–45 residues bracketed by Gly-His and Trp-Asp. Recently, the crystal structure of the Gß transducin was solved, and the WD repeats were shown to form structures designated ß-propellers (SONDEK et al. 1997 ). The protein sequence similarity and biochemical properties of other WD repeat proteins suggest that all WD repeats fold into a similar tertiary structure (GARCIA-HIGUERA et al. 1996 ). The WD repeats of Tup1 interact with the 2 repressor (KOMACHI et al. 1994 ) and may interact with other DNA-binding proteins such as the hypoxic repressor Rox1 or components of the RNA polymerase II holoenzyme.

The functional analyses of Tup1 carried out to date were done in vitro or in artificial systems. To identify residues important for Tup1 function in vivo, we developed a selection for tup1 mutations. We initially obtained three missense mutations, one in the Ssn6 interaction domain, and two in the WD repeat region. The mutation in the Ssn6 interaction domain eliminated Tup1/Ssn6 complex formation and caused derepression of both the hypoxic and glucose repressed regulons, but still formed Tup1 multimers and repressed the a-mating type genes. Although the effects of the two WD repeat mutants on the hypoxic and glucose-repressed genes were very similar, each had a different effect on the a-mating type genes. The differential repression displayed by these point mutations prompted a more detailed study of the role of the different WD repeats. In this paper we provide evidence that suggests that the WD repeats are not functionally redundant, and that they serve a function in the repression of at least two regulons.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Strains, cell growth and transformations:
The S. cerevisiae strain RZ53-6tup1 (MAT trp1-289 leu2-3, 112 ura3-52 ade1-100 tup1::ura3) was described previously (ZHANG et al. 1991 ). The following S. cerevisiae strains were constructed by standard methods of yeast genetics (ROSE et al. 1990 ): MZ14-29 (MATa/MAT trp1/trp1 leu2/leu2 ura3/ura3 lys2/lys2 tif51A::TRP1/tif51A::TRP1 tup1::URA3/tup1::URA3 ura3::AZ4/ura3::AZ4 gal-/gal-) and MZ12-16 (MATa trp1 leu2 lys2 his3 tup1::URA3 ura3::AZ4 gal-).

Yeast cells were transformed as described previously (CHEN et al. 1992 ).

Escherichia coli HB101 was maintained and transformed as described previously (AUSUBEL et al. 1994 ).

Enzymes and general methods for plasmid constructions:
Plasmid constructions were carried out according to standard protocols (AUSUBEL et al. 1994 ). Enzymatic reactions were carried out under the conditions recommended by the vendors. Most restriction enzymes and T4 DNA ligase were purchased from New England Biolabs (Beverly, MA) or Boehringer Mannheim (Indianapolis, IN). Taq polymerase was purchased from Perkin Elmer (Norwalk, CT). DNA sequence analyses were carried out by the dideoxy-chain termination method of SANGER et al. (1977) using sequenase (United States Biochemical, Cleveland, OH) and primer DNAs synthesized with an Applied Biosystems DNA Synthesizer.

General plasmids:
pBSTUP1 and YEp(181)TUP1 were constructed by insertion of the 4.2-kb HindIII-Sac I fragment from YCpTUP1 (ZHANG et al. 1991 ) into the polylinker site of pBSM13(+) (Stratagene, La Jolla, CA) and YEplac181 (GIETZ and SUGINO 1988 ), respectively.

pBSTUP1bbg was constructed as follows: the pBSTUP1 was subjected to site directed mutagenesis (ZOLLER and SMITH 1982 ) using the primer 5'-CTTCGTCTTCCAGATCTGGATC CGAGTTGTA-3' to generate BamHI and Bgl II sites at codon 439 of the TUP1 coding sequence. YEp(181)TUP1bbg was con-structed by insertion of the 4.2-kb HindIII-Sac I fragment of pBSTUP1bbg into the polylinker site of YEplac181.

YCpSSN6 was obtained from a YCp50 library and contains a large insert including the SSN6 gene. pBSSSN6 and YEp(112) SSN6 were constructed by insertion of the 6.1-kb XbaI-SphI fragment of YCpSSN6 into the polylinker of pBSM13(+) and YEplac112 (GIETZ and SUGINO 1988 ), respectively.

