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Budding yeast RSI1/APC2, a novel gene necessary for initiation of anaphase, encodes an APC subunit

1998; Springer Nature; Volume: 17; Issue: 2 Linguagem: Inglês

10.1093/emboj/17.2.498

1460-2075

Kate M. Kramer, Didier Fesquet, Anthony L. Johnson, Leland H. Johnston,

Photosynthetic Processes and Mechanisms

Article15 January 1998free access Budding yeast RSI1/APC2, a novel gene necessary for initiation of anaphase, encodes an APC subunit Kate M. Kramer Kate M. Kramer Division of Yeast Genetics, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA GB Search for more papers by this author Didier Fesquet Didier Fesquet Centre de Recherches de Biochimie Macromoleculaire, CNRS ERS 155, 1919 Route de Mende, 34293 Montpellier, France Search for more papers by this author Anthony L. Johnson Anthony L. Johnson Division of Yeast Genetics, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA GB Search for more papers by this author Leland H. Johnston Corresponding Author Leland H. Johnston Division of Yeast Genetics, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA GB Search for more papers by this author Kate M. Kramer Kate M. Kramer Division of Yeast Genetics, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA GB Search for more papers by this author Didier Fesquet Didier Fesquet Centre de Recherches de Biochimie Macromoleculaire, CNRS ERS 155, 1919 Route de Mende, 34293 Montpellier, France Search for more papers by this author Anthony L. Johnson Anthony L. Johnson Division of Yeast Genetics, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA GB Search for more papers by this author Leland H. Johnston Corresponding Author Leland H. Johnston Division of Yeast Genetics, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA GB Search for more papers by this author Author Information Kate M. Kramer1, Didier Fesquet2, Anthony L. Johnson1 and Leland H. Johnston 1 1Division of Yeast Genetics, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA GB 2Centre de Recherches de Biochimie Macromoleculaire, CNRS ERS 155, 1919 Route de Mende, 34293 Montpellier, France *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:498-506https://doi.org/10.1093/emboj/17.2.498 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info SIC1 is a non-essential gene encoding a CDK inhibitor of Cdc28-Clb kinase activity. Sic1p is involved in both mitotic exit and the timing of DNA synthesis. To identify other genes involved in controlling Clb-kinase activity, we have undertaken a genetic screen for mutations which render SIC1 essential. Here we describe a gene we have identified by this means, RSI1/APC2. Temperature-sensitive rsi1/apc2 mutants arrest in metaphase and are unable to degrade Clb2p, suggesting that Rsi1p/Apc2p is associated with the anaphase promoting complex (APC). This is an E3 ubiquitin-ligase that controls anaphase initiation through degradation of Pds1p and mitotic exit via degradation of Clb cyclins. Indeed, the anaphase block in rsi1/apc2 temperature-sensitive mutants is overcome by removal of PDS1, consistent with Rsi1p/Apc2p being part of the APC. In addition, like our rsi1/apc2 mutations, cdc23-1, encoding a known APC subunit, is also lethal with sic1Δ. Thus SIC1 clearly becomes essential when APC function is compromised. Finally, we find that Rsi1p/Apc2p co-immunoprecipitates with Cdc23p. Taken together, our results suggest that RSI1/APC2 is a subunit of APC. Introduction Orderly progression through the eukaryotic cell cycle is controlled by the regulated association of specific cyclins with a CDK (cyclin-dependent kinase). In the budding yeast, Saccharomyces cerevisiae, Cdc28 is the major CDK and is largely responsible for controlling cell cycle progression (reviewed in Nasmyth, 1996). G1 cyclins Clns 1, 2 and 3 are active during G1 up until S phase while B-type cyclins Clbs 1–6 control DNA synthesis (Schwob et al., 1994) and mitosis (Surana et al., 1991). The specific association of the appropriate cyclin with Cdc28 is achieved by cell cycle-controlled synthesis as well as controlled degradation of the cyclin at key stages of the cycle (reviewed in King