Sit4 Phosphatase Is Functionally Linked to the Ubiquitin-Proteasome System

Corresponding author: Wolfgang Hilt, Universität Stuttgart, Pfaffenwaldring 55, 70569 Stuttgart, Germany., hilt{at}po.uni-stuttgart.de (E-mail)

Communicating editor: F. WINSTON

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Using a synthetic lethality screen we found that the Sit4 phosphatase is functionally linked to the ubiquitin-proteasome system. Yeast cells harboring sit4 mutations and an impaired proteasome (due to pre1-1 pre4-1 mutations) exhibited defective growth on minimal medium. Nearly identical synthetic effects were found when sit4 mutations were combined with defects of the Rad6/Ubc2- and Cdc34/Ubc3-dependent ubiquitination pathways. Under synthetic lethal conditions, sit4 pre or sit4 ubc mutants formed strongly enlarged unbudded cells with a DNA content of 1N, indicating a defect in the maintenance of cell integrity during starvation-induced G₁ arrest. Sit4-related synthetic effects could be cured by high osmotic pressure or by the addition of certain amino acids to the growth medium. These results suggest a concerted function of the Sit4 phosphatase and the ubiquitin-proteasome system in osmoregulation and in the sensing of nutrients. Further analysis showed that Sit4 is not a target of proteasome-dependent protein degradation. We could also show that Sit4 does not contribute to regulation of proteasome activity. These data suggest that both Sit4 phosphatase and the proteasome act on a common target protein.

PROTEIN activity within the cell is controlled by three major mechanisms. Chemical modification of amino acid residues modulates the activity of proteins, while regulation of synthesis and selective proteolysis is implicated in controlling the cellular levels of proteins. Proteasomes are highly sophisticated protease complexes, which act as the major device for regulatory protein degradation in the cytoplasm and nucleus of the eukaryotic cell (for reviews see COUX et al. 1996 ; HILT and WOLF 1996 ; VOGES et al. 1999 ). The 26S proteasomes consist of a proteolytic core module, the 20S proteasome, and two 19S regulatory cap complexes attached to both ends of the 20S complex (PETERS 1994 ). The 20S proteasome is a hollow cylindrically shaped complex, which includes three different proteolytic activities. pre1-1 pre4-1 double mutants bearing mutations within ß-subunits in the center of the 20S proteasome show defective chymotrypsin-like and peptidyl-glutamyl-peptide-hydrolyzing (PGPH) activity and are strongly impaired in proteasome dependent proteolysis (HILT et al. 1993 ).

Nearly all proteins degraded via the 26S proteasome are marked by polyubiquitin chains. Ubiquitination of proteasomal substrates is performed by a complex enzyme system consisting of E1 (ubiquitin-activating enzymes), E2 (ubiquitin-conjugating enzymes), and, in many cases, E3 enzymes (ubiquitin-ligating enzymes; for reviews see JENTSCH 1992 ; CIECHANOVER 1994 ; HOCHSTRASSER 1996 ; SCHEFFNER et al. 1998 ; SOMMER 2000 ).

The ubiquitin-proteasome system is linked to a variety of different cellular pathways. Proteasomes are implicated in stress response by removal of abnormal proteins generated by heat stress, exposure of cells to amino acid analogs, or certain mutations (HEINEMEYER et al. 1991 , HEINEMEYER et al. 1993 ; HILT et al. 1993 ; HILLER et al. 1996 ; GERLINGER et al. 1997 ; PLEMPER et al. 1997 ). Proteasome-mediated destruction of defined substrate proteins is an essential regulatory step in many different cellular pathways, such as metabolic adaptation (MURAKAMI et al. 1992 ; KORNITZER et al. 1994 ; SCHORK et al. 1995 ), cell differentiation (CHEN et al. 1993 ; RICHTER-RUOFF et al. 1994 ), or cell cycle control (for reviews see DESHAIES 1995 ; HILT and WOLF 1996 ; KING et al. 1996 ; MANN and HILT 2000 ).

We performed a search for components that are physically or functionally linked to the proteasome system. We thereby discovered that a mutation residing in the SIT4 gene causes a synthetic effect when combined with proteasomal mutations. Originally, SIT4 was isolated in a screen for mutations that allowed expression of the HIS4 gene in the absence of its native transcription factors Bas1, Bas2, and Gcn4 (ARNDT et al. 1989 ). The SIT4/PPH1 gene codes for a serine/threonine protein phosphatase of the PP2A family (for overview see STARK 1996 ). Sit4 is implicated in the transcription of various genes and also has a function in control of the G₁ phase of the cell cycle (SUTTON et al. 1991A , SUTTON et al. 1991B ). A complex of Sit4 and the Tap42 protein, which acts as a regulator of Sit4 activity, is part of the TOR signaling pathway (DI COMO and ARNDT 1996 ; JIANG and BROACH 1999 ). The TOR pathway, which can specifically be inhibited by the drug rapamycin, controls protein translation in response to nutrient availability (HALL 1996 ; THOMAS and HALL 1997 ).

In some yeast strains the Sit4 phosphatase provides essential functions. Such vital necessity on SIT4 depends on the character of the polymorphous SSD1 gene (suppressor of sit4 deletion; SUTTON et al. 1991A ). Deletion of SIT4 is lethal in cells harboring an ssd1-d allele. In contrast, sit4 cells containing an SSD1-v allele are viable but grow with a significantly reduced rate. Phenotypic effects caused by the sit4-102 mutation also depend on the SSD1 allele present in the cell (SUTTON et al. 1991A ). The SSD1 gene codes for a 1250-amino-acid protein, whose exact function is still unclear. The SSD1 gene product has multisuppressor characteristics. Besides suppression of the sit4 deletion, SSD1-v alleles also partially cure defects that are caused by hyperactivation of the Ras-cyclase-cAPK pathway (SUTTON et al. 1991A ; WILSON et al. 1991 ), by ins1 (WILSON et al. 1991 ), by a bem2 mutation (KIM et al. 1994 ), or by failures of the cell integrity/protein kinase C (PKC1) pathway (COSTIGAN et al. 1992 ; LAPORTE et al. 1996 ). Ssd1 also functions in cell cycle control (SUTTON et al. 1991A ; CVRCKOVA and NASMYTH 1993 ; STETTLER et al. 1993 ; KIKUCHI et al. 1994 ; UESONO et al. 1997 ).