The epitope tagged version of SSN6 was constructed as follows. The synthetic DNA encoding the c-myc 9E10 epitope (5'- CGAACAAAAGCTTATTTCTGAAGAAGACTTGGG -3' and 5'-CAAGTCTTCTTCAGAAATAAGCTTTTGTTCGCC-3') was ligated into the unique Bsg I site at the 3' end of the coding sequence of SSN6 in pBSSSN6 creating pBSSSN6e. YEp(112) SSN6e was constructed by replacing the 2.2-kb MluI-XbaI fragment from YEp(112)SSN6 with the MluI-XbaI fragment from pBSSSN6e.

The lacZ fusion plasmids YCp(33)ANB1/lacZ (LOWRY et al. 1990 ) and pSL1169, containing a STE2/lacZ fusion on a 2 µm-based URA3 plasmid (HWANG-SHUM et al. 1991 ) were described previously.

Mutagenesis of TUP1:
(i) PCR mutant pools were generated as follows: A 2.2-kb fragment containing TUP1 5' flanking sequences plus codons 1–275 was amplified from plasmid YEp(181)TUP1 using the primers 5'-TCCCAGTCACGACGT-3' and 5'-CACCGCAGAAGGAGGAATCC-3'. PCR was carried out for 30 cycles with 10 ng of template under the conditions recommended by the vendor. Four separate reactions were performed to obtain independent pools. The reaction products were digested with Sac I and BamHI and fractionated on an agarose gel, and the desired product was purified and ligated into the Sac I-BamHI sites of YEp(181)TUP1.

A 1.8-kb fragment containing codons 226–713 plus 3' flanking sequences was amplified from YEp(181)TUP1 using the primers 5'-AACAGCTATGACCATG -3' and 5'-CCCTCTG TCAAGGCACCTGAATCTACG -3'. Reactions were carried out as above. The reaction products were digested with HindIII and BamHI and fractionated on an agarose gel, and the desired product was purified and ligated into the HindIII-BamHI sites of YEp(181)TUP1.

(ii) YEp(181)tup1-S593P was constructed as follows. A 2.8-kb fragment containing TUP1 5' flanking sequences plus codons 1–601 was amplified as recommended by the vendor for 15 cycles from 100 ng of the plasmid YEp(181)TUP1 using the primers 5'-CAACAGATCTATCTAATGAGCCGGGTA CAACGCTTTGTCC-3' and 5'-TCCCAGTCACGACGT-3'. The product contains a single base pair substitution (shown in bold) resulting in an amino acid change from serine to proline at codon 593. The reactions products were digested with Sac I and Bgl II, fractionated on an agarose gel, and ligated into the Sac I-Bgl II sites of YEp(181)TUP1.

(iii) YEp(181)tup1-T460P and YEp(181)tup1-T695P were constructed using the megaprimer method of site-directed mutagenesis (BARIK 1995 ) as follows.

YEp(181)tup1-T460P : A 420-bp fragment containing codons 325–465 was amplified from the plasmid YEp(181)TUP1 using the primers 5'-GTCTGTCTTCAGCACCTGGTGCCAAAAAT TTCCCATCTG - 3' and 5'-CAACCCGGCACTACC - 3'. The product contained a base pair substitution (shown in bold) resulting in an amino acid change from threonine to proline at codon 460 and was used as a megaprimer with the primer 5'-AACAGCTATGACCATG-3' and the template YEp(181)TUP1 to generate a 1-kb fragment containing codons 325–713 plus 3' flanking sequences. The reaction products were digested with Bgl II and HindIII and fractionated on an agarose gel, and the desired product was purified and ligated into the BamHI-HindIII sites of YEp(181)TUP1bbg.

YEp(181)tup1-T695P : A 440-bp fragment containing codons 577–698 was amplified from the plasmid YEP(181)TUP1 with the primers 5'-CCGCTACCAGGAGCAAAAACGTTATA-3' and 5'-ACTCTGTTTATAGCG - 3'. This product contained a base pair substitution (shown in bold) resulting in an amino acid change from threonine to proline at codon 695 and was used as a megaprimer with the primer 5'-AACAGCTATGACCATG -3' and the template YEp(181)TUP1 to generate a 730-bp fragment containing codons 577–713 plus 3' flanking sequences. The reaction products were digested with Bgl II and HindIII and fractionated on an agarose gel, and the desired product was purified and ligated into the Bgl II-HindIII sites of YEp(181)TUP1.