et al., 1996; Deshaies, 1997). The mitotic B-type cyclin Clb2p is active from late S phase until the end of mitosis when it is rapidly degraded by ubiquitin-mediated proteolysis (Surana et al., 1991; Irniger et al., 1995; Amon, 1997; Irniger and Nasmyth, 1997). Exit from mitosis and entry into the G1 phase of the next cell cycle requires the inactivation of the Clb2 protein; over-production of Clb2p which has been stabilized by removal of its destruction box causes cells to arrest in telophase with divided chromatin and an elongated spindle (Surana et al., 1993). E3 ubiquitin-protein ligases play an important role in specifying the selection of proteins for ubiquitin-mediated degradation (Ciechanover, 1994). Recently, studies in human, Xenopus and yeast systems have converged to reveal the E3 ubiquitin-protein ligase activity which confers specificity for cell cycle-regulated degradation of mitotic cyclin by the ubiquitin pathway (Irniger et al., 1995; King et al., 1995; Tugendreich et al., 1995). This E3 is a large complex of at least seven proteins, and has been called the cyclosome or anaphase promoting complex (APC). The APC is part of the essential cell cycle machinery whose components are evolutionarily conserved (Irniger et al., 1995; King et al., 1995; Tugendreich et al., 1995; Peters et al., 1996; Zachariae et al., 1996). In yeast CDC16, CDC23 CDC26, CDC27 and APC1 have been identified as genes coding for some of these components (Lamb et al., 1994; Irniger et al., 1995; Zachariae et al., 1996). As its name suggests, an active APC is required for cells to initiate anaphase; APC-defective yeast strains arrest in metaphase with replicated DNA but with unseparated sister chromatids (Lamb et al., 1994; Zachariae et al., 1996). While Clb2p degradation is normally initiated at anaphase onset (Surana et al., 1991; Irniger et al., 1995), its degradation is not required for the metaphase to anaphase transition (Surana et al., 1993) and some Clb2 protein persists until telophase (Irniger et al., 1995). Thus, the APC clearly has other critical targets. One of the early events required for the metaphase to anaphase transition is sister chromatid separation. Several proteins whose proteolysis is required for sister chromatid separation have recently been identified; the pimples product in Drosophila (Stratmann and Lehner, 1996), Cut2 in fission yeast (Funabiki et al., 1996) and Pds1p in budding yeast (Cohen Fix et al., 1996; Yamamoto et al., 1996a, b). Interestingly, yeast strains lacking the PDS1 gene are viable but in APC-deficient strains show a mixed terminal arrest phenotype with a large proportion of the cells in telophase, that is, with divided chromatin (Yamamoto et al., 1996a). This suggests that the critical requirement for the APC at the metaphase to anaphase transition is to ensure the proper separation of sister chromatids. Other known APC targets include Ase1p, a budding yeast protein involved in spindle elongation (Juang et al., 1997) whose destruction is required for proper spindle disassembly after the metaphase to anaphase transition. The final essential role of the APC in mitotic exit is the degradation of Clb2p (Surana et al., 1993; Irniger et al., 1995). However, there are clearly other factors which contribute to the control of Clb2p inactivation since the APC becomes active at metaphase but Clb2p levels persist until telophase. Another protein controlling Clb2p activity is Sic1p, a CDK inhibitor of both S phase and mitotic Clb–Cdc28 kinases (Donovan et al., 1994; Schwob et al., 1994; Amon, 1997). Sic1 protein regulates the timing of DNA synthesis by binding to and inhibiting S phase kinase Cdc28–Clb5 early in the cell cycle (Schwob et al., 1994). Low Clb-kinase activity at this stage of the cell cycle is critical for enabling replication proteins such as Cdc6 and Mcm2-7 to be loaded onto replication origins making the DNA competent for replication (Donovan et al., 1997), a process that is inhibited by Clb kinase activity (Dahmann et al., 1995; Tanaka et al., 1997). Shortly before S phase, Sic1p is degraded by the Cdc4/Cdc34/Cdc53-dependent ubiquitin-mediated proteolytic pathway, thereby activating