In this study we show that the Sit4 phosphatase and the proteasome are functionally connected. Because Sit4 does not control proteasomal activity and the proteasome does not degrade Sit4, this linkage seems to be indirect. The concerted action of both systems—which appears to be independent from the role of Sit4 in TOR-signaling—is required for cell maintenance during starvation-induced G₁ arrest.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Growth conditions:
Plasmids were amplified in Escherichia coli strains DH5, GM2163 (New England Biolabs, Beverly, MA), or DB6656. Bacteria were grown at 37° in LB medium, containing 50 µg/ml ampicillin for plasmid selection when necessary. Yeast cells were grown in YPD (1% yeast extract, 2% peptone, 2% glucose) or in synthetic minimal medium, SD (2% glucose, 0.67% Difco yeast nitrogen base without amino acids containing 20 µg/ml uracil, 20 µg/ml histidine, 30 µg/ml leucine as supplements). Ammonia-based synthetic complex dextrose (SC) media contained 120 mg/liter myo-inositol, 12 mg/liter para-aminobenzoic acid, and all amino acids at a concentration of 120 mg/liter with the exception of L-leucine, L-tryptophan, L-lysine, and L-histidine. If required for growth of strains containing auxotrophic mutations, SC medium was supplemented with 220 mg/liter L-leucine, 62.8 mg/liter L-histidine, 180 mg/liter L-lysine, 81.7 mg/liter L-tryptophan, 55.3 mg/liter adenine, or 22.4 mg/liter uracil. Selection for uracil auxotrophs was performed on SD medium containing 50 mg/liter uracil and 0.1% w/v 5-fluoroorotic acid (5-FOA). Yeast cells were sporulated in 1% potassium acetate medium.

General methods:
Transformation of E. coli cells, restriction reactions, DNA ligations, and other recombinant DNA techniques were performed following standard protocols as described in SAMBROOK et al. 1989 and AUSUBEL et al. 1990 . Mating of yeast cells, sporulation, tetrad dissection, plasmid segregation, and plasmid rescue were done as described (SAMBROOK et al. 1989 ; AUSUBEL et al. 1990 ; GUTHRIE and FINK 1991 ). Yeast transformations were performed by a modified version of the DMSO method (HEINEMEYER et al. 1991 ) or by the lithium acetate method (GIETZ et al. 1992 ).

Plasmids:
Plasmid pTS4 encoding the PRE1 and PRE4 genes was constructed by inserting a 1.15-kb EcoRI/BamHI fragment carrying PRE1 into the multicloning site of pDP83 [URA3 CEN14 ARS1] followed by the insertion of a 2-kb HindIII/SphI fragment carrying PRE4. Plasmid pTS5 was generated by insertion of a 1.5-kb EcoRI fragment containing the pre1-1 allele into the EcoRI site of plasmid pRS313 (SIKORSKI and HIETER 1989 ). Plasmid pTS6 was obtained by insertion of a 1.85-kb EcoRI/SacI fragment that contained the pre4-1 sequence into EcoRI/SacI sites of plasmid pRS313. Plasmid pTS9 [PRE1 PRE4 HIS3 CEN6 ARSH4] was made by consecutive insertion of a 1.9-kb EcoRI/SacI PRE4 fragment and a 1.5-kb EcoRI PRE1 fragment into plasmid pRS313. A 0.6-kb XbaI/EcoRV fragment containing the MET3 promoter was excised from plasmid pHAM8 (MOUNTAIN and KORCH 1991 ) and inserted into XbaI/EcoRV-digested pRS313 [HIS3 ARSH4 CEN6] (SIKORSKI and HIETER 1989 ), yielding plasmid pTS12. To generate plasmid pTS13, which contains PRE1 under the control of the MET3 promoter, a 0.6-kb fragment containing the PRE1 gene was amplified by PCR and inserted into the EcoRV site of pTS12. Plasmid pTS14 containing SIT4 controlled by the MET3 promoter was obtained by insertion of a 1-kb SIT4 fragment, which was amplified by PCR using p102/20 as the template. Plasmids pCK1 and pCK2 were obtained by insertion of a 2-kb SacII/PvuII fragment containing SIT4 into the SacII/SmaI sites of plasmid pRS305 [LEU2] and pRS315 [LEU2 ARSH4 CEN6], respectively (SIKORSKI and HIETER 1989 ). To construct plasmid pES1, which contains a truncated version of SIT4, plasmid pCK2 was digested with BglII/NruI, blunted, and religated. To generate pTS100 [sit4::URA3 LEU2], the HindIII site in the backbone of plasmid pCK2 was removed by digestion with HindIII, filling of the ends by treatment with Klenow enzyme, and religation. Thereafter, almost the complete SIT4 open reading frame (codons: 14–312 and 8 bp from the 3'-region) was excised by digestion with XbaI/SphI. Overhanging ends were blunted by treatment with Klenow enzyme and T4-DNA polymerase and, using a HindIII linker, a 1.2-kb HindIII fragment containing the URA3 gene was inserted, yielding pTS100. The plasmid pTS64 [SSD1-v LEU2 ARSH4 CEN6] was constructed by insertion of a 4.7-kb PvuII fragment containing the SSD1-v allele into the SmaI site of vector pRS315 (SIKORSKI and HIETER 1989 ). The plasmids CB239 [HA-SIT4 LEU2] and CB243 [SIT4-HA LEU2] containing epitope-tagged versions of SIT4 were a gift of K. Arndt. To isolate the sit4-51 allele, plasmid pES1 was gapped by XbaI/SphI digestion. The linear fragment that contained SIT4 5'- and 3'-flanking regions (572-bp SphI/PvuII fragments and 464-bp SacII/XbaI fragments) at its ends was used to transform the sit4-51 strain YTS98/5b. The repaired plasmid pES2 harboring the sit4-51 mutant sequence was isolated from leucine prototrophic transformants.

Yeast strains:
YTS40 cells were made by transforming WCG4a/ cells with a linear 1.98-kb SacII/SalI fragment derived from pTS100 that contained the sit4::URA3 allele. Correct integration of the sit4 sequence was verified by Southern analysis. A sit4 strain that was viable due to the presence of a plasmid-encoded SSD1-v allele (YTS43) was isolated from tetrads of YTS40 diploids that had been transformed with pTS64 (SSD1-v). WCG4 isogenic sit4-51 cells were made by one-step gene replacement. YTS43 cells were transformed with a linear 2-kb fragment harboring the sit4-51 mutant sequence isolated from the SacII/PvuII-digested pES2 plasmid. Cells that had lost the URA3 marker gene due to homologous recombination of the sit4-51 sequence with the chromosomal sit4::URA3 locus were selected on 5-FOA medium. Correct integration of the sit4-51 mutant gene was verified by Southern analysis. After growth on nonselective medium, cells were screened for clones that had lost the SSD1-v LEU2 encoding plasmid during mitotic division, yielding the sit4-51 single-mutant strain YTS100. WCG4 isogenic sit4-102 cells (strain YSH2) were obtained by a similar strategy. The sit4 pre1-1 pre4-1 triple-mutant YTS102 was constructed by dissection of tetrad spores from a diploid strain (YTS101) obtained by crossing strains YTS100 (sit4-51) and YHI29/14 (pre1-1 pre4-1). Both the sit4-51 single mutation and the pre1-1 pre4-1 double mutations cause temperature sensitivity (ts; HILT et al. 1993 ). Therefore, temperature-sensitive segregants derived from a nonparental ditype tetrad showing a 2:2 segregation for the ts phenotype should harbor all three mutations. Such clones were confirmed to contain the pre1-1 and pre4-1 mutations by measuring their defects for 20S proteasomal chymotrypsin-like and PGPH activity. The identity of a segregant with these properties, YTS102, as a sit4-51 pre1-1 pre4-1 triple-mutant strain was further confirmed by backcrossing with wild-type cells (WCG4). As expected, tetrads derived from such diploids showed 4:0, 3:1, and 2:2 segregation patterns for the ts phenotype. The sit4-102 pre1-1 pre4-1 mutant strain YSH3 was constructed following a similar strategy using diploid cells obtained by crossing YSH2 (sit4-102) with YHI30/14 cells (pre1-1 pre4-1). Strains YMHO17 [sit4 (HA-SIT4)] and YMHO18 [sit4 (SIT4-HA)] were derived from tetrad dissection of YTS40 diploids transformed with CB239 and CB243, respectively. cdc34-1 cells (strain YSH10) and rad6 cells (strain YSH12) with the genetic background of WCG4 were generated by backcrossing strains W432 and YWO62 four times with WCG4 cells. To generate sit4-51 ubc1 double mutants, YHI124/12D cells [ubc1- (UBC1, URA3)] were crossed with YTS100 cells, then sporulated, and dissected. Resulting sit4-51 ubc1 segregants were then selected for loss of the complementing UBC1 plasmid by streaking on 5-FOA plates. ubc10 sit4-51, ubc11 sit4-51, and ubc13 sit4-51 cells were obtained from a cross of YTS100 cells with Y04763 (ubc10::kanMX4), Y01636 (ubc11-kanMX4), and Y04027 (ubc13::kanMX4) strains (Euroscarf), respectively. The other sit4 ubc mutants were derived from crosses of sit4-51 cells with strains YWO23, W303BP, W303BQ, and 8b. All yeast strains are listed in Table 1.

