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MOLECULAR AND CELLULAR BIOLOGY, June 1996, p. 27442755 Vol. 16, No. 60270-7306/96/$04.000Copyright 1996, American Society for Microbiology

The SAPs, a New Family of Proteins, Associate and FunctionPositively with the SIT4 Phosphatase

MAY M. LUKE,1 FLAVIO DELLA SETA,1 CHARLES J. DI COMO,1,2 HANA SUGIMOTO,1

RYUJI KOBAYASHI,1

AND KIM T. ARNDT1

*Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724-2212,1 and Graduate Program in Genetics,

State University of New York, Stony Brook, New York 117942

Received 11 January 1996/Returned for modification 4 March 1996/Accepted 10 March 1996

SIT4 is the catalytic subunit of a type 2A-related protein phosphatase in Saccharomyces cerevisiae that isrequired for G

1cyclin transcription and for bud formation. SIT4 associates with several high-molecular-mass

proteins in a cell cycle-dependent fashion. We purified two SIT4-associated proteins, SAP155 and SAP190, andcloned the corresponding genes. By sequence homology, we isolated two additional SAP genes, SAP185 and

SAP4. Though such an association is not yet proven for SAP4, each of SAP155, SAP185, and SAP190 physicallyassociates with SIT4 in separate complexes. The SAPs function positively with SIT4, and by several criteria, theloss of all four SAPs is equivalent to the loss of SIT4. The data suggest that the SAPs are not functional in theabsence of SIT4 and likewise that SIT4 is not functional in the absence of the SAPs. The SAPs are hyper-phosphorylated in cells lacking SIT4, raising the possibility that the SAPs are substrates of SIT4. By sequence

similarity, the SAPs fall into two groups, the SAP4/SAP155 group and the SAP185/SAP190 group. Overex-pression of a SAP from one group does not suppress the defects due to the loss of the other group. Thesefindings and others indicate that the SAPs have distinct functions.

During the early G1 phase of the cell cycle, and in thepresence of nutrients, Saccharomyces cerevisiae cells synthesizecellular components and grow in size. Prior to reaching a pointin late G1 termed Start, the growing G1 cells can exit themitotic cell cycle, in response to either nutrient limitation orthe presence of mating pheromones (27). By contrast, cells thathave executed Start are committed to a round of cell division,independent of nutrients and the presence of mating phero-mones. After passage through Start, a cell initiates the pro-cesses required for the formation of the daughter cell, namely,DNA synthesis, bud formation, and duplication of the spindle

pole body.The execution of Start requires a sufficient level of G1 cyclin/

CDC28 protein kinase activity (6, 28, 29). The main determi-nants of the amount of G1 cyclin/CDC28 kinase activity are thelevels of the CLN1 and CLN2 proteins, which are the majorbudding yeast G1 cyclins (41). The CLN1 and CLN2 RNA andproteins are unstable, and it is thought that their rates oftranscription largely determine the rates and levels of CLN1and CLN2 protein accumulation during late G1 (41, 45). TheDNA-binding factor SWI4 and the associated factor SWI6have been implicated in the transcription ofCLN1 and CLN2(24, 25). However, CLN1 and CLN2 are transcribed at signif-icant levels in the absence of either SWI4 or SWI6 (7, 20, 37).

A major unanswered question is how nutrient growth signalsare transduced and integrated into an increase in CLN1 and

CLN2 transcription during late G1.The execution of Start also requires the function of SIT4,which is the catalytic subunit of a type 2A-related proteinphosphatase (1). Temperature-sensitive sit4 strains arrest inlate G1 at Start, prior to the initiation of DNA synthesis, budformation, and spindle pole body duplication (38). This Startarrest is due to the requirement for SIT4 for the transcription

ofCLN1 and CLN2, which is at least partly due to the involve-ment of SIT4 in SWI4 transcription (13). If CLN2 is providedfrom a SIT4-independent promoter, temperature-sensitive

sit4-102 cells arrest with replicated DNA but are still mostlyblocked for bud initiation (13). Therefore, in addition to beingrequired for G1 cyclin expression, SIT4 is required in late G1for bud formation.

How SIT4 performs its G1 functions is not yet clear. Thelevels of SIT4 protein are constant during the cell cycle. How-ever, SIT4 associates with two high-molecular-mass phospho-proteins, of 155 and 190 kDa, in a cell cycle-dependent manner

(38). In this report, we term these proteins SAP155 andSAP190, for SIT4-associated proteins. In G1 daughter cells,SIT4 is not associated with SAP155 and SAP190 but is foundmostly in a free monomeric form (38). In late G 1, SIT4 asso-ciates in separate complexes with SAP155 and SAP190 andremains associated until about the middle of mitosis (38). Pos-sibly, certain late G1 processes might be regulated via theassociation of the SAPs with SIT4.

To understand how the SAPs affect the functions of SIT4, wepurified SAP155 and SAP190 and cloned the correspondinggenes. Surprisingly, not only are SAP155 and SAP190 relatedproteins, but the cells also contain additional SAPs related toSAP155 and SAP190. At least one of these proteins, SAP185,also associates with SIT4. Our findings show that SIT4 associ-ates with a new family of high-molecular-mass proteins which

function positively with SIT4. Although the SAPs have se-quence similarity, they provide at least two distinct cellularfunctions.

MATERIALS AND METHODS

Strains and media. Table 1 lists the yeast strains used in this study. Rich (YP),synthetic complete (SC), and minimal (SD) media were constituted as describedelsewhere (33) except that the YP media also contained 10 mM KH2PO4, 0.1 gof tryptophan per liter, and 0.1 g of adenine per liter and the SC media contained0.2 g of leucine, 0.1 g of all other amino acids, 0.1 g of uracil, and 0.1 g of adenineper liter.

Purification of SAP155 and SAP190. CY202 cells, containing hemagglutinin(HA) epitope-tagged SIT4, were grown in YPD (D indicates 2% glucose) me-

* Corresponding author. Mailing address: Cold Spring Harbor Lab-oratory, P.O. Box 100, Cold Spring Harbor, NY 11724-2212. Phone:(516) 367-8836. Fax: (516) 367-8369. Electronic mail address:[email protected].

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dium. At an optical density at 600 nm of about 2.5, the cells were harvested,frozen, and stored at 70C. The remaining steps were done at 4C. Forty gramsof frozen cells, 35 ml of glass beads, and about 25 ml of breaking buffer (100 mMTris-HCl [pH 7.5], 200 mM NaCl, 1 mM EDTA, 5% glycerol, 1 mM dithiothre-itol [DTT], freshly added protease inhibitors to final concentrations of 0.2 mMphenylmethylsulfonyl fluoride and 1.25 g each of leupeptin, antipain, chymo-statin, and pepstatin [Sigma] per ml) were placed in a 100-ml container within theice water jacket of a Biospec bead beater set to go on for 10 s and off for 80 s until90% of the cells were broken (total time of about 60 to 90 min). The lysate wascleared by two successive 13,000 gspins of 20 and 10 min, giving about 35 mlof an extract with a protein concentration of about 30 mg/ml. The HA-taggedSIT4 protein was immunoprecipitated by incubation of the extract with 60 l ofascites fluid containing antibody 12CA5 (14) for 2 h and then binding theimmune complexes with 300 l of protein G-Sepharose beads (Pharmacia) withgentle rocking overnight. The protein G beads were washed 10 times in 20

volumes of a buffer containing 25% radioimmunoprecipitation assay buffer (50mM Tris-HCl [pH 7.5], 200 mM NaCl, 1% Triton X-100, 0.5% sodium deoxy-

cholate, 0.1% sodium dodecyl sulfate [SDS], 1 mM DTT, the protease inhibitorsspecified above) and 75% breaking buffer and rinsed once in a buffer containing50 mM Tris (pH 7.5), 50 mM NaCl, 1 mM DTT, and the protease inhibitor mix.

An equal volume of 2 Laemmli buffer was added to the beads, and the mixturewas boiled. The proteins in the immune complexes were resolved on an SDS8%polyacrylamide gel and visualized by Coomassie blue staining.

Peptide sequencing of SAP155 and SAP190. Peptide sequencing was doneessentially as described previously (5). Ten peptide sequences each for SAP155and SAP190 were obtained, and all matched the amino acid sequences predictedfrom the DNA sequences of SAP155 and SAP190.

Cloning and sequencing of the SAP genes. Degenerate primers were designedfrom peptide sequences of SAP155 and SAP190, taking into consideration thecodon usage of S. cerevisiae (34, 44) to minimize degeneracy. Genomic DNA ofCY202 was used as the template DNA for PCR. For SAP155, primers from threepeptides produced 0.5-, 0.7-, and 0.8-kb PCR products. For SAP190, sense and

antisense degenerate primers from three peptides produced PCR products of1.4, 0.7, and 2.1 kb. Southern blotting of these PCR products confirmed that the1.4- and 0.7-kb fragments are two parts of the 2.1-kb fragment. The SAP155 PCRproduct hybridized to clones 6341 and 4584 on chromosome VI, and the SAP190PCR product hybridized to clone 4163 on chromosome XI of the Olson libraryof yeast inserts in lambda phage (30). Although the SAP155 gene was obtainedfrom a YCp50 library by using the cloned SAP155 PCR product as a probe, theSAP155 gene that was sequenced and used for all of the experiments in thisreport was obtained from a library of yeast genomic inserts in YEp24 (3). ThisSAP155-containing YEp24 clone was isolated in a genetic screen for high-copy-number suppressors of the temperature-sensitive phenotype of a sit4-102 strain(7a). The full-length SAP190 gene was obtained from a library of yeast genomicinserts in YCp50 (32) by colony hybridization, using the cloned SAP190 PCRproducts as probes.