YEp(181)tup1-443-555 was constructed as follows. A 750-bp fragment containing TUP1 codons 556–713 plus 3' flanking sequences was amplified from the plasmid YCp(22)TUP1 using the primers 5'-AACAGCTATGACCATG -3' and 5'-TC CGGATCCGAGACCGGATTCTTGGTGGAA-3'. The reaction products were digested with BamHI and HindIII, fractionated on an agarose gel, and the desired product was purified and ligated into the Bgl II and HindIII sites of pBSTUP1BBg generating pBStup1-443-555. pBStup1-443-555 was then digested with Sac I and HindIII and the 3.9-kb fragment was ligated into the Sac I-HindIII sites of YEplac181.

In each of the above constructs, the presence of the desired mutation was verified by DNA sequence analysis.

Yeast protein extractions:
Yeast cells were grown in SD (ROSE et al. 1990 ) lacking specific growth requirements to mid-log phase, chilled to 0°, harvested by centrifugation for 10 min at 3000 g at 4°, washed once in extraction buffer [200 mM Tris-HCl (pH 8), 200 mM (NH₄)₂SO₄, 10 mM MgCl₂, 1 mM EDTA, 10% glycerol], resuspended in 0.5 ml of extraction buffer with protease inhibitors (1 mM pepstatin, 1 µg of leupeptin per ml, 1 mM phenylmethylsulfonyl fluoride), and broken by vigorous mixing with glass beads for seven cycles of one minute of mixing followed by one minute of cooling on ice. The extract was clarified by centrifugation for 1 hr at 5000 g at 4° (WILLIAMS et al. 1991 ). Protein concentration was determined by the Bradford assay (Bio-Rad, Richmond, CA).

Western (immunoblot) analysis:
(i) Denaturing gels: The yeast protein extracts were boiled with SDS-PAGE sample buffer and fractionated on SDS-10% polyacrylamide gels (PAGE) (AUSUBEL et al. 1994 ). Protein was electrophoretically transferred to nitrocellulose, and then analyzed by Western analysis (AUSUBEL et al. 1994 ) either with rabbit polyclonal antisera prepared against the Tup1 protein or with monoclonal antibody against the c-myc 9E10 epitope (Oncogene) for visualization of Ssn6. The primary antibodies were detected using goat anti-rabbit or goat anti-mouse IgG horseradish peroxidase conjugate (Bio-Rad) (AUSUBEL et al. 1994 ). The conjugated secondary antibodies were detected by enhanced chemiluminescence (ECL) reaction from Amersham (Arlington Heights, IL).

(ii) Nondenaturing gel:
The yeast protein extracts were fractionated on non-denaturing 5% polyacrylamide gels (WILLIAMS et al. 1991 ) and subsequently transferred to nitrocellulose electrophoretically. The protein pattern was analyzed by Western analysis as described above.

Enzyme assays:
For invertase assays glucose repressed cells were grown in SD (ROSE et al. 1990 ) lacking leucine and harvested in exponential phase as described previously (GOLDSTEIN and LAMPEN 1975 ). To measure cell density EDTA was added to 20 mM to disperse the cell clumps in flocculent cultures. Secreted invertase was assayed in whole cultures, and the activity was expressed as micromoles of glucose released per minute per 100 mg (dry weight) of cells. ß-galactosidase assays were carried out as described previously (ROSE et al. 1990 ). All enzyme assays were carried out multiple times on at least two different transformants.

Flocculence assays:
RZ53-6tup1 transformed with both the wild type and mutant TUP1 alleles was grown overnight in 5 ml of SD lacking leucine to late log phase. Each culture tube was vigorously mixed to disperse the aggregates, and 100 µl of culture was removed immediately. The cells were allowed to settle for five minutes, whereupon a second aliquot of 100 µl was removed from the aqueous portion of the culture. Each 100 µl sample was added to 900 µl of SD-leu with 20 mM EDTA, and the cell density was measured. Flocculence was determined by the equation: [A₆₀₀ at t₀ - A₆₀₀ at t_{5 min}]/[A₆₀₀ at t₀ x 5 min] = % culture settling out per minute.