Genetic mapping of the chromosomal sli1-1 locus:
A 2-kb SacII/PvuII complementing fragment (derived from plasmid pCK1) containing the SIT4 gene linked to the LEU2 auxotrophic marker gene was integrated in strain YTS91 at its native chromosomal locus. The resulting strain YTS93 was backcrossed with YTS21. All tetrads derived from the resulting diploids showed 4:0 segregation of viability on 5-FOA medium, evidencing that the sli1-1 mutation is allelic to the SIT4 gene. An identical genetic analysis using integration of the complementing SSD1-containing DNA resulted in both viable and nonviable spores on 5-FOA, confirming that sli1-1 is not allelic with SSD1.

Purification of the 20S proteasome and activity assays:
20S proteasomes were partially purified by gel filtration as described in FISCHER et al. 1994 . In vitro assays for proteasomal activities were done according to HILT and WOLF 1999 . Pulse-chase experiments for fructose-1,6-bisphosphatase (FBPase) degradation were done as described in GUECKEL et al. 1998 . For cycloheximide chase cells were grown in SC medium to an OD₅₇₈ of 1.5–2. To block protein synthesis, cycloheximide was added to a final concentration of 0.5 mg/ml. At indicated times, cells were harvested and suspended in 1 ml H₂O (OD₅₇₈ = 3) and thereafter lysed by the addition of 150 µl 1.85 M NaOH, 7.5% 2-mercaptoethanol solution (10 min incubation at 0°). Proteins were precipitated by addition of trichloroacetic acid (final concentration 5%). Precipitates were solubilized in 200 mM Tris/HCl pH 6.8, 8 M urea, 5% SDS, 0.1 mM EDTA, and 1.5% dithiothreitol and proteins were separated by electrophoresis on 10% SDS-polyacrylamide gels. After blotting onto nitrocellulose membranes, proteins were visualized using monoclonal mouse-anti-hemagglutinin (HA) antibodies (12CA5; Roche Diagnostics) by enhanced chemiluminescence detection (Amersham Pharmacia Biotech).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Mutations that cause synthetic lethality with an impaired proteasome:
To identify genes/proteins whose function is linked to the proteasome system we performed a screen for mutations that cause synthetic lethality when combined with impaired proteasomal proteolysis (sli mutations: synthetic lethality with impaired proteasome function). The yeast strain used for this screen (YTS21) harbored mutations in two chromosomal 20S proteasome ß-type genes (pre1-1 pre4-1) and the complementing plasmid pTS4 containing both wild-type genes PRE1 and PRE4 and the URA3 marker gene. As evidenced by activity tests for proteasome-mediated peptide cleavage, YTS21 cells exhibit wild-type activity. pre1-1 pre4-1 mutant cells are viable (HILT et al. 1993 ). Thus, on nonselective medium YTS21 cells are able to lose plasmid pTS4 during mitotic division. Cells can thereby switch from wild type to proteasome mutant background and become uracil auxotrophs. YTS21-derived mutants containing a sli mutation are expected to grow only when the proteasome is functional and therefore depend on the presence of plasmid pTS4. Thus sli mutants cannot lose pTS4 and can therefore be detected by their inability to grow on 5-FOA medium, which is toxic for uracil prototrophs.

YTS21 cells were mutagenized with ethyl methanesulfonate to a residual viability of 10% and plated on SC (Ura^-) plates. To enable mitotic loss of the plasmid pTS4, the cells were transferred to nonselective medium (YPD). Thereafter a total of 2500 clones were replica plated on 5-FOA containing minimal agar medium and screened for strains that could not grow under these conditions. Potential sli mutants were retested by streaking on 5-FOA medium, yielding 15 sli mutants. These mutants were backcrossed to their respective wild-type cells (YTS21) and the diploids were subjected to sporulation and tetrad analysis. By these means, seven mutants were proven to contain single gene mutations as shown for the sli1-1 (sit4-51) mutant YTS91 (Fig 1). Crossing of these sli mutants resulted in the identification of at least four complementation groups. To exclude the possibility that 5-FOA-induced lethality of the mutants was based on an undesired chromosomal integration of the URA3 gene, the sli mutants were transformed with plasmid pTS9 containing the PRE1 and PRE4 wild-type genes. With the exception of one clone, all pTS9 transformants regained the ability to lose plasmid pTS4, demonstrating that lethality was due to a new mutation in the genetic background of strain YTS21. To confirm that lethality of sli mutants was not based on undesired lethal mutations within the pre1 or pre4 coding sequences, we tested whether growth of the clones could be restored by complementation with plasmid-derived pre1-1 or pre4-1 alleles. After transformation with either pTS1 (pre1-1 HIS3) or pTS6 (pre4-1 HIS3), all clones remained unable to grow on 5-FOA medium, demonstrating that the sli mutations were not allelic with pre1-1 or pre4-1.

View larger version (57K): In this window In a new window Download PPT slide   Figure 1. YTS91 cells contain a single gene mutation that confers synthetic lethality with an impaired proteasome. Growth of yeast cells on SC-5-FOA medium (30°, 3-day incubation). YTS21 [pre1-1 pre4-1 (PRE1 PRE4 URA3)]: strain used for EMS mutagenesis and synthetic lethal screening; YTS91 [sli1-1 (sit4-51) pre1-1 pre4-1 (PRE1 PRE4 URA3)]: mutant strain isolated in the synthetic lethal screen. Tetrad spores derived from a cross of YTS21 x YTS91 show 2:2 segregation of synthetic lethality (shown is a typical tetrad: YTS98/1a/1b/1c/1d).