The location of the SAP155 gene on the yeast DNA insert was determined byhybridization using the cloned PCR product and by subcloning for the smallestcomplementing DNA fragment, taking advantage of the ability of high-copy-

number SAP155 to suppress the temperature-sensitive phenotype of a sit4-102strain. We sequenced both strands of our SAP155 clone from positions 481 to3224 (relative to the A of the ATG initiation codon).

The sequence of SAP190, corresponding to the open reading frame (ORF)YKR028, was reported previously (11). We sequenced from KpnI to HpaI,containing the amino terminus, and from EcoRV to BstBI, containing the car-boxy terminus, of our SAP190 clone. Relative to our SAP190 DNA sequence(which predicts a 1,033-amino-acid protein), the reported SAP190 sequence hasa frameshift at codon 1009 and predicts a 1,098-amino-acid protein.

The SAP185 gene was isolated from the YEp24 library (3) by colony hybrid-ization, using as a probe the 1.35-kb BstEII-PstI fragment from plasmid pCS3-1(a generous gift of B. Osmond). The sequence for SAP185 was published in areport describing the sequencing of a 9.7-kb fragment of chromosome X (re-ferred to as ORF J0840 in reference 23).

To isolate SAP4, 1 g of genomic DNA from a strain (CY4985) lacking

TABLE 1. Yeast strains used in this study

Strain Genotypea Reference

CY144 MATa sit4::HIS3 ssd1-d2 sit4-102/LEU2-CEN W303 38CY202 MATa sit4::HIS3 ssd1-d2 SIT4:HA-COOH/LEU2-CEN W303 38CY832 MATa ssd1::LEU2 W303 38CY1078 MAT sit4::HIS3 ssd1-d2 SIT4/YCp50 W303 38CY3938 MATa sit4::HIS3 SSD1-v1 W303 This study

CY4029 MATa SSD1-v1 W303 This studyCY4102 MAT cln3::URA3 SSD1-v1 W303 This studyCY4380 MATa sap190::TRP1 SSD1-v1 W303 This studyCY4796 MATa sit4::HIS3 sap190::TRP1 SAP190:HA/YCp50 SSD1-v1 W303 This studyCY4802 MATa sap190::TRP1 SAP190:HA/YCp50 SSD1-v1 W303 This studyCY4917 MATa sap185::ADE2 SSD1-v1 W303 This studyCY4985 MATa sap155::HIS3 sap185::ADE2 sap190::TRP1 SSD1-v1 W303 This studyCY4987 MAT sap155::HIS3 sap185::ADE2 sap190::TRP1 SSD1-v1 W303 This studyCY5001 MATa sap185::ADE2 sap190::TRP1 SSD1-v1 W303 This studyCY5067 MATa sap185::ADE2 sap190::TRP1 SAP185:HA/YCp50 SSD1-v1 W303 This studyCY5077 MATa sit4::HIS3 sap185::ADE2 sap190::TRP1 SAP185:HA/YCp50 SSD1-v1 W303 This studyCY5169 MATa sit4::HIS3 SIT4/LEU2-CEN SSD1-v1 W303 This studyCY5170 MATa sit4::HIS3 SIT4:PY/LEU2-CEN SSD1-v1 W303 This studyCY5196 CY5169 YEp24 This studyCY5201 CY5170 YEp24 This studyCY5206 MATa sit4::HIS3 sap155::HIS3 sap185::ADE2 sap190::TRP1 SIT4/LEU2-CEN YEp24 SSD1-v1

W303This study

CY5210 MATa sit4::HIS3 sap155::HIS3 sap185::ADE2 sap190::TRP1 SIT4/LEU2-CEN SAP4/YEp24SSD1-v1 W303 This study

CY5211 MATa sit4::HIS3 sap155::HIS3 sap185::ADE2 sap190::TRP1 SIT4:PY/LEU2-CEN YEp24SSD1-v1 W303

This study

CY5215 MATa sit4::HIS3 sap155::HIS3 sap185::ADE2 sap190::TRP1 SIT4:PY/LEU2-CEN SAP4/YEp24SSD1-v1 W303

This study

CY5217 MATa sap4::LEU2 SSD1-v1 W303 This studyCY5218 MATa sap155::HIS3 SSD1-v1 W303 This studyCY5220 MATa sap155::HIS3 sap4::LEU2 SSD1-v1 W303 This studyCY5224 MATa sap185::ADE2 sap190::TRP1 SSD1-v1 W303 This studyCY5234 MATa sap155::HIS3 sap185::ADE2 sap190::TRP1 SSD1-v1 W303 This studyCY5236 MATa sap155::HIS3 sap185::ADE2 sap190::TRP1 sap4::LEU2 SSD1-v1 W303 This studyCY5254 MATa sap155::HIS3 ssd1-d2 W303 This studyML58-4B MATa sis2::LEU2 SSD1-v1 ura3 leu2 his3 trp1 ade2 This studyML59-15A MAT bem2::LEU2 SSD1-v1 ura3 leu2 his3 trp1 ade2 This study

a The W303 strain background is ura3-1 leu2-3,112 his3-11,15 trp1-1 ade2-1 can1-100 and is normally ssd1-d2 (38). Plasmids are indicated by angled brackets.

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SAP155, SAP185, and SAP190 was used as the template DNA for PCR. Onesense (5-ATHMGKAARAAYAAYTCHGAYTAYGA-3) and two antisense(5-ADDNKCCRTTRAADTYTGYTG-3 and 5-TYDRYMAWRTGDCCCATRTADCC-3) degenerate primers (H represents ACT, D represents AGT, Mrepresents AC, W represents AT, R represents AG, Y represents CT, and Krepresents GT) were designed on the basis of three of the most conservedregions of the SAP155, SAP185, and SAP190 and used for PCR. Two DNAfragments, of 0 .7 and 0.92 kb, were amplified. The DNA sequences of these twoPCR products showed that they resulted from a single gene and that they

predicted a protein related to the other SAPs. The 0.92-kb PCR product hybrid-ized to clones 2985 and 3525 on chromosome VII of the Olson library of yeastinserts in lambda phage (30). The full-length SAP4 gene was isolated from boththe YCp50 (32) and the YEp24 (3) libraries by colony hybridization using thecloned PCR products as probes. A 5.1-kb ClaI-to-SfcI DNA fragment (fromabout 0.8 kb upstream of the SAP4 ATG initiation codon to about 1.8 kbdownstream of the SAP4 stop codon) was sequenced on both strands.

Epitope tagging of the SAPs. For SAP185 and SAP190, a NotI site was createdby oligonucleotide-directed mutagenesis (18) immediately upstream of the stopcodon (41). For SAP155, a 12-bp NotI linker was inserted into the BsaBI site thatoccurs 10 bp upstream of the stop codon. A pair of complementary oligonucle-otides that encode the HA epitope was inserted in-frame at the NotI site tocreate SAP190:HA, SAP185:HA, and SAP155:HA. The constructs were con-firmed by sequencing and Western blot (immunoblot) analysis. The SAP190:HA,SAP185:HA, and SAP155:HA genes fully complemented their respective SAPfunctions.

Deletion of the SAP genes. For sap190::TRP1, a NotI site was inserted byoligonucleotide-directed mutagenesis just after the translation initiation codonand just before the stop codon, allowing the deletion of DNA sequences forcodons 2 through 1033 (of a total of 1,033 codons) and replacement by a 0.83-kb

EcoRI-PstI fragment containing the TRP1 gene. For sap185::ADE2, the DNAsequences between the ClaI and NsiI sites containing codons 29 to 1024 (of atotal of 1,058 codons) were deleted and replaced with a 2.1-kb BglII fragmentcontaining the ADE2 gene. For sap155::HIS3, a NotI site was inserted just afterthe translational initiation codon by oligonucleotide-directed mutagenesis, andanother was inserted at the BsaBI site 10 bp upstream of the stop codon by NotIlinker insertion (see description of epitope tagging of SAP155, above), allowingthe deletion of DNA sequences for codons 2 to 997 (of a total of 1,000 codons)and replacement by a 1.8-kb BamHI fragment containing the HIS3 gene. Forsap4::LEU2, DNA sequences between the KpnI and SacI sites containingcodons 32 to 768 (of a total of 818 codons) were deleted and replaced with a2.2-kb XhoI-SalI fragment containing the LEU2 gene. All deletions were con-firmed by Southern analysis.

High-copy-number SAP plasmids. For SAP190, a 4.65-kb PvuII-SacI fragmentfrom 775 to 3880 relative to the A of the ATG initiation codon was clonedinto the BamHI site of YEp24. For SAP185, a 5.8-kb FspI-SalI fragment from1470 to 4342 relative to the A of the ATG initiation codon was cloned intothe BamHI site of YEp24. For SAP155, a 6.7-kb XhoI-PstI fragment from 2877to 3841 relative to the A of the ATG initiation codon was cloned into the

BamHI site of YEp24. The SAP155 fragment contains another ORF upstream ofthe SAP155 ORF. A frameshift mutation introduced at the SalI site (at codon187 of SAP155) renders the high-copy-number SAP155 plasmid inactive forsuppression of the temperature-sensitive phenotype of the sit4-102 strain. TheYEp24 plasmid carrying SAP4 is the original library clone containing about 10 kbof insert, including all of the sequenced region of SAP4.

Preparation of SIT4:PY. Duplex oligonucleotides were inserted into the NaeIsite of the SIT4 gene, changing the carboxyl terminus of the predicted SIT4protein from . . .RAGYFL to . . .RAGYFLMEYMPMEAGMEYMPMEA.Cells containing polyomavirus epitope-tagged SIT4 (SIT4:PY) as the only sourceof SIT4 grow slightly more slowly than isogenic SIT4 strains.