Cdc28–Clb5 kinase. Sic1p is then resynthesized in late mitosis (Donovan et al., 1994; Toyn et al., 1997). Three separate lines of evidence suggest a role for Sic1p in late mitosis. The first is the synthetic lethality between sic1Δ and hct1Δ. HCT1 is a recently described non-essential gene that directly controls Clb2p proteolysis (Schwab et al., 1997); hct1Δ mutants have high levels of Clb2p throughout the cell cycle and overexpression of HCT1 induces ectopic activation of Clb2 proteolysis. Second, SIC1 interacts genetically with a variety of late mitotic arrest mutants (for review see Deshaies, 1997) and multiple copies of SIC1 rescue temperature-sensitive mutants of the DBF2 group of kinases which all arrest in telophase with large budded cells, divided chromatin, an elongated spindle and high mitotic kinase activity (Toyn and Johnston, 1994; Deshaies, 1997). Third, sic1Δ dbf2Δ is a lethal combination and the double mutant has a late mitotic arrest terminal phenotype (Toyn et al., 1997). Taken together, these observations argue strongly that SIC1 and various other genes such as DBF2 and HCT1 are involved in functionally overlapping pathways controlling exit from mitosis. Because control of mitotic exit clearly involves redundant pathways, isolating mutations that are synthetically lethal with sic1Δ should identify other genes involved in mitotic exit. Accordingly, we carried out a screen for such mutants and describe here the isolation of a gene we call RSI1 (Requires Sic1 CDK Inhibitor). Although RSI1 is essential, we have characterized a particular allele which is viable but becomes lethal in a sic1Δ background. We show that Rsi1p is required for the metaphase to anaphase transition and also for Clb2p degradation at key cell cycle stages. Finally we show that Rsi1p interacts in vivo with the APC component Cdc23p. We conclude that RSI1 codes for an APC subunit and propose alternative models accounting for the rsi1 sic1Δ synthetic lethality. Results A genetic screen for mutants requiring SIC1 Mutagenesis was carried out in strain JD100 (Table I), a sic1Δ::TRP1 haploid strain containing SIC1 on a low copy number URA3 plasmid (pSIC1U). We exploited the toxicity of 5-fluoroorotic acid (FOA) in URA3 strains (see Materials and methods; Ota and Varshavsky, 1992) to isolate mutants requiring SIC1 for growth; such mutants will not be able to lose pSIC1U and therefore will be unable to grow on FOA. For the mutagenesis, we used a yeast genomic library, generously provided by Dr M.Snyder, Yale University (Burns et al., 1994), in which wild-type sequences are randomly disrupted by LEU2 insertions. Disrupted yeast sequences are cut out of the library vector and integrated into the target genome by transplacement. This effectively replaces wild-type genomic DNA with LEU2 disrupted sequences. We felt disruption to be an appropriate mutagenesis since late mitotic pathways are clearly redundant (see Introduction), hence, at least some genes are expected to be dispensable in a wild-type background. We screened 80 000 LEU+ transformants and only a single mutation lethal with sic1Δ was identified. This lay in a previously uncharacterized gene we are calling RSI1 (see above). We refer to this mutant allele as rsi1-1. Table 1. Yeast strainsa Strain name Genotype Source CG378 MATa ura3 leu2 trp1 ade5 C.Giroux, (Wayne State University, Detroit, Michigan) CG379 MATα ura3 leu2 trp1 his7 C.Giroux JD100 MATα sic1Δ::TRP1 leu2 ura3 his7 Donovan et al. (1994) KTM100U MATα rsi1::LEU2b sic1Δ::TRP1 his7 pSIC1Uc this study KTM100H MATα rsi1::LEU2 sic1Δ::TRP1 pSIC1Hd this study KTM100sΔH MATα rsi1::LEU2 sic1Δ::TRP1 ura3 psic1ΔHe this study KTM110 MATa rsi1::LEU2 trp1 ura3 ade5 this study KTM208 MATa rsi1Δ::HIS7 ura3 leu2 prsi1-8f this study CG378CH MATa CLB2HA3 ura3 leu2 trp1 ade5 this study KTM208CH MATa CLB2HA3 rsi1Δ::HIS7 ura3 leu2 prsi1-8f this study CG378GCH CG378 pGAL-CLB2HA3g this study KTM208GCH MATa rsi1Δ::HIS7 ura3 leu2 prsi1-8f pGAL-CLB2HA3 this study 2351 MATa cdc23-1 ura3 leu2 ade2 his3 K.Nasmyth, IMP, Vienna 2351W MATa cdc23-1 ura3 leu2 trp1 ade2 his3 this study KTM308 MATa rsi1Δ::HIS7 pds1Δ::URA3 leu2 prsi1-8 this study CG378 