SIT4/PPH1 and SSD1 complement the synthetic growth defect of sli1 mutants:
A sli1-1 mutant strain (YTS98/5b) was used to isolate complementing DNA. Cells were transformed with a YCplac111-derived Saccharomyces cerevisiae genomic DNA library. Screening of 25,000 transformants yielded 50 clones with restored viability on 5-FOA medium. To avoid isolation of library plasmids harboring a complementing PRE1 gene, plasmids were isolated from transformants that still showed defective proteasomal chymotrypsin-like activity. In this way two plasmids, p102/20 and p102/25, which contained independent genomic inserts, were obtained.

Plasmid p102/20 harbored a 2.8-kb genomic fragment and contained the entire open reading frame of SIT4/PPH1, which by subcloning was proven to be responsible for complementation of the synthetic defect of the sli1-1 mutants (YTS91 cells). SIT4 codes for a serine/threonine phosphatase involved in gene expression and cell cycle control (ARNDT et al. 1989 ; POSAS et al. 1991 ; SUTTON et al. 1991A ; FERNANDEZ et al. 1992 ). The second plasmid, p102/25, harbored a genomic insert of 12 kb in length. Subcloning restricted the complementing DNA to a 4.7-kb PvuII fragment, containing the complete open reading frame of the SSD1 gene. SSD1 was known to function as a low-copy suppressor of certain Sit4-related defects (SUTTON et al. 1991A ).

A genetic analysis (ROSE and BROACH 1991 ) was performed to uncover the chromosomal location of the mutation conferring the synthetic growth defect to YTS91 cells (for details see MATERIALS AND METHODS). This analysis demonstrated that sli1-1 resides within the SIT4 locus and is nonallelic with SSD1. Therefore, SSD1-mediated complementation of the synthetic growth defect of YTS91 could be assigned to a low-copy suppressor effect. Deletion of SIT4 in the wild-type strain WCG4 resulted in lethality. This result confirms that YTS91 cells, which are derived from WCG4, harbor an ssd1-d allele. The lethality caused by the deletion of SIT4 in WCG4 was cured after transformation of the cells with the centromeric plasmid pTS64 (LEU2 CEN6), which contained the SSD1 allele isolated from the YCplac111-derived genomic library. Therefore, we conclude that pTS64 contains an SSD1-v allele. These data clearly evidence that the synthetic defects of YTS91 cells depend on the presence of an ssd1-d allele and are suppressed by low-copy expression of an SSD1-v allele.

The sit4-51 mutation causes temperature sensitivity:
The sit4 mutant allele was rescued from strain YTS91 by "gap repair" (ROTHSTEIN 1991 ). Sequencing of the sit4 mutant allele revealed a single point mutation within the SIT4 coding region (guanine 152 was mutated to adenine, resulting in replacement of cysteine 51 by tyrosine). This mutation, referred to as sit4-51 hereafter, resides in a region that is highly conserved among Ser/Thr-phosphatases in yeast and other species (Fig 2). Moreover, this cysteine residue is conserved in many members of the protein phosphatase family (Fig 2). To study the phenotypic effects of the sit4-51 mutation we constructed a sit4-51 mutant strain (YTS100) with the genetic background of WCG4a by one-step gene replacement (see MATERIALS AND METHODS). Similar to the previously described sit4-102 mutation (SUTTON et al. 1991A ), sit4-51 caused ts under certain conditions. YTS100 cells show normal growth on YPD at 30°. However, when shifted to 37°, sit4-51 mutants grow at reduced rates (Table 2). On minimal medium YTS100 cells can form colonies at 30° but do not grow at elevated temperatures (37°; Table 2). The sit4-51 mutation is recessive and sit4-51-induced temperature sensitivity can be fully complemented by a plasmid-encoded SIT4 wild-type gene. The temperature sensitivity of sit4-51 mutants could also be complemented by expression of a plasmid-derived SSD1-v gene (data not shown), demonstrating that, as previously shown for sit4-102 (SUTTON et al. 1991A ), sit4-51 causes temperature sensitivity only in the ssd1-d background.

View larger version (32K): In this window In a new window Download PPT slide   Figure 2. The sit4-51 mutation is located in a highly conserved region among protein phosphatases, marked by an arrowhead. Cysteine 51 is conserved within many protein phosphatases. Sequences are arranged according to decreasing overall homology of the respective protein with the Sit4 sequence. Schizosaccharomyces pombe (ppe1+, dis2+) and human (protein phosphatase X, calcineurin -chain) proteins are indicated. The others are S. cerevisiae proteins.

pre1-1 pre4-1 sit4-51-induced lethality depends on limitation of nutrients:
We performed shut-off experiments to determine the terminal phenotype of sit4-51 pre1-1 pre4-1 cells under synthetic lethal conditions. For this purpose, we constructed sit4-51 pre1-1 pre4-1 mutant cells kept viable by the presence of plasmids encoding wild-type SIT4 or PRE1 under the control of repressible promoters. Surprisingly, no synthetic growth defect was observed when sit4-51 pre1-1 pre4-1 cells harboring a plasmid encoding the PRE1 gene under the control of the GAL1 promoter (strain YTS94) were shifted to repressive conditions (glucose-containing media). By recording the proteasomal chymotrypsin-like activity under repressive conditions we found that YTS94 cells exhibited weak residual activity compared with pre1-1 cells containing an empty vector. This result indicated that GAL1-controlled expression of the plasmid-encoded PRE1 gene could not completely be repressed and that the presence of low amounts of wild-type Pre1 protein were sufficient to complement the synthetic defect of sit4-51 pre1-1 pre4-1 mutants. Therefore, promoter shut-off experiments were performed with sit4-51 pre1-1 pre4-1 cells that contained plasmid-encoded SIT4 or PRE1 wild-type genes under the control of the MET3 promoter (strains YTS95 and YTS96). When the plasmid-encoded SIT4 or PRE1 genes were repressed on minimal medium (SD), corresponding to the conditions used during the synthetic lethality screen, YTS95 and YTS96 cells showed the expected lethality. However, YTS95 and YTS96 cells formed colonies on YPD or synthetic complete (SC) media, indicating that viability of sit4-51 pre1-1 pre4-1 mutants depended on the availability of nutrients. These results suggested that the triple mutants might grow on rich medium. In agreement with this idea we were able to generate haploid sit4-51 pre1-1 pre4-1 and sit4-102 pre1-1 pre4-1 triple-mutant strains by crossing the sit4 single mutants with pre1-1 pre4-1 double-mutant strains (for details see MATERIALS AND METHODS). As expected, the sit4-51 pre1-1 pre4-1 (YTS102) and also sit4-102 pre1-1 pre4-1 (YSH3) cells obtained were able to grow on rich media—YPD and SC medium—but not on SD (Table 2). YTS102 cells did not grow on minimal medium at temperatures >30°. YSH3 cells showed lethality on minimal medium even at 28°, indicating that, compared to sit4-51, sit4-102 leads to a slightly stronger defect of Sit4 function. This finding was further confirmed by the stronger effects observed when the sit4-102 mutation was combined with the proteasomal single mutations (Table 2). Interestingly, slight synthetic effects occurred even when sit4 mutations were combined with the pre4-1 mutation (Table 2). Until now it was thought that this proteasomal mutation, like other mutations that solely affect the 20S proteasomal PGPH activity, does not cause any detectable defects in proteasome-dependent protein degradation or in vivo proteasome function (HILT et al. 1993 ; GUECKEL et al. 1998 ).