Construction of a SSD1-v1 W303 strain. Strain CY832 (MATa ssd1::LEU2W303) was transformed with a mixture of the 6-kb BamHI-SphI yeast DNAfragment containing the entire SSD1-v1 gene (from pCB881 [38]) and a TRP1CEN plasmid at a molar ratio of 100:1. Trp transformants were screened forthose that were also Leu, presumably as a result of a recombination event thatreplaced the LEU2 marker with the intact SSD1-v1 gene. To test for SSD1-v1,several Trp Leu transformants were crossed to CY1078 (ssd1-d2 W303sit4::HIS3 plus a SIT4/YCp50 plasmid to maintain v iability). After loss of the

SIT4/YCp50 plasmid, the diploids were sporulated and dissected. Because sit4is lethal in the absence of an SSD1-v allele, any diploid that gave viable His

(sit4) haploid progeny must have the SSD1-v1 gene contributed from the Trp

Leu parent. Crosses were used to obtain MATa and MAT SSD1-v1 W303strains. Interestingly, SSD1-v1 W303 strains do not mate and sporulate as well as

ssd1-d2 W303 strains. Of the laboratory strains surveyed, about half contain anssd1-d allele and about half contain an SSD1-v allele (38). Possibly, sequentialrounds of mating and sporulation would select for ssd1-d variants.

35S labeling, immunoprecipitation, and Western blot analysis. 35S labeling ofproteins, immunoprecipitation, and Western blot analysis were performed es-sentially as described previously (38).

Phosphatase treatment of immunoprecipitated SAPs. SAP155:HA, SAP185:HA, and SAP190:HA were immunoprecipitated from strains with and without adeletion of SIT4. The SAPs from the sit4 strains were phosphatase treatedessentially as described previously (42) except that the reactions were carried out

with 2 U of calf alkaline phosphatase (Boehringer Mannheim) in a 50-l reactionvolume at 30C for 15 min. The phosphatase inhibitor cocktail used was exactlyas described previously (42).

Antibodies. A monoclonal antibody for SAP155 (10/2-63) was generated byusing SAP155 (from large-scale SIT4:HA immunoprecipitates) as the antigen.

Antibody 12CA5, against the HA epitope (14), and an antibody against thepolyomavirus middle T epitope (15) were from ascites fluid.

Nucleotide sequence accession numbers. The sequence data reported havebeen assigned GenBank accession numbers U50560 (SAP155), U50561 (SAP190),and U50562 (SAP4).

RESULTS

Isolation of the SAP155 and SAP190 genes. The SAP155 andSAP190 proteins were purified by large-scale immunoprecipi-tation of SIT4:HA followed by SDS-polyacrylamide gel elec-trophoresis (see Materials and Methods). From amino acidsequences of SAP155 and SAP190 peptides, degenerate oligo-nucleotides were designed and used for PCR amplification ofgenomic yeast DNA (see Materials and Methods). The PCRproducts were from the SAP155 and SAP190 genes becausetheir DNA sequences predicted other sequenced SAP155 andSAP190 peptides that were not used to design the PCR prim-ers. The SAP155 and SAP190 PCR products were used toobtain the SAP155 and SAP190 genes by colony hybridization

to libraries of yeast genomic inserts in YCp50 (32) or YEp24(3).

We sequenced the SAP155 gene (see Materials and Meth-ods). The SAP190 gene (ORF SCYKR028w) was sequenced aspart of the chromosome XI sequencing project (11). However,our SAP190 clone contains a frameshift at codon 1009 relativeto the reported SAP190 sequence (see Materials and Meth-ods). On the basis of our SAP190 DNA sequence, we preparedthe SAP190:HA gene, which encodes a SAP190 protein withthe HA epitope at the carboxyl terminus. This construct ex-presses a fusion protein detectable by Western blotting withthe anti-HA monoclonal antibody. Interestingly, the predictedSAP155 and SAP190 proteins are similar to each other, with26% identity over their entire lengths (Fig. 1 and 2).

Identification and isolation of the SAP185 gene. Searches of

the databases with SAP190 identified an ORF (SCYJL098w)next to BCK1 on chromosome X (ORF J0840 in reference 23)that could encode a protein with 14% identity to SAP155 and42% identity to SAP190 (Fig. 1 and 2). We isolated this gene,

which we call SAP185, and prepared a strain containing achromosomal deletion of the gene (see Materials and Meth-ods).

If SAP185 associates with SIT4, it might be detectable inSIT4 immunoprecipitates. We previously showed that whenextracts prepared from cells containing either SIT4 or SIT4:HA

were immunoprecipitated with anti-HA antibody 12CA5,SAP155 and SAP190 specifically coimmunoprecipitated withSIT4:HA (38). However, the immunoprecipitates contained a185-kDa protein (called p188 in reference 38) regardless of

whether SIT4 or SIT4:HA extracts were used. Moreover, thepresence of the 185-kDa band was dependent on the additionof antibody 12CA5. Therefore, we had thought that, unlikeSAP155 and SAP190, the 185-kDa protein was not a specificSIT4-associated protein.

Interestingly, when SAP185 was deleted, the 185-kDa bandwas no longer present in 12CA5 immunoprecipitates of eitherSIT4 or SIT4:HA extracts (data not shown). Moreover, whenthe SAP185 gene was present on a high-copy-number plasmid,the 185-kDa band increased in levels when the anti-HA anti-body 12CA5 was used for immunoprecipitation of either SIT4-or SIT4:HA-containing extracts (data not shown). These find-ings indicate that SAP185 is recognized by the anti-HA anti-body 12CA5. Therefore, to determine if SAP185 associates

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with SIT4, we prepared a strain containing SIT4:PY and usedan anti-polyomavirus epitope antibody for the immunoprecipi-tations (15) (see Materials and Methods). A 185-kDa protein

was specifically present in the immunoprecipitates when theextracts were prepared from a SIT4:PYstrain but not when theextracts were prepared from a SIT4 strain (Fig. 3A, lanes 3 and4). Moreover, the 185-kDa band was absent from the SIT4:PYimmunoprecipitates when the extracts were prepared fromstrains containing a deletion of SAP185 (lane 5). Therefore,SAP185 specifically coimmunoprecipitates with SIT4:PY and isthe third high-molecular-mass SAP identified.

Identification and isolation of the SAP4 gene. To determineif additional SAP-related genes are present within the S. cer-

evisiae genome, we designed degenerate oligonucleotide PCRprimers based on conserved regions of SAP155, SAP185, andSAP190 (see Materials and Methods). A discrete PCR product

was obtained by using genomic DNA from a sap155 sap185sap190 strain (CY4985) as the template. The translated DNA

sequence of this PCR product predicts a SAP-related protein.The PCR product was used as a probe for colony hybridizationto obtain the full-length gene, which we call SAP4 (we do notcurrently know the apparent molecular mass of SAP4 on SDS-polyacrylamide gels). The sequence of the SAP4 gene predictsan 818-amino-acid protein related to the other three SAPs(Fig. 1 and 2).

The SAP genes encode a novel family of proteins whosemembers specifically associate with SIT4. Alignment of thefour predicted SAPs shows that they belong to the same familyof related proteins. The similarity is moderate in the amino-terminal regions, highest in the central regions, and least in thecarboxyl-terminal regions of the proteins (Fig. 1). Moreover,on the basis of their sequence similarity, the SAPs fall into twogroups: the SAP4/SAP155 group and the SAP185/SAP190group (Fig. 1 and 2). Although the SAPs are related to eachother, they are not significantly similar to any other proteins inthe current databases. All four SAPs are acidic (Fig. 2A). In

FIG. 1. The SAPs are a new family of related proteins. Alignment of the predicted SAP protein sequences is shown. First, SAP185 was aligned with SAP190 andSAP155 was aligned with SAP4. Then, the alignment of all four proteins was determined by the alignment of SAP155 with SAP190. Identical amino acid residues arein reverse font. The predicted SAP proteins are based on DNA sequences as described in Materials and Methods. , residue conserved among all four SAPs.



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addition, SAP155 has a high asparagine content (Fig. 2A).Interestingly, SAP155, SAP185, and SAP190 migrate onSDS8% polyacrylamide gels as proteins with molecularmasses of 155, 185, and 190 kDa, which are larger than theirpredicted molecular masses of 114.8, 121.3, and 117 kDa, re-spectively.

SAP155, SAP185, and SAP190 are the major proteins thatspecifically coimmunoprecipitate with SIT4:PY (Fig. 3A).SAP155, SAP185, and SAP190 were absent from the immuno-precipitates when the extracts were prepared from a sap155sap185 sap190 strain (Fig. 3A). We could not detect SAP4as a discrete band in SIT4:PY immunoprecipitates from 35S-labeled extracts, even when the SAP4 gene was present on ahigh-copy-number plasmid (Fig. 3B) and the SAP155, SAP185,

and SAP190 genes were deleted (Fig. 3A). Therefore, if SAP4does associate with SIT4, then either SAP4 is a very minor SAPunder these growth conditions (minimal medium with 2% glu-cose) and/or SAP4 comigrates with a nonspecific band so thatit is not detectable as a separate band on the gels.

We also examined if SAP155, SAP185, and SAP190 associ-ate with the type 1 or the type 2A protein phosphatase catalyticsubunit, to which SIT4 is related. Under conditions in whichthe individual SAPs were detected as intense bands in SIT4immunoprecipitates (by Western analysis using antibodies di-rected against the individual SAPs), we were not able to detectSAP155, SAP185, or SAP190 in immunoprecipitates of eitherepitope-tagged GLC7 (the single type 1 phosphatase catalyticsubunit in S. cerevisiae [12, 26, 39]) or epitope-tagged PPH21or PPH22 (the two type 2A phosphatase catalytic subunits in S.

cerevisiae [31, 36]) (data not shown). Therefore, the SAPsspecifically associate with the SIT4 phosphatase but not withthe catalytic subunit of either type 1 or type 2A phosphatase.