RSI1Myc3 CG378 pRS306-RSI1myc3h this study CG378 RSI1Myc3, CG378 pRS306-RSI1myc3 pRS329-CDC23HA2i this study CDC23HA2 CG378 CDC23HA2 CG378 pRS329-CDC23HA2 this study a All strains except cdc23-1 are in a CG378/CG379 background. cdc23-1 is in a W303 background. b rsi1::LEU2 is referred to as rsi1-1 in the text. c pSIC1U is a CEN-based plasmid containing SIC1 and URA3. d pSIC1H is an ARS-based plasmid containing SIC1 and HIS7. e psic1ÆH is as pSIC1H except SIC1 is deleted. f prsi1-8 is a CEN-based plasmid containing TRP1 and a temperature-sensitive mutation in RSI1. g pGAL-CLB2HA3 is a CEN-based plasmid containing CLB2HA3 under control of the the GAL10 promoter and URA3. h pRS306-RSI1Myc3 is an integrative plasmid containing RSI1Myc3 and URA3. i pRS329-CDC23HA2 is a CEN-based plasmid containing TRP1 and expressing CDC23HA2 under its own promoter (a gift from the Phil Hieter laboratory). sic1Δ rsi1-1 synthetic lethality The rsi1-1 mutant was isolated by its inability to grow on FOA plates in a sic1Δ pSIC1U genetic background (KTM100U, Table I and Figure 1A). Providing wild-type SIC1 on a plasmid vector with HIS7 as a selectable marker (KTM100H, Table I) allowed growth on FOA whereas sic1Δ on the same plasmid vector (KTM100sΔH, Table I) did not. To confirm the synthetic lethality of rsi1-1 and sic1Δ, we crossed KTM100U with wild-type CG378 (Table I). Out of eight tetrads dissected, all LEU+ (rsi1-1) TRP+ (sic1Δ) spore clones were also URA+ (pSIC1U) and unable to grow on FOA. Furthermore, a subsequent cross between an rsi1-1 SIC1 haploid (KTM110, Table I) and JD100, the original RSI1 sic1Δ strain used for the mutagenesis, yielded no LEU+ (rsi1-1) TRP+ (sic1Δ) viable spore clones from 18 tetrads dissected, confirming that rsi1-1 requires a wild-type copy of SIC1 for viability. Non-viable spores had germinated but arrested as large budded cells after one or several divisions. Figure 1.(A) sic1Δ rsi1-1 synthetic lethality. Cells were streaked onto plates containing either no FOA or 1mg/ml FOA and incubated for 3 days at 30°C. The strain sic1Δ rsi1-1 pSIC1U carries SIC1 on a URA3 marked plasmid (Table I). Strains sic1Δ rsi1-1 pSIC1H and sic1Δ rsi1-1 psic1ΔH are identical to sic1Δ rsi1-1 pSIC1U but carry either SIC1 or sic1Δ on a HIS7 marked plasmid. (B) The heavy line indicates the RSI1 2.5 kb open reading frame located on the left arm of CHR XII. The rsi1-1 mutation is a LEU2 -transposon insertion 425 bp upstream of the initiating ATG. The XhoI–NdeI fragment was replaced by the HIS7 gene in rsi1Δ strains. (C) Sequence alignment showing the homologous region between Rsi1 and cullin family members. DDBJ/EMBL/GenBank accession numbers are indicated in brackets. Sc, S.cerevisiae Rsi1; Sp, S.pombe hypothetical protein; Dm, D.melanogaster Lin-19; Hs, Homo sapiens Cul-1. Download figure Download PowerPoint The sequences around the LEU2 insertion site in rsi1-1 were cloned by integrating a single copy of a bacterial/yeast shuttle vector at that site in KTM100H (see Materials and methods). Sequence analysis revealed that the LEU2 insertion in rsi1-1 is in the promoter of a gene on chromosome XII (Figure 1B) we have named RSI1 (see above). RSI1 encodes a 853 amino acid protein of 99 kDa (DDBJ/EMBL/GenBank Z73299; YPD YLR127C) containing several potential Cdc28 phosphorylation sites. Database searches reveal a domain of weak homology (23–28% identity, 49–55% similarity) to Drosophila and and human genes belonging to the cullin family (Jackson, 1996; Kipreos et al., 1996) as well as to a Schizosaccharomyces pombe hypothetical protein (Figure 1C). Cullin family members appear to have a role in cell cycle regulation and include S.cerevisiae CDC53, which controls G1 cyclin degradation (Jackson, 1996; Willems et al., 1996). A null allele of RSI1 was created by replacing one copy of the RSI1 coding sequences with the HIS7 gene (Figure 1B) in diploid strain CG378×CG379 (Table I). Sporulation followed by dissection of 18 tetrads gave a segregation pattern of 2 viable:2 dead in all cases and the viable spores were all his− (RSI1). RSI1 is therefore an essential gene. The non-viable spores had germinated, entered the cell cycle and