Mutant selection scheme:
Missense mutations in TUP1 were obtained using random mutagenesis in the following selection scheme. The aerobically expressed TIF51A gene and the heme-repressed ANB1 gene are homologs encoding isoforms of the essential protein eIF-5a. Cells carrying a null allele of TIF51A cannot grow aerobically unless ANB1 is expressed constitutively as in a tup1, ssn6, or rox1 deletion strain. Strain MZ14-29 is a tif51a tup1 leu2 homozygous diploid containing an ANB1/lacZ disruption of the URA3 gene. This strain could not be transformed to leucine prototrophy by a 2µ plasmid containing the LEU2 and TUP1 genes because the TUP1 gene resulted in aerobic repression of ANB1 and subsequent cell death due to a lack of eIF-5a. However, using a plasmid pool in which mutations were targeted to the TUP1 gene, leu+ transformants were obtained, presumably from plasmids bearing tup1 mutations. Constitutive expression of ANB1 in these transformants was confirmed using the ANB1/lacZ fusion by testing for blue color on X-gal plates. To screen out frame shift and nonsense mutations or missense mutations leading to highly unstable protein, whole yeast extracts were prepared from colonies which were constitutively expressing ANB1, and Western analysis was performed using Tup1 polyclonal antisera. Those colonies that produced stable, full-length Tup1 were examined further. Approximately 500 putative tup1 mutants were screened by Western analysis. The plasmid DNA was recovered from each mutant and used to retransform MZ12-16 (tup1,TIF51A) to confirm the mutant phenotype was plasmid borne.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Isolation of TUP1 mutations:
Utilizing the selection described in MATERIALS AND METHODS, the following three missense mutations in TUP1 were isolated: L62R, S595P, and S647P. The latter two mutations each altered a conserved serine at different positions in the fourth and fifth WD repeats, respectively (Figure 1), and, as documented below, each affected the Tup1 repressed genes differently. Such differential effects could arise from either a difference in the role of the two different serines within a WD repeat or a difference in the function of the two WD repeats. To distinguish between these possibilities, three new mutant alleles were generated by site-directed mutagenesis: T460P, S593P, and T695P. Each contained a serine or threonine to proline substitution in the residue corresponding to S647 in the first, fourth, and sixth WD repeat, respectively (Figure 1). We also constructed a deletion mutation that precisely excised the first three WD repeats (443-555). The cellular levels of the mutant proteins were determined by Western analysis. As demonstrated in Figure 2, L62R, S593P, S595P, and S647P (Figure 2A) levels were similar to wild type, but T460P (Figure 2B) and T695P (Figure 2C) protein accumulated to lower levels. The 443-555 mutant did not accumulate detectable levels of protein (data not shown). This deficiency was not due to the inability of the antibody to detect the deletion protein, since truncated versions of Tup1 lacking all sequences after residue 291 were readily detected (data not shown).

View larger version (21K): In this window In a new window Download PPT slide   Figure 1. The alignment of the Tup1 WD repeats. Regions of the WD repeats of Tup1 are aligned to maximize homology. The numbers refer to the first residue in each repeat and the dashed lines represent gaps. The residues that were changed to prolines in this study are shown in bold. The last line represents the consensus for WD repeats where B represents hydrophobic residues and the dashed lines represent gaps.

View larger version (27K): In this window In a new window Download PPT slide   Figure 2. Cellular levels of mutant Tup1 proteins. Western blots of denaturing gels were carried out with 25 µg of crude protein extract prepared from the tup1 strain MZ12-16 grown aerobically in SD-leucine media and carrying the following plasmids: (A) YEp(181)TUP1 (Wt), YEplac181 (tup1), YEp (18)tup1-L62R, YEp(181)tup1-S593P, YEp(181) tup1- S595P, and YEp(181) tup1-S6 47P. (B) YEp(181)TUP1 (Wt), YEplac181(tup1), and YEp(181)tup1-T460P. (C) YEp(181)TUP1 (Wt), YEplac181(tup1), and YEp (181)tup1-T695P. The position of the Tup1 is indicated by the arrow on the right side of the panel. The blot was probed with polyclonal antisera raised against Tup1 and developed using the ECL system (Amersham).