The SAPs associate with SIT4 in separate complexes. Whenyeast whole cell extracts are fractionated on gel filtration col-umns, about one-half (38) and sometimes as much as 90%(20a) of the SIT4 is present in high-molecular-mass SIT4-SAPcomplexes. The SIT4-SAP155 complex can be partially re-solved from the SIT4-SAP190 complex during gel filtration(38). Moreover, we cannot detect any SAP155 in large-scaleSAP190:HA immunoprecipitates (data not shown), further in-dicating that the SAP190-SIT4 complex is separate from theSAP155-SIT4 complex.

SAP155 and SAP190 are present in excess relative to SIT4.For example, 5 to 10 times more SAP155 was present in thecell extracts compared with the amount of SAP155 that coim-munoprecipitated with SIT4 (data not shown). Therefore, it ispossible that in vivo, SAP155 and SAP190 compete with eachother for binding to SIT4. Overexpression of either SAP155 orSAP190 caused an increase in the amount of that particularSAP in the SIT4:PY immunoprecipitates (Fig. 3B). Impor-tantly, the increased levels of either SAP155 or SAP190 de-creased the levels of the other SAP in the SIT4:PY immuno-precipitates (Fig. 3B). Western analysis of unlabeled extractsshowed that overexpression of SAP155 did not decrease thelevels of SAP190 and that overexpression of SAP190 did notdecrease the levels of SAP155 in the extracts (data not shown).Therefore, SAP155 and SAP190 compete for binding to SIT4.

Overexpression of any of the SAPs suppresses the temper-ature-sensitive phenotype of a sit4-102 strain. The first indica-tion that the SAPs act positively with SIT4 was that any one ofthe four SAP genes (SAP4, SAP155, SAP185, or SAP190),

FIG. 2. Molecular data for the SAPs. The molecular data (A), percent iden-tity (B), and similarity tree (C) were calculated on the basis of the proteinsequences shown in Fig. 1, using the GeneWorks or IG Suite program fromIntelliGenetics, Inc. For the similarity tree (dendrogram), the horizontal distanceis proportional to the relative sequence difference (cost of alignment) and wascalculated by the GeneWorks program. The vertical distances are arbitrary.

FIG. 3. The SAPs associate with SIT4 in separate complexes. (A) Immuno-precipitation of 35S-labeled proteins from strains containing untagged SIT4(lanes 1 to 3) or SIT4:PY (lanes 4 to 6), using a monoclonal antibody against thepolyomavirus epitope. Strains for lanes 3 and 4 have wild-type SAPgenes, whilethose for lanes 1, 2, 5, and 6 lack SAP155, SAP185, and SAP190. The strains forlanes 1 and 6 contain SAP4/YEp24, while the strains for lanes 2 through 5 haveYEp24. The strains used were CY5210 (lane 1), CY5206 (lane 2), CY5196 (lane3), CY5201 (lane 4), CY5211 (lane 5), and CY5215 (lane 6). A 50-kDa protein,

which is present at higher levels in lane 4, is present in both the SIT4 andSIT4:PY lanes and varies in amount from lane to lane between experiments. (B)Immunoprecipitation of 35S-labeled proteins from strains containing untagged

SIT4 (lanes 1 through 5) or SIT4:PY (lanes 6 through 10), using a monoclonalantibody against the polyomavirus epitope. The strains contained either YEp24(lanes 5 and 6), SAP190/YEp24 (lanes 4 and 7), SAP185/YEp24 (lanes 3 and 8),SAP155/YEp24 (lanes 2 and 9), or SAP4/YEp24 (lanes 1 and 10) and weretransformants of CY5169 (SIT4; lanes 1 through 5) or CY5170 ( SIT4:PY; lanes6 through 10). Why increased levels of SAP185 do not seem to compete withSAP155 and SAP190 for binding to SIT4 is not currently known. For all strains,the growth medium was SD containing tryptophan, adenine, and 2% glucose.



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when present on a high-copy-number plasmid, partially sup-pressed the temperature-sensitive phenotype of a sit4-102 mu-tant (Fig. 4C). Therefore, even though we have not yet beenable to detect physical association of SAP4 with SIT4 in 35S-labeled extracts, high-copy-number SAP4 is equivalent to high-copy-number SAP155, SAP185, and SAP190 in terms of sup-pression of the sit4-102 mutation.

Loss of the SAP genes causes either a slow growth rate orlethality. The second indication that the SAPs act positively

with SIT4 was that, like cells without SIT4, cells without theSAPgenes either had a slow growth rate or were inviable. Theeffects due to sit4 mutations are dependent on the SSD1 gene.

In ssd1-d strains, deletion ofSIT4 is lethal and sit4-102 mutantsare temperature sensitive and arrest in late G1. In SSD1-vstrains, sit4 mutants are viable but grow very slowly and

sit4-102 mutants are not temperature sensitive for growth (38).Because of the genetic interactions between SIT4 and SSD1, itis important to determine the phenotypes of the sap mutants inboth SSD1-v and ssd1-d backgrounds. We prepared isogenic

MATa and MAT SSD1-v1 derivatives of strain W303 (40),which normally has an ssd1-d2 allele (38) (see Materials andMethods) (interestingly, the SSD1-v1 W303 strains do notmate and sporulate as well as ssd1-d2 W303 strains). Strainscontaining deletions of the SAP genes and/or the SIT4 gene

FIG. 4. Effects due to the loss or gain ofSAPfunction on growth rates. (A and B) Strains of the indicated genotypes were streaked either onto YPD plates (froma fresh streakout of the same strains on a YPD plate) or onto YPGE plates (from a fresh streakout of the same strains on a YPGE plate) and grown for the indicatednumber of days (D) at 30C. For panel A, the times (1.5 days for YPD plates and 2.7 days for YPGE plates) were chosen so that the colony sizes of the wild-type (wt)strain were about the same on the YPGE plates as on the YPD plates. For panel B, the time (2.2 days) was chosen to compare growth of the sap185 sap190 strainon the YPD plate with that on the YPGE plate. The strains for panels A and B were all SSD1-v1 W303: CY4029 (wild type) CY5218 (sap155), CY4917 (sap185),CY4380 (sap190), CY5224 (sap185 sap190), CY5220 (sap155 sap4), CY5217 (sap4), CY3938 (sit4), CY5236 (sap155 sap185 sap190 sap4), and CY5234(sap155 sap185 sap190). (C) Strain CY144 (sit4-102 ssd1-d2) was transformed with either YEp24 or the indicated SAPgenes on YEp24. The transformants werepatched onto a SC-uracil plate, grown overnight at 24C, and replica plated onto a YPD plate. Growth is shown after 1.5 days at 37 C.



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were prepared: one set in the SSD1-v1 W303 background anda second set in the ssd1-d2 W303 background.

In the SSD1-v1 background, deletion of SAP185 alone orSAP190 alone caused no significant growth rate defect on ei-ther YPD or YPGE (GE represents 2% glycerol plus 2%ethanol) medium (Fig. 4A). However, cells with deletions ofboth SAP185 and SAP190 had a moderate slow-growth-ratephenotype on YPD medium (Fig. 4A). This finding suggests

that SAP185 and SAP190 have full or nearly full functionaloverlap and that their combined function is important whenthe cells are grown on YPD medium. By contrast, on YPGEmedium, the sap185 sap190 cells had only a slight defect ingrowth rate (Fig. 4A and B). In fact, in contrast to wild-typecells, the sap185 sap190 cells grew almost as fast on theYPGE plates as on the YPD plates (Fig. 4B). Therefore,SAP185 and SAP190 are especially important when the cellsare grown with glucose as the carbon source.

Deletion of SAP155 (in the SSD1-v1 background) caused aslight to moderate growth rate defect on both YPD and YPGEmedia (Fig. 4A). Combining a deletion of SAP155 with a de-letion of either SAP185 or SAP190 caused a slightly slowergrowth rate than deletion of SAP155 alone, but the effect wasnot as severe as deletion of both SAP185 and SAP190 (data not

shown). Therefore, either the function of SAP155 overlapsslightly with that of SAP185 and SAP190 or the cells requirefull SAP185/SAP190 activity when SAP155 is absent. In addi-tion, on either YPD or YPGE medium, deletion of SAP4 didnot alter the growth rate of an otherwise wild-type strain (Fig.4A) or ofsap155 strains and sap185 sap190 strains (datanot shown). However, the loss of SAP4 did cause a sap155strain (but not a sap185 sap190 strain) to grow more slowlyon SC-glycerol-ethanol (SC-GE) medium (data not shown).This finding suggests that, consistent with the sequence simi-larity, the function of SAP4 may be closer to that of SAP155than to that of SAP185 or SAP190.

The quadruple sap mutant (sap4 sap155 sap185 sap190SSD1-v1) grew as slowly as the isogenic sit4 mutant on eitherYPD or YPGE plates (Fig. 4A). This finding is consistent with

the idea that the SIT4 pathway is nonfunctional in the absenceof all four SAPs. When SAP4 was the only intact SAP gene, aslightly different result was obtained: the strain (sap155sap185 sap190 SSD1-v1) grew nearly as slowly as the sit4mutant on YPD medium but significantly faster than the sit4mutant on YPGE medium (Fig. 4A). Therefore, as assayed inthe absence of SAP155, SAP185, and SAP190, SAP4 is moreimportant when the cells are grown on glycerol-ethanol me-dium than when they are grown on glucose medium.