The synthetic growth defect of pre1-1 pre4-1 sit4-51 cells is based on an osmosensitivity during starvation of cells:
The terminal phenotype of sit4-51 pre1-1 pre4-1 cells (YTS102) exposed to synthetic lethal conditions was determined. As shown by fluorescence-activated cell sorter (FACS) analysis, the vast majority of sit4-51 pre1-1 pre4-1 triple-mutant cells—as found for wild-type, pre1-1 pre4-1, and sit4-51 mutant cells—switched to a state with 1 N DNA content during prolonged incubation in liquid minimal medium. Twenty-four hours after shift to SD medium, most YTS102 cells (>90%) were arrested as single unbudded cells. As previously observed for mutants deficient in proteasomal activity, the asynchronous cultures of pre1-1 pre4-1 cells cultivated in rich medium harbored a higher amount of 2 N DNA-containing cells (GUECKEL et al. 1998 ). Nevertheless, these cells switched to a 1 N DNA content during incubation in minimal medium. After >48 hr incubation, a shoulder appeared at the 1 N DNA peaks of pre1-1 pre4-1 cultures. We attribute this to the formation of abnormally elongated cells after sustained incubation. At all time points examined, pre1-1 pre4-1 sit4-51 triple-mutant cells showed the same 1 N/2 N DNA ratios as measured for the sit4-51 single-mutant cells. (Fig 3). On the basis of these data we conclude that sit4-51 pre1-1 pre4-1 mutant cells, like wild type, sit4 single-mutant, and even pre1-1 pre4-1 double-mutant cells, are able to induce a G₁ arrest when they run out of nutrients. However, after 36 hr of incubation on minimal medium, strong morphological changes became visible specifically for the sit4-51 pre1-1 pre4-1 triple-mutant cells. The majority of the cells appeared to be greatly enlarged, had lost their normal oval shape, and contained abnormal large vacuoles (Fig 4), which often collapsed when cells were exposed to even slight mechanical stress (e.g., application of coverslips; data not shown). This morphology led us to the idea that under lethal conditions mutant cells may be prone to unregulated water uptake from the medium and, therefore, to be extremely sensitive to low osmotic pressure. To test this idea, cells were transferred onto SD agar media containing high concentrations of salt (NaCl >0.3 M or KCl >0.5 M) or sorbitol (>0.5 M). Indeed, these conditions of high osmotic pressure restored growth of sit4-51 pre1-1 pre4-1 cells (Fig 5). Cells cured by high salt or sorbitol concentrations had normal size and shape (Fig 4). Taken together, these data indicate that the growth defects of mutants impaired in Sit4 and proteasome function are based on an osmosensitivity occurring under limiting nutrients.

View larger version (27K): In this window In a new window Download PPT slide   Figure 3. sit4-51 pre1-1 pre4-1 mutants undergo G1 arrest when incubated in minimal medium. Wild-type (WCg4a), pre1-1 pre4-1 double-mutant (YHI29/14), sit4 single-mutant (YTS100), and sit4-51 pre1-1 pre4-1 triple-mutant (YTS102) cells were grown in YPD medium to logarithmic phase (30°) and then transferred to liquid SD medium. After different incubation times samples were taken, DNA was stained with propidium iodide, and cells were analyzed for their DNA content by FACS. The vertical axes indicate the cell count, the horizontal axes the DNA content.

View larger version (83K): In this window In a new window Download PPT slide   Figure 4. Morphology of synthetic lethal and sorbitol-cured sit4-51 pre1-1 pre4-1 mutants. sit4-51 pre1-1 pre4-1 mutants (YTS102) grown on YPD were transferred to SD plates containing (A) no sorbitol (seen are four enlarged unbudded cells, two of which are accidentally positioned near each other) or (B) 0.5 M sorbitol and incubated at 30° for 36 hr. Cells were scraped off the plates, dissolved in liquid SD medium, mounted on slides, and inspected with a microscope (Zeiss axioscop). Bar, 22 µm.

View larger version (43K): In this window In a new window Download PPT slide   Figure 5. The growth defect of sit4-51 pre1-1 pre4-1 mutants on SD medium is rescued by high concentrations of salt or sorbitol. Wild-type (WCG4a), sit4-51 single-mutant (YTS100), pre1-1 pre4-1 double-mutant (YHI29/14), and sit4-51 pre1-1 pre4-1 triple-mutant (YTS102) cells were streaked on plates with SD (A), SD containing 0.5 M sodium chloride (B), or SD containing 0.5 M sorbitol (C) and incubated at 30° for 50 hr.

sit4-51 and sit4-102 mutants show different behavior after rapamycin-induced inhibition of the TOR pathway:
Mutations sit4-102 (SUTTON et al. 1991A ) and sit4-51 both cause temperature sensitivity and synthetic effects when combined with proteasomal mutations. However, when the TOR pathway is blocked (HALL 1996 ; THOMAS and HALL 1997 ) by the presence of the TOR specific inhibitor rapamycin, sit4-51 and sit4-102 mutants behave differently. As already known, sit4-102 cells are hypersensitive against rapamycin. In contrast, compared to wild-type cells, sit4-51 mutants exhibit significant resistance against the inhibitor (Fig 6).

View larger version (35K): In this window In a new window Download PPT slide   Figure 6. sit4-102 and sit4-51 mutants behave differently against rapamycin. Wild-type cells (WCG4a), sit4-51 (YTS100), and sit4-102 mutants (YSH2) were streaked on YPD agar plates (30°) containing 50 µg/ml rapamycin.

Sit4 does not regulate the proteasome:
A possible explanation for the Sit4-proteasome interaction may be that the proteasome is regulated by a Sit4-mediated dephosphorylation step. In agreement with this idea it is known that proteasomes from different species contain subunits that are phosphorylated (ARRIGO and MEHLEN 1993 ; ETLINGER et al. 1993 ; CASTANO et al. 1996 ; MASON et al. 1996 ). Additionally, potential phosphorylation sites have been identified in some yeast 20S proteasome subunits (HEINEMEYER et al. 1994 ). To test whether Sit4 phosphatase is involved in regulation of proteasomal activity, we compared the in vitro peptide-cleaving activities of 20S proteasomes partially purified from sit4-51 mutants (YTS100) and isogenic wild-type cells, but we did not observe any deviations from 20S proteasomal peptidase activity profiles (Fig 7). To substantiate these results, we also tested the in vivo degradation rates of well-defined 20S proteasomal substrates in sit4-51 mutant and wild-type cells. The kinetics of glucose-induced degradation of FBPase (SCHORK et al. 1994 , SCHORK et al. 1995 ) was not altered in YTS100 cells (sit4-51) compared to wild-type cells (Fig 8A and Fig B). Moreover, presence of the sit4-51 mutation did not influence FBPase stability in the pre1-1 pre4-1 proteasome mutant background (Fig 8C). Also, the short-lived substrates of the N-end-rule pathway, Leu-ß-Gal and Arg-ß-Gal (BACHMAIR et al. 1986 ), as well as the short-lived Ub-Pro-ß-Gal protein, were degraded at wild-type rates in sit4-51 cells (data not shown). These data demonstrate that neither the in vitro peptidase activity of the 20S proteasome nor the in vivo proteolytic activity of the 26S proteasome are influenced by the sit4-51 mutation. These data, in addition, indicate that Sit4 does not influence the cellular concentration of proteasomes.