Interactions of mutant Tup1 with Ssn6:
One function of Tup1 is to associate with Ssn6, thus we investigated this complex formation by the mutant proteins. Yeast extracts were prepared from cotransformants carrying YEp(112)SSN6e, an epitope-tagged SSN6 allele, and each of the TUP1 mutant plasmids. The extracts were fractionated on a nondenaturing polyacrylamide gel, and complex formation was analyzed by Western analysis with primary monoclonal antibody against the c-myc 9E10 epitope. The results are presented in Figure 3A. Ssn6 was visualized rather than Tup1 because the differential migration between free versus complexed protein is greater for Ssn6 than for Tup1; Tup1 itself forms a multi-meric complex that migrates almost as slowly as the Tup1/Ssn6 complex. The Tup1/Ssn6 complex can be visualized as a slowly migrating band as seen in a comparison of the migration of free Ssn6 in a tup1 strain (lane 8) with that of the complex in a strain containing wild-type Tup1 (lane 1). The N-terminal mutant, L62R was unable to form a complex with Ssn6 (lane 7). The three WD repeat mutants, S593P, S595P, and S647P (lanes 3, 4, and 5), were all capable of interacting with Ssn6. Surprisingly, the Tup1/Ssn6 complex was neither detected in T460P (lane 2) nor T695P (lane 6) extracts. This may be directly due to the inability of the T460P and T695P mutants to form complexes with Ssn6, but we believe, given the repression activity of these mutant proteins described below, that these results reflect the low Tup1 levels in these mutants, which left most of the Ssn6 free and made detection of the complex difficult.

View larger version (52K): In this window In a new window Download PPT slide   Figure 3. Tup1-Ssn6 and Tup1-Tup1 interactions of mutant proteins. (A) 25 µg of crude protein extract was prepared from tup1 strain MZ12-16 cotransformed with YEp(112)SSN6e and each of the following Tup1 alleles: YEp(181)TUP1 (Wt), YEp(181)tup1-T460P, YEp(181)tup1-S593P, YEp(181)tup1-S595P, YEp(181)tup1-S647P, YEp(181) tup1-T695P, YEp(181)tup1-L62R, and YEplac181 (tup1). Extracts were fractionated on a nondenaturing gel and probed with a monoclonal antibody against the c-myc epitope tag of Ssn6. The Tup1/Ssn6e complex is indicated by the filled triangle (); the free Ssn6e is indicated by the arrow (). (B) Crude protein extract (25 µg) was prepared from the tup1 strain MZ12-16 cotransformed with YEp(112)SSN6e and each of the following TUP1 alleles: YEp(181)TUP1 (Wt), YEplac181 (tup1), and YEp(181)tup1-L62R. Extracts were fractionated on a non-denaturing gel and probed with polyclonal antisera raised against Tup1. The complex is indicated by the arrow.

The L62R allele is able to form multimers:
The Tup1/Ssn6 repression complex is composed of three or four Tup1 subunits and one Ssn6 subunit (VARANASI et al. 1996 ; REDD et al. 1997 ). Both the multimerization domain and the Ssn6 interaction domain of Tup1 were reported to reside in the first 72 amino acids of Tup1 (WILLIAMS et al. 1991 ; TZAMARIAS and STRUHL 1994 ; TZAMARIAS and STRUHL 1995 ; VARANASI et al. 1996 ). As shown in Figure 3A, the N-terminal L62R mutant was unable to complex with Ssn6. To examine the ability of this mutant to multimerize, we performed non-denaturing Western analysis with polyclonal antisera against Tup1 and compared the size of the wild-type complex to that formed by the L62R mutant. As seen in Figure 3B, the mutant protein (lane 3) migrated only slightly faster than did the wild type (lane 1).