In the ssd1-d2 W303 background, deletion ofSIT4 is lethal(38). Significantly, deletion of the three major SAP genes(SAP155, SAP185, and SAP190) or deletion of all four SAPgenes (SAP4, SAP155, SAP185, and SAP190) was also lethal inthe ssd1-d2 background (data not shown). Cells containing anyother combination ofsap deletions in the ssd1-d2 background

were viable and grew at a rate similar to that of cells with thesame combination ofsap deletions in the SSD1-v1 background(data not shown). In summary, in both the ssd1-d2 andSSD1-v1 backgrounds, loss of all SAPfunction is equivalent toloss of SIT4 function by growth rate and viability tests.

Deletion of the SAP genes causes a G1 delay. Compared with

wild-type SIT4 cells, sit4 SSD1-v1 cells have a defect in CLN1and CLN2 G1 cyclin accumulation, spend a longer time in G1,and have a delay in the initiation of DNA replication and budformation (13). For exponentially growing wild-type cultures,most of the cells have a 2n DNA content, about 80% of thecells are budded, and most of the 1n cells are small (Fig. 5),indicating that the G1 phase is short relative to the other stages

of the cell cycle. By contrast, sit4 SSD1-v1 cells are delayed inG1: there are more 1n than 2n cells, there is a marked reduc-tion in the percentage of budded cells, and there are many verylarge 1n cells (Fig. 5). The large 1n sit4 SSD1-v1 cells aremost likely from two sources: (i) the large 1n mother cells thathave just separated from the daughter cell and (ii) the newdaughter cells that grow to a large cell size (as determined bymicroscopic analysis of pedigrees [13]) before they form a budand replicate their DNA.

As the SAPgenes were progressively removed, the cells wereincreasingly delayed in G1 (Fig. 5). Importantly, the loss of allfour SAPgenes gave rise to similar increases in 1n cells relativeto 2n cells, similar reductions in the percentage of budded

FIG. 5. Effects due to the loss ofSAP function on percentage budded cells,cell size, and DNA content. Strains of the indicated genotypes, which were thesame as those used for Fig. 4, were grown to exponential phase in SC-glucosemedium and prepared for flow cytometry analysis as described previously (13).The percentage of budded cells (upper right corner in the panels on the left) wasdetermined, by counting at least 400 cells for each of three independent cultures,from the sample of cells used for the flow cytometry. For the plots on the left, cellcount (vertical axis) is plotted versus fluorescence intensity, which is proportionalto DNA content (horizontal axis). For the plots on the right, the vertical axis isDNA content, the horizontal axis is scattering, which is proportional to cell size,and the intensity and density of the datum points are proportional to the cellcount.



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cells, and as many large 1n cells as seen for the sit4 SSD1-v1cells (Fig. 5). In summary, loss of all SAPfunction causes a G1delay very similar to that caused by the loss of SIT4.

A strain without the SAP genes is sensitive to the loss ofCLN3, SIS2, or BEM2. Like a strain without SIT4, a strain

without the SAPgenes is sensitive to the loss ofCLN3, SIS2, orBEM2. We have previously shown that SIT4 is required for thenormal expression of the CLN1 and CLN2 G1 cyclins (13).CLN3 also provides a pathway for activating the expression ofCLN1 and CLN2. Cells containing a deletion of CLN3 have a

normal growth rate, but they accumulate CLN1 and CLN2RNAs at slower than normal rates and initiate bud formationand DNA synthesis at a larger than normal cell size (9, 41).CLN3 and SIT4 provide additive pathways for CLN1 andCLN2 expression (13), so that cln3 sit4 SSD1-v1 segregantsfrom a cross of a cln3 strain to a sit4 strain either areinviable or grow extremely slowly (1 week to form a very smallcolony). Moreover, the synthetic phenotypes resulting fromcombining the loss ofCLN3 with the loss ofSIT4 is due to lowG1 cyclin levels because it is cured by expressing CLN2 at near

wild-type levels from a heterologous SIT4-independent pro-moter (Schizosaccharomyces pombe ADH:CLN2 [13]).

From a cross of a sap155 sap185 sap190 SSD1-v1 strainto a cln3 SSD1-v1 strain, the sap155 sap185, the sap155sap190, and the sap185 sap190 segregants were not verysensitive to the loss of CLN3. However, the sap155 sap185sap190 cln3 SSD1-v1 segregants either were inviable orgrew extremely slowly (Table 2). Moreover, the synthetic phe-notypes resulting from combining the loss of CLN3 with theloss of the three major SAPgenes is due to low G1 cyclin levelsbecause it was cured by expressing CLN2 from a heterologousSIT4-independent promoter (S. pombe ADH:CLN2) (data notshown). Therefore, by this test of a requirement for G1 cyclinexpression, the loss of the three major SAPs is similar to theloss of SIT4.

In sit4 SSD1-v strains, overexpression of SIS2 stimulatesthe growth rate and increases the expression of SWI4, CLN1,and CLN2 (8). Importantly, high-copy-number SIS2 increased

the growth rate of a sap4 sap155 sap185 sap190 SSD1-v1strain to the same extent as it did for a sit4 SSD1-v1 strain(data not shown). Moreover, sis2 sit4 SSD1-v1 cells areinviable (8). In a cross of a sap155 sap185 sap190 SSD1-v1strain to a sis2 SSD1-v1 strain (which has a wild-type growthrate [8]), all seven of the predicted sis2 sap155 sap185sap190 segregants were inviable (Table 2). Even the sap155sap185, the sap155 sap190, and the sap190 strains grew

significantly more slowly in the absence ofSIS2. Therefore, likecells without SIT4, cells without the SAPs are very sensitive tothe loss of SIS2, and their growth rate defect is partially sup-pressed by overexpression of SIS2.

In addition to G1 cyclin expression, SIT4 is also requiredduring late G1 for bud formation (13). Moreover, sit4SSD1-v1 cells are inviable in the absence of BEM2 (10, 17).BEM2 is involved in bud emergence, but cells are normally

viable in the absence of BEM2 (2, 16). Significantly, like cellswithout SIT4, cells without SAP155, SAP185, and SAP190 areinviable in the absence of BEM2 (Table 2). Therefore, the lossof SAP function is equivalent to the loss of SIT4 with respectto the genetic interactions with BEM2. The loss of BEM2 incombination with only certain deletions of the SAP genes isdiscussed below. In summary, these genetic interactions with

CLN3, SIS2, and BEM2 further demonstrate that the SAPsfunction positively with SIT4.

In vivo, SAP function requires SIT4, and conversely, SIT4function requires the SAPs. The growth rate of a sap4sap155 sap185 sap190 strain on YPD medium was notstimulated by overexpression of SIT4 (from its own promoteror the ADH1 promoter, both on a high-copy-number plasmid)(data not shown). In addition, overexpression of eitherSAP155, SAP185, or SAP190 (the given SAP gene on a high-copy-number plasmid) did not increase the growth rate of asit4 SSD1-v1 strain on YPD medium (data not shown). Whendeletions of SAP155, SAP185, and SAP190 were combined

with a deletion of SIT4 (when SAP4 was present), the straingrew at the same rate as a sit4 SSD1-v1 strain (on either YPDor YPGE medium) (data not shown). Therefore, by this test,

SAP155, SAP185, and SAP190 do not have important func-tions in the absence of SIT4. In addition, this finding showsthat SAP4 is not able to stimulate the growth rate and may benonfunctional in the absence of SIT4. Further evidence sug-gesting that SIT4 combines with the SAPs to provide a functionnot provided by either SIT4 alone or the SAPs alone is fromthe effects due to the overexpression of both SIT4 and eitherSAP155 or SAP190 (all expressed from the ADH1 promoter ona 2m plasmid). Overexpression of either SIT4 alone, SAP155alone, or SAP190 alone caused either no effect or a very slightslow-growth-rate phenotype (data not shown). By contrast,overexpression of both SIT4 and SAP155 or of both SIT4 andSAP190 caused a very strong defect in the growth rate (datanot shown). In summary, these findings suggest that the func-tion of SIT4 requires the SAPs and that, in turn, the functionsof the SAPs require SIT4.

The SAPs provide distinct functions. By sequence similarity,the SAPs fall into two groups: the SAP4/SAP155 group and theSAP185/SAP190 group (Fig. 1 and 2). The first indication thatthe SAPs provide distinct functions was the effect due to theloss of certain SAP combinations on the growth rates of thecells (Fig. 4). Another indication that the SAPs provide distinctfunctions was the sensitivity of a bem2 strain to the loss ofSAP function. In 55 tetrads from a cross of a sap155 sap185sap190 SSD1-v1 strain to a bem2 SSD1-v1 strain, thesap155, the sap155 sap185 (SAP190 only, not consideringSAP4), and the sap155 sap190 (SAP185 only) segregantsgrew only slightly more slowly if they also had a deletion of

TABLE 2. Sensitivity of a sap155 sap185 sap190 SSD1-v1 strainto the loss ofCLN3, SIS2, or BEM2a

Mutation

Growth rate of strain with genotype:

SAP SIT4 sap155sap185sap190

sap155sap185sap190

sit4

cln3 Wild type Slightly

slower

Slightly

slower

Lethalb Lethalb

sis2 Wild type Slower Slower Lethal Lethalbem2 Moderatec Slightly

slowerLethald Lethale Lethal

a The effects due to the loss of CLN3, SIS2, or BEM2 on the growth rates ofstrains without the indicated SAP genes were determined. Slightly slowerindicates that the particular sap segregants grew very slightly but detectably moreslowly if they also had a cln3 or bem2 mutation. The strains used for thecrosses were CY4985 and CY4102 for the cln3 cross, ML58-4B and CY4987 forthe sis2 cross, and CY4985 and ML59-15A for the bem2 cross. All strains wereSSD1-v.

b Of the 18 sap155 sap185 sap190 cln3 segregants, 13 were inviable and5 grew extremely slowly. Of the 40 sit4 cln3 segregants, 22 were inviable and18 grew extremely slowly.

c The growth rate of a bem2 strain is dependent on SSD1. In ssd1-d back-grounds, bem2 causes a slow growth rate. In SSD1-v backgrounds, bem2causes a small defect in the growth rate.

d Of the five sap185 sap190 bem2 segregants, four were inviable and onegrew extremely slowly and papillated to faster growth.

e Of the 12 sap155 sap185 sap190 bem2 segregants, 11 were inviable and1 grew extremely slowly.