View larger version (21K): In this window In a new window Download PPT slide   Figure 7. In vitro 20S proteasome peptide-cleaving activities are not altered in sit4-51 mutant cells. Crude extracts of cells grown to exponential phase (30°) were fractionated by gel filtration on sepharose CL4B and peptide-cleaving activities were determined using artificial substrates: (A) The chymotrypsin-like activity was determined using Suc-Leu-Leu-Val-Tyr-AMC; (B) the trypsin-like activity was determined using Cbz-Ala-Arg-Arg-MoßNA; and (C) the PGPH activity was determined using Cbz-Leu-Leu-Glu-ßNA.

View larger version (20K): In this window In a new window Download PPT slide   Figure 8. Fructose-1,6-bisphosphatase is degraded at wild-type rates in sit4-51 mutants. (A) Pulse-chase analysis of FBPase degradation in wild-type (WCG4a) and sit4-51 (YTS100) mutant cells. Cells were pulse labeled during derepression of FBPase in ethanol-containing medium and chased with the addition of glucose followed by extraction, immunoprecipitation, and SDS-PAGE. (B) Quantification of A. (C) FBPase turnover during catabolite inactivation was followed in wild-type (WCG4a), sit4-51 single-mutant (YTS100), pre1-1 pre4-1 double-mutant (YHI29/14), and sit4-51 pre1-1 pre4-1 triple-mutant (YTS102) cells by immunoblotting using the method of SCHORK et al. 1995 .

Sit4 is not a proteasomal substrate:
The sit4-51 and sit4-102 mutations are recessive and are therefore believed to result in loss of Sit4 function. In such a case proteolytic stabilization of the sit4-51 and sit4-102 gene products is expected to cause suppressor, and not synthetic, effects when combined with mutations that impair proteasome function. Therefore, the genetic data indicate that Sit4 is not a target for proteasome-mediated degradation. To confirm this idea we tested whether Sit4 is a stable protein and not a substrate of the proteasome. Plasmid-encoded Sit4 containing an immunoepitope at the carboxyl terminus (Sit4-HA) or amino terminus (HA-Sit4; SUTTON et al. 1991A ) was expressed in wild-type and proteasome mutant strains that contained a deletion of the chromosomal SIT4 gene. Therefore, the plasmid-encoded HA-tagged SIT4 genes comprised a single source of Sit4 protein in these cells. Both N-terminally and C-terminally HA-tagged Sit4 (Fig 9; data not shown) were found to be long lived in wild-type cells. As expected, there was no difference in Sit4 stability in proteasome mutant strains (Fig 9). Due to the following facts we can exclude that the immunoepitopes influenced the proteolytic stability of the tagged Sit4 protein: (1) Concerning proteolytic stability, both epitope-tagged versions, Sit4-HA and HA-Sit4, behaved in the same manner; (2) both epitope-tagged versions of Sit4 used for determination of Sit4 stability were proven to be fully functional (SUTTON et al. 1991A ); and (3) in contrast to pre1-1 pre4-1 cells containing a sit4-51 or sit4-102 mutation, proteasomal mutants expressing either Sit4-HA or HA-Sit4 showed normal growth on minimal medium. This result evidences that the immunoepitope does not influence the functional interaction between Sit4 and the proteasome. Taken together, the data clearly show that Sit4 is a stable protein and not a target of proteasome-mediated destruction.

View larger version (17K): In this window In a new window Download PPT slide   Figure 9. Sit4 is proteolytically stable. Wild-type (YMHO27), pre1-1 (YMHO29), and pre1-1 pre4-1 (YMHO33) mutant cells expressing plasmid (LEU2 CEN)-derived C-terminally epitope-tagged Sit4-HA protein as the only source of Sit4 were grown to logarithmic phase at 30°. Protein synthesis was blocked by the addition of cycloheximide (0.5 mg/ml) and Sit4-HA protein monitored at different chase times by immunoblotting using anti-HA antibodies. C, negative control; wild-type cells (WCG4) containing no SIT4-HA-encoding plasmid.

Sit4 function is linked to Rad6- and Cdc34-dependent ubiquitination:
Sit4-related synthetic effects are specific for impaired proteasome-mediated proteolysis. No effects were found when the sit4 mutations were combined with defective vacuolar proteolysis due to mutated proteinase A (data not shown). In most cases proteasome-dependent degradation requires targeting of substrate proteins by ubiquitination. If Sit4 phosphatase function is linked to defined proteasome-mediated degradation pathways, synthetic effects should be found when sit4 mutations are combined with mutations causing defects in certain ubiquitination pathways. To test this idea, we generated sit4 mutant strains that harbored mutations in genes coding for ubiquitin-conjugating (E2) enzymes. We tested the complete set of yeast E2 genes. Striking synthetic effects were found when sit4 mutations were combined with the ubc3/cdc34-1 or the ubc2/rad6 mutation (Table 3; see also Fig 10D). No synthetic effects were detected with mutations of the other S. cerevisiae Ubc enzymes. No detectable synthetic effect was seen even for a sit4-51 ubc4 ubc6 ubc7 quadruple mutant.

View larger version (73K): In this window In a new window Download PPT slide   Figure 10. Synthetic defects of sit4 pre and sit4 ubc mutants are suppressed by the addition of certain amino acids to the medium. Wild-type (WCG4a), pre4-1 sit4-102 (YSH5), pre1-1 sit4-102 (YSH4), pre1-1 pre4-1 sit4-102 (YSH3), pre1-1 pre4-1 sit4-51 (YTS102), rad6 sit4-51 (YSH13), cdc34-1 sit4-102 (YSH19), and cdc34-1 sit4-51 (YSH11) cells were streaked on minimal media (SD ura+, leu+, his+) that contained serine (A), alanine (B), or cysteine (C). Control plate without additional amino acids (D).

On SD medium at 30°, sit4-51 cdc34-1 double mutants showed very poor growth, whereas sit4-102 cdc34-1 double mutants exhibited synthetic lethality. Under such conditions sit4-102 cdc34-1 cells appeared to be significantly enlarged, highly resembling the morphological phenotype of sit4-51 mutants that contained pre1-1 pre4-1 mutations. Moreover, as for sit4 pre mutants, impaired growth of sit4-51 cdc34-1 double mutants, as well as synthetic defects of sit4-102 cdc34-1 double mutants on SD medium, was cured by applying high osmotic pressure (1 M sorbitol; data not shown).