arrested as large budded cells after only a few divisions. The rsi1-1 strain is hypersensitive to Clb2p levels Strains defective in mitotic exit, including sic1Δ strains, are hypersensitive to increased levels of Clb2p (Shirayama et al., 1994; Toyn et al., 1997); a single copy of GAL-CLB2 prohibits growth on galactose. We reasoned that if the sic1Δ rsi1-1 synthetic lethality is due to a defect in Clb2p kinase inactivation, then the rsi1-1 mutation on its own might be sensitive to increased levels of Clb2p. To examine this, a single copy of GAL-CLB2 was integrated at the URA3 locus in the rsi1-1 mutant and also in congenic wild-type cells. The copy number of the integrated GAL-CLB2::URA3 was confirmed by Southern analysis (data not shown). Figure 2 shows that while all strains can grow on 2% glucose, the rsi1-1 mutation, like sic1Δ, prohibits growth on 2% galactose. Furthermore, sensitivity to GAL-CLB2 expression in the rsi1-1 strain is overcome by providing either RSI1 or SIC1 on a low copy plasmid. These results taken together with the sic1Δ rsi1-1 synthetic lethality strongly suggest that Rsi1p and Sic1p function in parallel pathways controlling the inactivation/degradation of Clb2p. Figure 2.The rsi1-1 mutant is hypersensitive to CLB2 levels. A single copy of GAL-CLB2 was integrated at the URA3 locus in the rsi1-1 mutant as well as in congenic sic1Δ and wild-type strains. Cells were streaked onto plates containing either 2% glucose or 2% galactose and incubated for 3 days at 30°C. Download figure Download PowerPoint Cell cycle phenotype of rsi1 temperature-sensitive mutants To characterize the function of RSI1 in more detail, we generated a bank of temperature-sensitive alleles using mutagenic PCR and in vivo 'gap repair' (Connelly and Hieter, 1996). All of the mutant alleles showed a similar terminal arrest phenotype as exemplified by the rsi1-8 allele in strain KTM208 (Table I) described below. When a log phase culture of rsi1-8 cells was shifted from growth at 25°C to 37°C for 3 h, the cells displayed a cell cycle phenotype, arresting with large buds, an undivided nucleus positioned at the neck of the bud and a short mitotic spindle (Figure 3A). FACS analysis showed them to contain fully replicated DNA (Figure 3B). This phenotype is identical to that reported for cdc23-1, a temperature-sensitive allele of CDC23 which encodes an APC subunit (Sikorski et al., 1993). Figure 3.Terminal arrest phenotype of rsi1-8 temperature-sensitive mutants. rsi1-8 cells were grown to log phase at 25°C then shifted to 37°C for 3 h. (A) Chromatin was visualized by DAPI staining and spindles were visualized by anti-tubulin staining. (B) FACS analysis. Cells were stained with propidium iodide and 20 000 were analyzed for each sample. Download figure Download PowerPoint The rsi1-8 strain is defective in Clb2p degradation cdc23-1 mutants are defective for Clb2p degradation, therefore we assessed the Clb2-Cdc28-specific H1 kinase activity as well as Clb2p stability in the rsi1-8 mutant. The H1 kinase activity was measured using anti-HA immunoprecipitated Clb2HA3. First, we examined the kinase activity from a log phase culture of rsi1-8 cells containing an integrated copy of CLB2HA3, that had been shifted from growth at 25°C to 37°C for 3 h. In this experiment the tagged CLB2 replaced the endogenous gene and was fully functional. This was compared with the same activity from congenic wild-type cells also containing CLB2HA3 and treated for 3 h either in nocodazole (a microtubule-depolymerizing drug), to induce an M phase arrest where Clb2–Cdc28-specific H1 kinase activity is high (Amon et al., 1994), or with α-factor to induce an early G1 arrest where the kinase activity is undetectable. FACS analysis confirmed that the predicted arrests had occurred (Figure 4A). As expected, the α-factor arrested wild-type cells had no detectable Clb2–Cdc28-specific H1 kinase activity (Figure 4A, lane 1). In marked contrast, the extracts from rsi1-8 mutant cells had a high Clb2–Cdc28-specific H1 kinase activity at 37°C (Figure 4A, lane 3), comparable with the nocodazole arrested wild-type cells (Figure 4A, lane 2). Moreover, Clb2p was readily detectable