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BEM2 (Table 2 and data not shown). By contrast, of the fivepredicted sap185 sap190 bem2 segregants, four were invi-able and one grew extremely slowly and papillated to fastergrowth (Table 2). Therefore, the function of SIT4 that is es-sential in the absence of BEM2 requires either SAP185 orSAP190. This function of SIT4 cannot be provided by SAP155.

These effects are not simply due to the difference in thequantity of the remaining SAP activity. We determined if over-expression of a SAP from one group could cure the growthdefects due to the absence of the other group. The growth rateof a sap185 sap190 strain was not stimulated by either theSAP155 gene or the SAP4 gene on a high-copy-number plas-mid (Fig. 6A). By contrast, either the SAP185 or the SAP190gene on a high-copy-number (Fig. 6A) or a low-copy-numberCEN plasmid (data not shown) caused the sap185 sap190strain to grow like wild-type cells. In the strains containinghigh-copy-number SAP155, SAP155 was overexpressed at least10-fold (as determined by Western analysis of serial dilutionsof extracts [data not shown]). Therefore, overexpression ofSAP155 cannot compensate for the loss of SAP185/SAP190function.

Similarly, neither the SAP185 gene nor the SAP190 gene ona high-copy-number plasmid stimulated the growth rate of asap155 strain (Fig. 6B). Although the sequence of SAP4 ismore related to that of SAP155 and the loss ofSAP4 caused asap155 strain to grow more slowly on SC-GE medium, high-copy-number SAP4 was not able to stimulate the growth rate of

a sap155 strain (Fig. 6B). This finding suggests either thatSAP4 cannot substitute for SAP155 or that high-copy-numberSAP4 does not provide enough SAP155-like function to bedetected by this assay. In summary, SAP155 provides a cellularfunction that cannot be provided by either SAP185 or SAP190and the SAP185/SAP190 group provides a cellular functionthat cannot be provided by SAP155.

The SAPs are hyperphosphorylated in the absence of SIT4.We had previously shown that the SAPs which coimmunopre-cipitate with SIT4 are phosphoproteins (38). With reagents todetect the SAPs independently of SIT4, we can now address

whether the presence or absence of SIT4 affects the phosphor-ylation state of the SAPs. The SAP155, SAP185:HA, andSAP190:HA in extracts from sit4 SSD1-v1 strains all haveforms that migrate more slowly on SDS-polyacrylamide gelsthan the proteins from isogenic SIT4 strains (Fig. 7A). More-over, the more slowly migrating forms of the SAP185:HA pro-tein (Fig. 7B) and the SAP155:HA and SAP190:HA proteins(data not shown) were converted to a faster-migrating formafter treatment with alkaline phosphatase, indicating that thereduced mobility of the SAPs from sit4 cells is due to in-creased phosphorylation. In summary, the loss of SIT4 causesSAP155, SAP185, and SAP190 to become hyperphosphory-lated. In experiments in which we added SIT4 (from either a

wild-type strain or a strain lacking SAP155, SAP185, andSAP190) to the hyperphosphorylated SAPs obtained from asit4 strain, we were not able to detect dephosphorylation ofthe SAPs by SIT4 in vitro (data not shown). However, in these

FIG. 6. SAP185 and SAP190 provide a function that is distinct from thatprovided by SAP155. Strain CY5001 (sap185 sap190) (A) and strain CY5254(sap155) (B) were transformed with either YEp24 (control) or the indicatedSAPgene on YEp24 (HC# SAP; see Materials and Methods for descriptions ofplasmids). The transformants were streaked onto plates containing SC medium

without uracil and with 2% glucose and grown for 2 days at 30C.

FIG. 7. The SAPs are hyperphosphorylated in the absence of SIT4. (A)Western analysis of SAP155, SAP185:HA, and SAP190:HA in extracts preparedfrom isogenic wild-type SIT4 (WT) or sit4 () SSD1-v1 strains grown in SCmedium without uracil and with 2% glucose. The strains were CY4796 ( sit4)and CY4802 (SIT4) for the SAP190:HA samples and CY5067 (SIT4) and

CY5077 (sit4) for the SAP185:HA and SAP155 samples. Three lanes wereincluded for the SAP190 samples to demonstrate the small mobility change.Interestingly, the levels of SAP155 were about threefold higher and the levels ofSAP155 RNA (which do not vary in the cell cycle) were about two- to threefoldhigher in sit4 SSD1-v1 strains than in isogenic SIT4 strains (data not shown).Perhaps the cells detect the loss of SIT4 and induce the expression of SAP155.The SAP185:HA and SAP190:HA blots were probed with monoclonal antibody12CA5 (anti-HA ascites fluid), and the SAP155 blot was probed with an anti-SAP155 monoclonal antibody. (B) SAP185:HA was immunoprecipitated fromeither a wild-type SIT4 strain (CY5067; lane 1) or a sit4 strain (CY5077; lanes2 to 5). For lanes 1 and 2, the immune complexes were kept on ice withoutfurther treatment. For lanes 3 through 5, the immune complexes were treated for15 min at 30C with either calf alkaline phosphatase (lane 3), a phosphataseinhibitors mix plus the calf alkaline phosphatase (lane 4), or the phosphataseinhibitors mix only (lane 5) as described in Materials and Methods.



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experiments, the hyperphosphorylated SAPs did not bind toSIT4, so that whether SIT4 can dephosphorylate the SAPs in

vivo remains unknown.

DISCUSSION

The SIT4 protein phosphatase has a unique subunit com-position. SIT4 is about 55% identical to the catalytic subunit of

type 2A phosphatases and about 45% identical to the catalyticsubunit of type 1 phosphatases (1). In side-by-side comparisonsof immunoprecipitates of the S. cerevisiae SIT4, type 1 (GLC7),and type 2A (both PPH21 and PPH22) phosphatases, the in

vitro activity (toward lysozyme [Sigma L 1526] phosphorylatedby protein kinase A) of SIT4 was at least as sensitive to low[nanomolar] concentrations of okadaic acid as the PPH21 andPPH22 type 2A phosphatases (20a). Therefore, by proteinsequence and okadaic acid sensitivity, SIT4 is a type 2A-likephosphatase. However, SIT4 is clearly distinct from the type2A phosphatases. First, SIT4 provides an essential (in ssd1-dbackgrounds) or very important (in SSD1-v1 backgrounds) cel-lular function in late G1 that is distinct from the essentialfunctions of the budding yeast type 2A phosphatases (19, 31,36). Second, SIT4 differs from the type 2A phosphatases in

subunit composition. In S. cerevisiae, as in other organisms (4),the type 2A catalytic subunits (PPH21 and PPH22) associate

with two regulatory subunits, an A subunit (a 65-kDa proteinencoded by TPD3 [43]) and a B subunit (a 60-kDa proteinencoded by CDC55 [16]). Although we are able to detect TPD3and CDC55 as very intense bands in Western analysis of eitherPPH21:HA or PPH22:HA immunoprecipitates, we have notbeen able to detect TPD3 or CDC55 in SIT4:HA immunopre-cipitates (7a). Instead, SIT4:HA associates with at least threeSAPs with apparent molecular masses of 155, 185, and 190kDa. Moreover, we have not been able to detect the SAPs inimmunoprecipitates of HA-tagged type 2A (or type 1) catalyticsubunits. Therefore, SIT4 associates with the SAPs whereasthe type 2A catalytic subunits associate with the TPD3 andCDC55 subunits.

SIT4 homologs have been found and characterized in S.pombe and in Drosophila melanogaster(21, 22, 35). These find-ings suggest that, like the conserved type 1 and type 2A proteinphosphatases, SIT4 and the SIT4 homologs define a uniqueclass of protein phosphatase catalytic subunit that are con-served during evolution. In this report, we identified the high-molecular-weight SAPs as a new protein family whose mem-bers uniquely associate with SIT4. We suggest that the SAPsare also a conserved class of proteins whose members bind toand function positively with the SIT4 homologs in other eu-karyotic organisms.

Physical association of the SAPs with SIT4. Previously, weshowed that SAP155 and SAP190 associate with SIT4 in a cellcycle-dependent manner (38). Reexamination of the data ofSutton et al. (38) shows that SAP185, which we thought at thattime was a nonspecific band, also associates with SIT4 in a cellcycle-dependent manner. Several pieces of evidence show thatthe SAPs are in separate complexes with SIT4: (i) the SAP190-SIT4 complexes can be partially resolved from the SAP155-SIT4 complexes on gel filtration columns (38), (ii) we cannotdetect any SAP155 in SAP190:HA immunoprecipitates (datanot shown), (iii) the binding of SAP155 and SAP185 to SIT4does not require the presence of SAP190 (data not shown),and (iv) SAP155 and SAP190 compete with each other forbinding to SIT4 (Fig. 3B). These findings suggest that there isprobably a single SAP binding site on SIT4, and because of thesimilarity of the SAPs to one another, there may be commonsequences within the SAPs that specify binding to SIT4.