The strongest synthetic effect was observed when we combined sit4-51 with a deletion of the RAD6 gene. sit4-51 rad6 double mutants could not be directly made by dissection of tetrads obtained from heterozygous diploids. Spores containing the sit4-51 rad6 double mutation stopped growth at a size of 30 cells even when grown on YPD medium (data not shown), indicating that sit4-51 rad6 mutants are able to germinate but cannot continue growth under these conditions. Therefore, sit4-51 rad6 spores that in addition contained a complementing SIT4-encoding LEU2 plasmid (pCK2) were generated. After germination these spores were grown to colonies with normal size. After further growth on nonselective medium, clones that had lost the SIT4-encoding plasmid during mitosis, thereby yielding sit4-51 rad6 double mutants (strain YSH13), were detected. To prove whether these sit4-51 rad6::HIS3 cells had acquired a suppressor mutation that enabled their growth on rich medium, strain YSH13 was backcrossed to a sit4-51 single-mutant strain. After sporulation tetrads were dissected and analyzed. As expected, in each tetrad two sit4-51 single-mutant clones were found. However, in addition to nonviable rad6 sit4 cells, histidine prototrophic sit4-51 rad6 double-mutant spore clones that grew up to colonies were obtained. These sit4-51 rad6 double-mutant clones grew considerably slower than the sit4-51 single mutants. These results clearly prove that combination of sit4-51 with rad6 causes genuine synthetic lethality even on rich medium.

The suppressor mutation present in YSH13 cells does not cure the synthetic growth defects of sit4-51 rad6 cells on minimal medium. YSH13 cells exhibited synthetic lethality on minimal medium at 23° and even at 18° (data not shown), representing the strongest synthetic effect of all mutants inspected in this work. Moreover, under these conditions sit4-51 rad6 cells arrested with 1N DNA content and developed the expected morphology of enlarged round cells (data not shown). Lethality of sit4-51 rad6 cells on SD medium could not be cured by high osmotic pressure (1 M sorbitol or 1.5 M KCl; data not shown) but rather by supplementation with the same set of single amino acids (see next section). On the basis of these findings we suggest that the suppressor mutation enabling growth of YSH13 cells on rich medium does not have a significant effect on the phenotypic behavior of YSH13 cells on minimal medium.

One prominent function of Rad6 is targeting of substrates of the N-end-rule pathway (DOHMEN et al. 1991 ; VARSHAVSKY 1997 ). In this pathway Ubr1 functions as the E3 enzyme required for substrate recognition (BARTEL et al. 1990 ; VARSHAVSKY 1996 ). No synthetic effect was observed when sit4 mutations were combined with a deletion of UBR1 (data not shown), proving that the proteasome-related function of Sit4 is independent of Ubr1-mediated pathways. Taken together, these data evidence that Sit4 function is linked to the proteasome system via Ubc2- and Ubc3-mediated pathways, but exclude Ubr1 as a contributor to that function.

The sit4-induced synthetic growth defect is suppressed by the presence of certain amino acids:
sit4 pre- and sit4 ubc-induced synthetic effects were observed mainly on minimal medium. We were interested to know whether viability of cells defective in the Sit4 phosphatase and the ubiquitin-proteasome system depended on the general availability of nutrients or on the presence of certain amino acids. To answer this question, growth of sit4 pre or sit4 ubc mutants was tested on agar media that, in addition to the amino acids necessary for complementation of auxotrophic mutations, were supplemented with a single amino acid. Interestingly, addition of certain amino acids led to suppression of sit4 pre- or sit4 ubc-induced growth defects. The amino acids tested could be sorted into three classes depending on their ability to restore viability of the mutants. Addition of asparagine and serine caused strong suppression of sit4 pre- or sit4 ubc-induced growth defects. These amino acids could even rescue the sit4-51 rad6-induced synthetic growth defects of YSH13 cells (Fig 10A, Table 4). The second class of amino acids (Ala, Ile, Phe, Thr) exhibited lesser suppressor effects. These amino acids cured only synthetic defects of mutants harboring combinations of sit4 and pre or ubc mutations that cause weak synthetic effects (for instance, sit4-102 pre1-1 or sit4-51 cdc34-1 mutations; Fig 10B, Table 4). The third class of amino acids led to almost no suppression of sit4 pre- or sit4 ubc-induced lethality. Growth of sit4 pre or sit4 ubc mutants was not restored even when SD medium was supplemented with mixtures of several class III amino acids (Table 4). Taken together, these data clearly demonstrate that the viability of cells bearing defects of the Sit4 phosphatase and the ubiquitin-proteasome system depends on the presence of certain amino acids. No direct relationship was found between the biosynthetic pathways of class I and class II amino acids that restored growth of the mutants.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

sit4 mutations cause synthetic growth defects when combined with proteasomal mutations:
We isolated mutants that showed lethality in combination with proteolytically impaired proteasomes. Complementation of one of the mutants using a plasmid-based library yielded two genes, SIT4 and SSD1. The mutation conferring synthetic lethality was found to reside within the SIT4 gene, whereas SSD1 functioned as a low-copy suppressor. We suggest that sit4 mutations conferring synthetic effects with impaired proteasomes lead to a general deficiency of Sit4 protein phosphatase function and not to a defect that is restricted to the proteasome-associated function of Sit4. This conclusion is based on the following observations: (1) Synthetic effects with proteasomal mutations are not limited to the sit4-51 allele but can also be induced with the previously described sit4-102 allele, which leads to multiple defects in Sit4-related functions (SUTTON et al. 1991A ); (2) sit4-51-induced synthetic lethality, as well as sit4-51- and sit4-102-induced temperature sensitivity, were suppressible by low-copy expression of a SSD1-v gene, which is capable of rescuing cells from complete loss of Sit4 function; and (3) cysteine 51, which in the mutated Sit4-51 protein is replaced by tyrosine, is conserved in many other protein phosphatases. This residue resides within a region that is highly conserved among protein phosphatases and even human calcineurin (Fig 2). The cysteine residue of the calcineurin chain A corresponding to cysteine 51 of Sit4 is located in the center of this structurally well-defined phosphatase in the vicinity of the active site. On the basis of these findings we suggest that the sit4-51 mutation might affect Sit4 phosphatase activity.

The sit4-102 mutation induced slightly stronger synthetic phenotypes than sit4-51 did, indicating that this mutation may affect Sit4 function to a larger extent. Interestingly, although sit4-51 and sit4-102 cells showed similar effects with defects of the ubiquitin-proteasome system, they responded differently to inhibition of the TOR-signaling pathway. sit4-51 caused resistance against rapamycin, whereas sit4-102 caused hypersensitivity against this drug. Therefore, the two mutations seem to influence differently the TOR-related function of Sit4. Moreover, on the basis of these results we can exclude a connection between the ubiquitin-proteasome-related function of Sit4 described in this study and the TOR-signaling pathway.