in the rsi1-8 cells at 37°C (Figure 4A, lane 3) suggesting that it was stabilized. Figure 4.rsi1-8 temperature-sensitive mutants arrest with high Clb2p kinase activity and have a defect in Clb2p degradation at the restrictive temperature. (A) Strain KTM 208CH, rsi1-8, congenic wild-type CG378 and strain CG378CH containing integrated CLB2HA3 were grown to log phase at 25°C. The wild-type cells were treated for 3 h with α-factor or with nocodazole. The rsi1-8 cells were shifted to 37°C for 3 h. Extracts were prepared and Clb2p levels and associated H1 kinase activity was measured. Lanes 1 and 2, wild-type CLB2HA3 cells arrested with α-factor and nocodazole, respectively; lane 3, 37°C arrested rsi1-8 CLB2HA3 cells; lane C, nocodazole-arrested wild-type cells that do not contain CLB2HA3. (B) Strain KTM208GCH, rsi1-8, and a congenic wild-type strain, CG378GCH, both containing GAL–CLB2HA3 on a low copy number plasmid, were grown to log phase at 25°C in 2% raffinose medium. α-factor was added and after 4 h galactose was added to 2% and the culture was simultaneously shifted to 37°C. Extracts were prepared from cells after 0, 25, 60 and 100 min growth at 37°C. Additional α-factor was added at intervals to ensure continued G1 arrest. Equal amounts of protein were analyzed by SDS–PAGE and Western blotting using a monoclonal anti-HA antibody (12CA5, upper panel). The nitrocellulose membrane was stained with Ponceau S and is shown as a loading control (lower panel). At the same time points, cells were also removed for FACS analysis. Download figure Download PowerPoint To confirm this we examined Clb2p stability in rsi1-8 cells that had been arrested in G1 with α-factor. Clb2p normally has a half-life of <1 min in G1 (Amon et al., 1994) but is stabilized in G1-arrested cdc23-1 cells (Irniger et al., 1995). We used a GAL-CLB2 fusion construct with a triple HA epitope tag, pGAL-CLB2HA3, to examine Clb2p stability in G1-arrested rsi1-8 and isogenic wild-type cells. Log phase cultures were exposed to the mating pheromone α-factor during growth in raffinose at 25°C. Once the cells were arrested in G1, as determined by the absence of buds and the formation of mating projections, galactose was added to 2% to induce high levels of ectopic Clb2p expression and simultaneously the cultures were shifted to 37°C. In the strain carrying the rsi1-8 mutation, Clb2 protein was detectable by Western blotting after 25 min with Clb2p levels increasing throughout the 100 min induction (Figure 4B). In contrast, in the wild-type strain, we observed no such Clb2 protein accumulation. Thus, in α-factor-arrested cells, Rsi1p is required for Clb2p degradation. It is significant that in the rsi1-8 mutant after 100 min of galactose-induced CLB2 expression at 37°C, FACS analysis showed that the cells had entered S phase (Figure 4). In contrast, the wild-type cells maintained a G1 DNA content throughout the experiment. It is important to note that the rsi1-8 cells had not initiated DNA synthesis due to escape from the α-factor holding; they retained the mating projection induced by α-factor and remained unbudded. Clb kinase is required for entry into S phase (Schwob et al., 1994) but is kept low during G1 by the presence of the Sic1p CDK inhibitor and persistent APC function (Irniger and Nasmyth, 1997). In the mutant, however, sufficient levels of Clb kinase had accumulated to initiate S phase despite the presence of α-factor. This phenomenon has been previously observed in mutants of APC components (Zachariae et al., 1996; Irniger and Nasmyth, 1997) and supports a direct association of Rsi1p with the APC. cdc23-1 is also synthetically lethal with sic1Δ The phenotypic similarities between rsi1-8 and cdc23-1 led us to look for a possible genetic interaction between CDC23 and SIC1. We crossed a cdc23-1 mutant with a sic1Δ::TRP1 strain and examined segregation of temperature sensitivity (cdc23-1) with the TRP1 (sic1Δ) marker. Out of 27 tetrads dissected, no spore clones were temperature-sensitive (cdc23-1) and TRP+ (sic1Δ), indicating that the sic1Δ cdc23-1 combination is lethal in haploids. We also examined spores where the sic1Δ cdc23-1 combination