Effects of the TUP1 mutants on different Tup1 regulated genes:
The effects of these mutants on oxygen repression (ANB1/lacZ ), catabolite repression (invertase activity), cell type regulation (STE2/lacZ), and flocculence were assessed and the results are presented in Table 1 and summarized in Figure 4. The L62R mutant was completely derepressed for ANB1 expression and showed little repression of SUC2 expression (25%) and flocculence (18%). In contrast, this mutant repressed STE2 expression to 83% of the wild type level. This ability to repress STE2 expression in the absence of the Tup1-Ssn6 interaction probably arises from the ability of the 2 repressor to bind both Tup1 and Ssn6 (KOMACHI et al. 1994 ; SMITH et al. 1995 ). Consequently, the repression complex may still be localized to the STE2 locus.

View larger version (37K): In this window In a new window Download PPT slide   Figure 4. The Tup1 mutants exhibit differential repression. Percent repression for the three different genes were obtained from the data in Table 1. The mutants are indicated below. The hatched, filled, and unfilled bars represent the percent repression of Tup1 mutants of ANB1, STE2, and SUC2, respectively.

The WD repeat mutants exhibited different effects on the different regulons. All of the mutants, including the deletion of the first three repeats, repressed expression of the SUC2 and flocculence genes to near wild-type levels. On the other hand, ANB1 expression was derepressed in the T460P (first repeat) and 443-555 mutants but repressed to significant levels in the S593P and S595P (fourth repeat), S647P (fifth repeat), and T695P (sixth repeat) mutants. The repression of STE2 expression showed a different pattern. Expression was completely derepressed in the T460P (first repeat), S647P (fifth repeat), and 443-555 mutants, but repressed to near wild-type levels in the S593P and S595P (fourth repeat) and T695P (sixth repeat) mutants. Thus, as more easily visualized in Figure 4, the mutations in the WD repeats had differential effects on the repression of the four regulons assayed.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In this study we report the isolation and characterization of six single amino acid substitutions in the general repressor protein Tup1 acquired through both random mutagenesis and site directed mutagenesis.

The N-terminal mutant L62R was unable to interact with Ssn6, but retained the ability to homo-oligomerize. This demonstrates that interaction with Ssn6 is not necessary for Tup1 oligomerization. This is in agreement with both two-hybrid data and in vitro studies with truncated Tup1 (TZAMARIAS and STRUHL 1994 ; TZAMARIAS and STRUHL 1995 ). This mutant was constitutive for the expression of the hypoxic ANB1 gene and the glucose-repressed SUC2 gene and was flocculent, but repressed expression of the a-mating type STE2 gene. Since 2 can interact with both Tup1 and Ssn6, it may serve to stabilize the mutant complex. If so, the lack of repression in the other regulons suggests that the Rox1 and Mig1 repressor interact with Tup1 and Ssn6 in a different manner.

All the remaining mutations fell within the WD repeats, and perhaps the most intriguing finding to emerge from our analysis is that these mutations affected the regulons studied here differently. The WD repeat region of Tup1 is required for full Tup1-dependent repression of at least two of the four regulons studied here. Deletion of the first three repeats resulted in full derepression of ANB1 and STE2. However, SUC2 was only partially derepressed (57%), and the cells exhibited only mild flocculence, and even this partial loss of repression may have been due to the severely reduced amount of mutant Tup1 protein in the cell. The requirement of the WD repeats for STE2 repression is not surprising given that the 2 repressor has been demonstrated to interact with this region. On the other hand, the Rox1 repressor of the hypoxic genes has been shown to interact with Ssn6, but it is possible that it has some weak interaction with Tup1 also. Alternatively, the requirement of hypoxic gene repression for the WD repeats could stem from an interaction between these repeats and the general transcriptional machinery to achieve repression.

It has been proposed (GARCIA-HIGUERA et al. 1996 ) that the structure of the Tup1 WD region is homologous to that derived for the WD repeats of Gß transducin where each repeat forms the blade of a propeller-like structure. One of our original mutations, S647P, fell within what appeared to be a critical residue for the structural integrity of the ß-sheet structure of the fifth WD repeat. This same mutation was generated in the first, fourth, and sixth repeats on the assumption that each repeat has the same basic structure. For the purposes of the discussion below, we assume, based upon the Gß transducin structure, that the mutated serines themselves are buried within the blade structure and, therefore, are not directly involved in interactions with other proteins. Consequently, we interpret the loss of activity of any of our mutant proteins as resulting from a loss of the structural integrity of the mutated WD repeat rather than a loss of a specific interaction with the mutated serine. This assumption allows us to make general conclusions about the roles of the specific WD repeats.