The SAPs function positively with SIT4. Any one of the fourSAPgenes in high copy number can suppress the temperature-sensitive phenotype ofsit4-102 strains. In addition, the loss ofSAP function is, by every test that we have done, equivalent tothe loss of SIT4. These findings suggest that the SAPs functionpositively with SIT4 and that the four SAPs together are in-

volved in the same set of cellular functions as SIT4. Moreover,the SAPs and SIT4 require each other to function in vivo.

Although overexpression of any SAP can suppress the temper-ature-sensitive phenotype ofsit4-102 strains, it cannot increasethe growth rate of a sit4 SSD1-v1 strain. Similarly, overex-pression of SIT4 cannot suppress the defects due to loss of theSAPs (either the loss of SAP155 alone or loss of both SAP185and SAP190, of SAP155, SAP185, and SAP190, or of all fourSAPs). Therefore, overexpression of SIT4 cannot bypass therequirement for the SAPs and likewise, overexpression of theSAPs cannot bypass the requirement for SIT4. Furthermore,combining a deletion ofSIT4 with deletions of the three majorSAPgenes (SAP155, SAP185, and SAP190) is equivalent to thedeletion ofSIT4 alone. By these criteria, the SAPs do not haveimportant functions in addition to their functions with SIT4,and SIT4 does not have significant functions, except possiblythose with SAP4, in addition to its functions with SAP155,

SAP185, and SAP190.The role of SAP4. We have not yet been able to detect SAP4

as a discrete band in SIT4 immunoprecipitates of 35S-labeledextracts. However, the genetic interactions of SAP4 suggestthat it functions as a SAP. High-copy-number SAP4 partiallysuppresses the temperature-sensitive phenotype of a sit4-102strain. Also, in the absence of SAP155, SAP185, and SAP190,SAP4 stimulates the growth rate slightly on YPD medium andmoderately on YPGE medium (Fig. 4A). Moreover, this abilityof SAP4 to stimulate the growth rate is dependent on SIT4because a sit4 SAP4 sap155 sap185 sap190 SSD1-v1strain grows at the same rate as either a sit4 SSD1-v1 strain ora sap4 sap155 sap185 sap190 SSD1-v1 strain. Together,these findings suggest that SAP4 functions as a SIT4-associatedprotein.

The SAPs provide at least two distinct cellular functions. Asmentioned above, the SAPs together are involved in most or allof the functions that are provided by SIT4. The combined lossof the SAP genes, like the loss of SIT4, causes either lethalityor a slow growth rate with a G1 delay. In addition, the loss ofSAP155, SAP185, and SAP190 is very similar to the loss ofSIT4in terms of the genetic interactions with the loss of eitherCLN3, SIS2, or BEM2. The genetic interactions with CLN3show that, like SIT4, the SAPs are required for normal G1cyclin expression.

However, some of the functions of SIT4 are provideduniquely via SAP155 (function B in Fig. 8A) because the lossof SAP155 causes a growth defect that is not cured by overex-pressing either SAP185 or SAP190. SAP185 and SAP190 prob-ably have significant or total functional overlap: deletion ofeither SAP185 or SAP190 does not cause a detectable growthrate defect, but deletion of both SAP185 and SAP190 causes asignificant growth rate defect. Moreover, this growth rate de-fect ofsap185 sap190 strains is not cured by the overexpres-sion of SAP155. Therefore, SAP185 and SAP190 together pro-

vide a unique SIT4-dependent function (function A in Fig.8A). This SAP185-SAP190 function is required for the SIT4activity that is essential in the absence of BEM2 (Table 2),raising the possibility that the SAP185/SAP190-dependentSIT4 function is involved, directly or indirectly, in budding.

The loss of SAP155 combined with the loss of SAP185 andSAP190 causes inviability in ssd1-d backgrounds and a veryslow growth rate in SSD1-v backgrounds (an effect more severe



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than either deletion of SAP155 alone or deletion of bothSAP185 and SAP190). This effect can be explained by one oftwo possibilities. For one possibility, SAP155 has no functionaloverlap with SAP185 and SAP190. SAP155 provides only func-tion B, while SAP185 and SAP190 together provide only func-tion A. The synthetic growth defect or lethality of combiningthe loss of SAP185/SAP190 with the loss of SAP155 is due onlyto the loss of both functions A and B. In this case, if function

A and function B are in separate processes, then the abnormaloperation of both processes resulting from the absence of bothfunctions A and B causes ssd1-d cells to be inviable. Alterna-tively, function A and function B may be required at twodifferent steps within the same process, so that in the absenceof both functions, this single process is so defective that ssd1-dcells are inviable.

For the second possibility, which is difficult to exclude,SAP155 shares a common function (function C in Fig. 8A) withSAP185 and SAP190. This common function would be distinctfrom functions A and B. The finding that a sap155 strain (butnot a SAP155 strain) grows slightly more slowly in the absenceof either SAP185 or SAP190 could be either due to SAP155having a slight functional overlap with SAP185 and SAP190 ordue to a sap155 strain having a greater requirement than aSAP155 strain for a fully active A function. If such a commonfunction exists, then the synthetic defects due to the absence ofSAP155, SAP185, and SAP190 could be due to the loss offunction C alone (and unrelated to the loss of functions A andB) or due to any combination of losses of functions A, B,and/or C.

Do the SAPs activate SIT4 or does SIT4 activate the SAPs?We envision two models for the function of the SAPs relativeto SIT4. In model 1 (Fig. 8B), SIT4 regulates the SAPs. In thismodel, the SAPs have a different activity, localization, or func-tion when they are activated by SIT4, either via association intoa complex with SIT4 or via dephosphorylation by SIT4. Inmodel 2, the SAPs regulate SIT4. In this model, the SAPs

could either physically target SIT4 to the specific SIT4 sub-strates (similar to the glycogen-targeting subunit of type 1phosphatases [4]) or regulate the phosphatase activity of SIT4so that SIT4 is much more active for dephosphorylating thespecific SIT4 substrates (which are not the SAPs themselves).For the latter case, SIT4 would be a SAP-dependent phos-phatase for the in vivo substrates, similar to the cyclin-depen-dent kinases such as CDC28. Both SIT4 and CDC28 are

present at relatively constant levels throughout the cell cycle,but their activities may require the association with other pro-teins, and these associations are regulated in the cell cycle.

That the SAPs are hyperphosphorylated in the absence ofSIT4 is consistent with either model. In model 1, the SAPs

would be the relevant physiological substrates of SIT4 andbecome hyperphosphorylated and inactive in the absence ofSIT4. In model 2, the SAPs regulate the activity of SIT4 towardother substrates. However, the SAPs might become hyperphos-phorylated either because sit4 cells spend a longer time in G1(when SAP kinases might be more active) or because they donot undergo their normal cell cycle-regulated association withSIT4. Distinguishing between these models will require thedetermination of the downstream processes or componentsthat are either regulated via dephosphorylation by SIT4 or

regulated by the SAPs.For either model, the activity of the SAPs or the activity of

SIT4 would be regulated by the association of the SAPs withSIT4. We previously showed that while the levels of SIT4 areconstant during the cell cycle, the association of SIT4 with theSAPs is regulated during the cell cycle (38). Moreover, ourinitial characterizations show that the amount of the SAPs thatare associated with SIT4 is regulated by cellular growth signals:by carbon source (glycerol-ethanol versus glucose), by the pres-ence or absence of amino acids in the growth medium, andduring saturation of the cultures (20b). Possibly, the SAPs areinvolved in transducing nutrient growth signals via their asso-ciation with SIT4, linking growth signals with G1 cyclin expres-sion, bud formation, and other late G1 processes. The eluci-dation of such a signal transduction pathway will require the

determination of how the association of the SAPs with SIT4 isregulated and what downstream processes are regulated by theSAPs and SIT4.

ACKNOWLEDGMENTS

We thank P. Burfeind for flow cytometry analysis, C. Bautista, andM. Falkowski for monoclonal antibodies, M. Christman for the cell lineexpressing the anti-PY antibody, J. Thorner for the OCR roman fontused in Fig. 1, B. Osmond for plasmid pCS3-1, and A. Doseff, B.Futcher, D. Shevell, and A. Sutton for comments on the manuscript.

This work was supported by Human Frontier postdoctoral fellow-ship LT-268/92 to F.D.S. and NIH grant GM45179 to K.T.A.

M.M.L. and F.D.S. contributed equally to this work.

REFERENCES

1. Arndt, K. T., C. A. Styles, and G. R. Fink. 1989. A suppressor of a HIS4

transcriptional defect encodes a protein with homology to the catalytic sub-unit of protein phosphatases. Cell 56:527537.

2. Bender, A., and J. R. Pringle. 1991. Use of a screen for synthetic lethal andmulticopy suppressee mutants to identify two new genes involved in mor-phogenesis in Saccharomyces cerevisiae. Mol. Cell. Biol. 11:12951305.

3. Carlson, M., and D. Botstein. 1982. Two differentially regulated mRNAswith different 5 ends encode secreted and intracellular forms of yeast in-vertase. Cell 28:145154.

4. Cohen, P. 1989. The structure and regulation of protein phosphatases. Annu.Rev. Biochem. 58:453508.

5. Collins, K., R. Kobayashi, and C. W. Greider. 1995. Purification of Tetra-hymena telomerase and cloning of genes encoding the two protein compo-nents of the enzyme. Cell 81:677686.

6. Cross, F. R. 1990. Cell cycle arrest caused by CLN gene deficiency inSaccharomyces cerevisiae resembles START-I arrest and is independent of

FIG. 8. Models of SIT4 and SAP function. (A) Different functions providedby the various SAPs. (B) SIT4 as a regulator of the SAPs (model 1) versus theSAPs as regulators of SIT4 (model 2).