could be predicted based on the genotype of the viable spore clones. Of 16 such spores, all but one had germinated and arrested as large budded cells after one or a few divisions. Because this is identical to the phenotype observed in the sic1Δ rsi1-1 spores described above we considered the possibility that Rsi1p is an APC subunit and thus shares a common role in cell cycle progression with Cdc23p. Rsi1p interacts with the APC component Cdc23p in vivo As a conclusive indication that RSI1 encodes a subunit of APC we looked for a direct association of Rsi1p with Cdc23p in vivo. A triple Myc epitope-tagged version of RSI1, RSI1Myc3, was integrated in wild-type haploid CG378 at the RSI1 locus. In this strain the only copy of RSI1 with a promoter is the RSI1Myc3 version of the gene; hence this is the only source of Rsi1p. RsiMyc3p was detected as a 100 kDa protein by Western blotting of crude extracts from log phase cells (Figure 5B). The RSI1Myc3 strain and the corresponding isogenic parental strain were transformed with a low copy plasmid containing a double HA epitope-tagged CDC23, CDC23HA2. Extracts prepared from cells expressing various combinations of these tagged proteins were subjected to immunoprecipitation with anti-Myc antibodies and probed with an anti-HA antibody to look for co-precipitation of Cdc23p with Rsi1p (Figure 5A). As expected, Cdc23HA2p was specifically co-precipitated with Rsi1Myc3p. This in vivo interaction between Cdc23p and Rsi1p strongly supports our evidence that Rsi1p is indeed a newly identified component of APC. Rsi1p is likely to be the previously detected 100 kDa component of APC (Zachariae et al., 1996). Figure 5.The in vivo interaction between Rsi1p and Cdc23p. Isogenic strains containing either RSI1Myc3 or CDC23HA2 alone or with both constructs together were grown to log phase and cell extracts were prepared. (A) Protein from crude extracts (left panel) and from extracts immunoprecipitated using a monoclonal anti-Myc antibody (9E10, right panel) were electrophoresed and probed with a monoclonal anti-HA antibody (12CA5) to detect Cdc23HA2p. (B) The anti-HA antibody was stripped from the blot shown in (A) and it was re-probed with monoclonal anti-Myc antibody to detect Rsi1Myc3p. *indicates remaining traces of the previous HA probing. Download figure Download PowerPoint The pre-anaphase block in rsi1-8 mutants can be overcome in the absence of Pds1p A unique and characteristic phenotype of mutations in APC components is manifested in strains lacking Pds1p. Because APC-mediated proteolysis of Pds1p is essential for sister chromatid separation (Cohen Fix et al., 1996; Yamamoto et al., 1996a, b), a high proportion of APC-deficient pds1Δ cells enter anaphase and arrest in telophase with divided chromatin (Yamamoto et al., 1996a). Accordingly, we examined the terminal arrest phenotype in a rsi1-8 pds1Δ double mutant compared with a rsi1-8 PDS1 control strain. Log phase cultures of both strains growing at 25°C were shifted to growth at 37°C for 3 h. FACS analysis showed that after 3 h both strains had stopped growing and had arrested with fully replicated DNA (Figure 6). However, examination of chromosome segregation in DAPI stained cells showed a dramatic difference in the arrest phenotype. The number of cells with divided chromatin in the rsi1-8 pds1Δ double mutant had increased from 12% in the log phase culture (0 h, Figure 6) to 51% after 3 h at 37°C. In contrast, the number of cells with divided chromatin in the rsi1-8 PDS1 control strain showed a significant decrease from 12% in log phase to 5% after 3 h at 37°C, these cells having accumulated in metaphase. The percentage of cells with divided chromatin in the rsi1-8 pds1Δ double mutant after 3 h at 37°C is in good agreement with the previously observed percentage in terminally arrested cdc16 pds1 and cdc23 pds1 double mutants (Yamamoto et al., 1996a). Thus, rsi1 behaves in the same way as cdc16 and cdc23 in a pds1Δ genetic background. The high proportion of cells with divided chromatin in the rsi1-8 pds1Δ double mutant shows that those cells which were able to initiate anaphase had subsequently arrested in telophase demonstrating,