Sit4 function is specifically linked to deficient proteasomal proteolysis. No synthetic effects were observed when the sit4-51 mutation was combined with a general defect in vacuolar proteolysis. sit4-51 and sit4-102 mutations cause synthetic growth defects when combined with pre1-1 pre4-1 double mutations or with a pre1-1 single mutation. Both pre1-1 pre4-1 and pre4-1 lead to significant defects in proteasome-mediated protein degradation. Surprisingly, even moderate synthetic effects were found when sit4 was combined with pre4-1. So far, neither a defect of proteasomal substrate degradation nor any other cellular phenotype has been identified in mutants defective in proteasomal PGPH activity including pre4-1 mutants (HILT et al. 1993 ; HEINEMEYER et al. 1997 ; GUECKEL et al. 1998 ). Therefore, it was thought that lack of the PGPH activity does not cause impairment of in vivo proteasomal protein degradation and is dispensable for proteasomal in vivo function (HILT et al. 1993 ; HEINEMEYER et al. 1997 ; GUECKEL et al. 1998 ). This view is now challenged by the synthetic effect observed in pre4-1 sit4 mutants.

How is Sit4 linked to proteasome function? Different models might explain the link between the Sit4 phosphatase and the ubiquitin-proteasome system: (1) Sit4 may regulate the activity or the concentration of the proteasome; (2) Sit4 may be a substrate of proteasome-mediated degradation; (3) peptides produced by proteasomal degradation may provide a nutritional signal that feeds into a Sit4-mediated pathway; and (4) Sit4 and the proteasome may share a common target.

Several findings lead to the exclusion of models 1 and 2. The sit4-51 mutation did not cause any alteration of the 20S proteasomal peptidase activities in vitro. In addition, well-defined model substrates of the proteasome pathway were degraded at wild-type rates in sit4-51 mutant cells, demonstrating that the sit4-51 mutation does not influence the in vivo activity of the proteasome. Therefore, we conclude that Sit4 is not implicated in controlling the activity or the concentration of the proteasome. We also demonstrated clearly that the Sit4 phosphatase is proteolytically stable. Hence, Sit4 is not a target of proteasome-mediated degradation. Thus, we suggest that Sit4 does not directly interact with the proteasome.

Could Sit4 respond to a nutritional signal that depends on peptides produced by proteasome-mediated protein destruction? Such a model is supported by the clear dependence of the synthetic effects of the sit4 pre1-1 pre4-1 mutant on the availability of certain amino acids in the growth medium. However, there are also strong arguments against this model. Under starvation conditions, proteins are turned over mainly by vacuolar proteolysis (TEICHERT et al. 1989 ; LANG et al. 2000 ). Therefore, at least under such conditions it is the vacuole and not the proteasome system that is the major endogenous source for peptides and amino acids. However, no synthetic effects were observed when the vacuole-dependent pathway of protein degradation was blocked in a sit4 mutant. Moreover, if peptides generated by proteasomal degradation were the source of an internal signal acting on Sit4, one would expect induction of this signal not to be limited to defects of Rad6- and Cdc34-dependent ubiquitination. Nevertheless, the possibility exists that Sit4 phosphatase and the proteasome system may execute a concerted function in sensing of external nutrients and our data may support such a model.

The finding that sit4-induced synthetic effects are restricted to defined ubiquitination pathways indicates that Sit4 phosphatase is functionally connected to a protein that was degraded via the proteasome in a Rad6/Cdc34-dependent way. Because sit4-related synthetic effects were found in rad6 null mutants, Sit4 phosphatase can be excluded from contribution to Rad6-mediated substrate targeting; in such a case epistatic, but not synthetic, effects should have been observed. Hence, at least for the Rad6-dependent pathway, we can exclude the requirement of Sit4-dependent dephosphorylation of a proteasomal substrate as a signal for its proteolytic destruction.

Only weak synthetic effects were observed when sit4-51 or sit4-102 mutations were expressed together with the conditional cdc34-1 mutation. In this case, the sit4-induced synthetic effects were measured at temperatures permissive for growth of the cdc34-1 cells. Under these conditions, cells are expected to possess residual Cdc34 activity. Consequently, at these temperatures, cdc34-1-related synthetic effects are expected to be weak.

Even though each has specific functions—Rad6/Ubc2 is involved in DNA repair, whereas Cdc34/Ubc3 has an essential role in the cell cycle—both ubiquitin-conjugating enzymes, Rad6 and Cdc34, are closely related. They exhibit strong sequence similarity (HAAS and SIEPMANN 1997 ) and both localize to the nucleus. These data suggest that Rad6 and Cdc34 may also share common cellular functions. Indeed, it was reported that both enzymes (the S. cerevisiae and the human homologs) exhibit overlapping functions in substrate targeting (KORNITZER et al. 1994 ; PAGANO et al. 1995 ; TAM et al. 1997 ; PATI et al. 1999 ). Therefore, we suggest that Rad6 and Cdc34 are involved in ubiquitination of a defined substrate protein, functionally connected to Sit4 phosphatase.

Sit4-induced synthetic effects depended on nutrient availability and osmotic conditions. Interestingly, Ubr1, which acts as an E3 enzyme contributing to Rad6-dependent ubiquitination, is required for peptide uptake (ALAGRAMAM et al. 1995 ; BYRD et al. 1998 ; TURNER et al. 2000 ) and linked to osmoregulation (OTA and VARSHAVSKY 1993 ; POSAS et al. 1996 ). Thus, it seemed probable that Sit4 function was related to Ubr1-mediated degradation. However, this model is clearly excluded because no synthetic effects were observed when sit4 mutations were combined with a UBR1 deletion.

Models of Sit4-proteasome interaction:
How could Sit4 and the proteasome mechanistically act on a common target protein? Both systems may redundantly contribute to the inactivation of a common target. Alternatively, Sit4 may control the cellular level of a common target by influencing its expression. Mutation of Sit4 protein was found to result in both induction and repression of gene expression. In light of these findings, a promising model for the Sit4-proteasome interaction is that sit4 mutations cause ectopic expression or expression to abnormally high levels of a protein whose concentration is controlled by proteasomal degradation. This model is supported by the fact that the synthetic growth defect of sit4-51 pre mutants was restored by expression of SSD1-v. Due to the capability of SSD1-v alleles to restore correct expression of G₁ cyclins (SUTTON et al. 1991A ) and because Ssd1 can bind mRNA, we can speculate that SSD1-v alleles may cause correction of sit4-derived alteration of gene expression.

	FOOTNOTES

¹ These authors contributed equally to this work.

	ACKNOWLEDGMENTS

Thanks go to W. Heinemeyer, W. Seufert, S. Jentsch, C. Mann, and K. Arndt for plasmids or strains, to F. Cvrckova and K. Nasmyth for genomic libraries, and to K. D. Entian for antibodies. The authors thank Z. Kostova for helpful comments on the manuscript. Thanks go to D. H. Wolf for support and E. Schmidl and J. Kost for technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft and the EU.

Manuscript received November 14, 2002; Accepted for publication April 4, 2003.

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