12/12

the mating-pheromone signalling pathway. Mol. Cell. Biol. 10:64826490.7. Cross, F. R., M. Hoek, J. D. McKinney, and A. H. Tinkelenberg. 1994. Role

of Swi4 in cell cycle regulation of CLN2 expression. Mol. Cell. Biol. 14:47794787.

7a.Di Como, C. J. Unpublished results.8. Di Como, C. J., R. Bose, and K. T. Arndt. 1995. Overexpression of SIS2,

which contains an extremely acidic region, increases the expression ofSWI4,CLN1, and CLN2 in sit4 mutants. Genetics 139:95107.

9. Dirick, L., T. Bohm, and K. Nasmyth. 1995. Roles and regulation of Cln-Cdc28 kinases at the start of the cell cycle of Saccharomyces cerevisiae.

EMBO J. 14:48034813.10. Doseff, A. I., and K. T. Arndt. 1995. LAS1 is an essential nuclear protein

involved in cell morphogenesis and cell surface growth. Genetics 141:857871.

11. Dujon, B., D. Alexandraki, B. Andre, W. Ansorge, V. Baladron, J. P. Ballesta,A. Banrevi, P. A. Bolle, M. Bolotin-Fukuhara, P. Bossier, et al. 1994. Com-plete DNA sequence of yeast chromosome XI. Nature (London) 369:371378.

12. Feng, Z. H., S. E. Wilson, Z. Y. Peng, K. K. Schlender, E. M. Reimann, andR. J. Trumbly. 1991. The yeast GLC7 gene required for glycogen accumu-lation encodes a type 1 protein phosphatase. J. Biol. Chem. 266:2379623801.

13. Fernandez-Sarabia, M. J., A. Sutton, T. Zhong, and K. T. Arndt. 1992. SIT4protein phosphatase is required for the normal accumulation of SWI4,CLN1, CLN2, and HCS26 RNAs during late G1. Genes Dev. 6:24172428.

14. Field, J., J. Nikawa, D. Broek, B. MacDonald, L. Rodgers, I. A. Wilson, R. A.Lerner, and M. Wigler. 1988. Purification of a RAS-responsive adenylylcyclase complex from Saccharomyces cerevisiae by use of an epitope additionmethod. Mol. Cell. Biol. 8:21592165.

15. Grussenmeyer, T., K. H. Scheidtmann, M. A. Hutchinson, W. Eckhart, andG. Walter. 1985. Complexes of polyoma virus medium T antigen and cellularproteins. Proc. Natl. Acad. Sci. USA 82:79527954.

16. Healy, A. M., S. Zolnierowicz, A. E. Stapleton, M. Goebl, A. A. DePaoli-Roach, and J. R. Pringle. 1991. CDC55, a Saccharomyces cerevisiae geneinvolved in cellular morphogenesis: identification, characterization, and ho-mology to the B subunit of mammalian type 2A protein phosphatase. Mol.Cell. Biol. 11:57675780.

17. Kim, Y.-J., L. Francisco, G.-C. Chen, E. Marcotte, and C. S. M. Chan. 1994.Control of cellular morphogenesis by the Ipl2/Bem2 GTPase-activating pro-tein: possible role of protein phosphorylation. J. Cell Biol. 127:13811394.

18. Kunkel, T. A. 1985. Rapid and efficient site-specific mutagenesis withoutphenotypic selection. Proc. Natl. Acad. Sci. USA 82:488492.

19. Lin, F. C., and K. T. Arndt. 1995. The role of Saccharomyces cerevisiae type2A phosphatase in the actin cytoskeleton and in entry into mitosis. EMBO J.14:27452759.

20. Lowndes, N. F., A. L. Johnson, L. Breeden, and L. H. Johnston. 1992. SWI6protein is required for transcription of the periodically expressed DNAsynthesis genes in budding yeast. Nature (London) 357:505508.

20a.Luke, M. M. Unpublished results.20b.Luke, M. M., F. Della Seta, and K. T. Arndt. Unpublished results.21. Mann, D. J., V. Dombradi, and P. T. Cohen. 1993. Drosophila protein

phosphatase V functionally complements a SIT4 mutant in Saccharomycescerevisiae and its amino-terminal region can confer this complementation toa heterologous phosphatase catalytic domain. EMBO J. 12:48334842.

22. Matsumoto, T., and D. Beach. 1993. Interaction of the pim1/spi1 mitoticcheckpoint with a protein phosphatase. Mol. Biol. Cell 4:337345.

23. Miosga, T., E. Boles, I. Schaaff-Gerstenschlager, S. Schmitt, and F. K.Zimmermann. 1994. Sequence and function analysis of a 9.74 kb fragment ofSaccharomyces cerevisiae chromosome X including the BCK1 gene. Yeast10:14811488.

24. Nasmyth, K., and L. Dirick. 1991. The role of SWI4 and SWI6 in the activityof G1 cyclins in yeast. Cell 66:9951013.

25. Ogas, J., B. J. Andrews, and I. Herskowitz. 1991. Transcriptional activation

ofCLN1, CLN2, and a putative new G1 cyclin (HCS26) by SWI4, a positiveregulator of G1-specific transcription. Cell 66:10151026.

26. Ohkura, H., N. Kinoshita, S. Miyatani, T. Toda, and M. Yanagida. 1989. Thefission yeast dis2 gene required for chromosome disjoining encodes one oftwo putative type 1 protein phosphatases. Cell 57:9971007.

27. Pringle, J. R., and L. H. Hartwell. 1981. The Saccharomyces cerevisiae cellcycle, p. 97142. In J. N. Strathern, E. W. Jones, and J. R. Broach (ed.), Themolecular biology of the yeast Saccharomyces, life cycle and inheritance.Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

28. Reid, B. J., and L. H. Hartwell. 1977. Regulation of mating in the cell cycle

of Saccharomyces cerevisiae. J. Cell Biol. 75:355365.29. Richardson, H. E., C. Wittenberg, F. Cross, and S. I. Reed. 1989. An essen-

tial G1 function for cyclin-like proteins in yeast. Cell 59:11271133.30. Riles, L., J. E. Dutchik, A. Baktha, B. K. McCauley, E. C. Thayer, M. P.

Leckie, V. V. Braden, J. E. Depke, and M. V. Olson. 1993. Physical maps ofthe six smallest chromosomes of Saccharomyces cerevisiae at a resolution of2.6 kilobase pairs. Genetics 134:81150.

31. Ronne, H., M. Carlberg, G. Z. Hu, and J. O. Nehlin. 1991. Protein phos-phatase 2A in Saccharomyces cerevisiae: effects on cell growth and budmorphogenesis. Mol. Cell. Biol. 11:48764884.

32. Rose, M. D., P. Novick, J. H. Thomas, D. Botstein, and G. R. Fink. 1987. ASaccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. Gene 60:237243.

33. Rose, M. D., F. Winston, and P. Hieter. 1990. Laboratory course manual formethods in yeast genetics. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.

34. Sharp, P. M., T. M. Tuohy, and K. R. Mosurski. 1986. Codon usage in yeast:cluster analysis clearly differentiates highly and lowly expressed genes. Nu-cleic Acids Res. 14:51255143.

35. Shimanuki, M., N. Kinoshita, H. Ohkura, T. Yoshida, T. Toda, and M.Yanagida. 1993. Isolation and characterization of the fission yeast proteinphosphatase gene ppe1 involved in cell shape control and mitosis. Mol.Biol. Cell 4:303313.

36. Sneddon, A. A., P. T. Cohen, and M. J. Stark. 1990. Saccharomyces cerevisiaeprotein phosphatase 2A performs an essential cellular function and is en-coded by two genes. EMBO J. 9:43394346.

37. Stuart, D., and C. Wittenberg. 1994. Cell cycle-dependent transcription ofCLN2 is conferred by multiple distinct cis-acting regulatory elements. Mol.Cell. Biol. 14:47884801.

38. Sutton, A., D. Immanuel, and K. T. Arndt. 1991. The SIT4 protein phos-phatase functions in late G1 for progression into S phase. Mol. Cell. Biol.11:21332148.

39. Sutton, A., F. Lin, and K. T. Arndt. 1991. The SIT4 protein phosphatase isrequired in late G1 for progression into S phase. Cold Spring Harbor Symp.Quant. Biol. 56:7581.

40. Thomas, B. J., and R. Rothstein. 1989. Elevated recombination rates intranscriptionally active DNA. Cell 56:619630.

41. Tyers, M., G. Tokiwa, and B. Futcher. 1993. Comparison of the Saccharo-

myces cerevisiae G1 cyclins: Cln3 may be an upstream activator of Cln1, Cln2and other cyclins. EMBO J. 12:19551968.

42. Tyers, M., G. Tokiwa, R. Nash, and B. Futcher. 1992. The Cln3-Cdc28 kinasecomplex of S. cerevisiae is regulated by proteolysis and phosphorylation.EMBO J. 11:17731784.

43. van Zyl, W., W. Huang, A. A. Sneddon, M. Stark, S. Camier, M. Werner, C.Marck, A. Sentenac, and J. R. Broach. 1992. Inactivation of the proteinphosphatase 2A regulatory subunit A results in morphological and transcrip-tional defects in Saccharomyces cerevisiae. Mol. Cell. Biol. 12:49464959.

44. Wada, K., Y. Wada, H. Doi, F. Ishibashi, T. Gojobori, and T. Ikemura. 1991.Codon usage tabulated from the GenBank genetic sequence data. Nucleic

Acids Res. 19(Suppl.):19811986.45. Wittenberg, C., K. Sugimoto, and S. I. Reed. 1990. G1-specific cyclins of S.

cerevisiae: cell cycle periodicity, regulation by mating pheromone, and asso-ciation with the p34CDC28 protein kinase. Cell 62:225237.


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