The flocculent phenotype and SUC2 regulation were only minimally affected by any of the WD repeat missense mutations. These results demonstrate that the Tup1 mutant proteins are functional and that despite the low levels of protein in the mutants T460P and T695P, the protein that is made can associate with Ssn6. If there is any requirement for the WD repeats at all by these two regulons, no repeat is uniquely required for regulation. Also, the retention of repression activity of these regulons indicates that none of the mutated repeats is uniquely required for interaction with the transcriptional machinery. The Mig1 repressor of the glucose-repressed genes has been reported to interact with TPR (tetratricopeptide) repeats seven through ten of Ssn6, but complete repression requires that TPR repeats one to three be present to interact with Tup1 (TZAMARIAS and STRUHL 1995 ). Thus it is possible that no Mig1-Tup1 interaction occurs.

Unlike the case with flocculence and SUC2 repression, the mutations in either the first or fifth repeats resulted in complete derepression of STE2. The 2 repressor is known to interact with the WD repeats of Tup1, and it is tempting to speculate that it binds to blades one and five of the propeller. But these blades separated by the sixth blade, where mutations do not affect STE2 repression dramatically. However, contact could be made from the top or bottom surface (as is the case for G and G for Gß transducin) skipping over or spanning the sixth repeat.

As for STE2, the mutation in the first repeat caused complete derepression of ANB1, but unlike the case of STE2 only partial loss of ANB1 repression resulted from a mutation in the fifth repeat. Rox1 has been reported to require Ssn6 TPR repeats four through seven for specific repression as well as TPR repeats one through three, presumably for their interactions with Tup1 (TZAMARIAS and STRUHL 1995 ). Rox1 has not been demonstrated to contact the Tup1, but our results raise the possibility that some interaction might occur. Alternatively, although seemingly less likely, the first repeats might be involved in some general repression mechanism that is required for repression of the a-mating type and hypoxic genes but not by glucose-repressed or flocculent genes.

Summarizing these results, the regulons studied here were affected differently by the battery of mutations in the TUP1 gene. It appears that the Tup1/Ssn6 repression system is both an economical and flexible method of regulation. The DNA binding proteins that are known to be involved in repression of the different systems (Mig1, Rox1, and 2) show no sequence or structural similarities, but all have recruited the Tup1/Ssn6 complex to achieve repression. Our demonstration here that the same mutation in different WD repeats has deferential effects on repression further reinforces the view that each regulon evolved independently, recruiting the repression complex through different interactions.

Note: While this manuscript was in revision, KOMACHI and JOHNSON 1997 reported the isolation of 12 missense mutations in the WD repeat region of Tup1 which disrupt specific interactions between the WD repeats of Tup1 and 2. Their mutants were different than the mutants described here. The effect of their mutations on MFA2 (an a-mating type gene) expression was more allele specific than WD repeat specific. Six of their mutations were tested for repression of SUC2 and ANB1, and none exhibited a signficant effect. The differences between their findings and those reported here probably reflect a difference in the selections. Since they selected for a disruption of the 2-Tup1 interaction but maintenance of general repression activity, all their mutations lay clustered on one surface of the WD propellar as they suggested from the presumed homology between the Tup1 and Gß structure. Therefore, most of their mutations are not expected to seriously disrupt the structure of a given blade of the propellar as is the case with the residues mutated in this present study. Thus these two studies are complementary rather than duplicative.

	ACKNOWLEDGMENTS

We thank ALEXANDER KASTANIOTIS and MING ZHANG for help in some plasmid constructions and MARGYE ZITOMER for strain constructions. We thank GEORGE SPRAGUE for the STE2/lacZ plasmid and PETER SHERWOOD for advice for the invertase assays. This study was supported by National Institute of General Medical Science grant 26061 to R.S.Z. and the Burke Fellowship to P.M.C.

Manuscript received September 3, 1997; Accepted for publication October 28, 1997.

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