key: cord-0960355-qaurriwg authors: Connelly, Carla; Hieter, Philip title: Budding Yeast SKP1 Encodes an Evolutionarily Conserved Kinetochore Protein Required for Cell Cycle Progression date: 1996-07-26 journal: Cell DOI: 10.1016/s0092-8674(00)80099-9 sha: e31fe1509bb347d50c56545d284cf09b6febfdb9 doc_id: 960355 cord_uid: qaurriwg The budding yeast SKP1gene, identified as a dosage suppressor of a known kinetochore protein mutant, encodes an intrinsic 22.3 kDa subunit of CBF3, a multiprotein complex that binds centromere DNA in vitro. Temperature-sensitive mutations in SKP1 define two distinct phenotypic classes. skp1-4 mutants arrest predominantly as large budded cells with a G2 DNA content and short mitotic spindle, consistent with a role in kinetochore function. skp1-3 mutants, however, arrest predominantly as multiply budded cells with a G1 DNA content, suggesting an additional role during the G1/S phase. Identification of Skp1p homologs from C. elegans, A. thaliana, and H. sapiens indicates that SKP1 is evolutionarily highly conserved. Skp1p therefore represents an intrinsic kinetochore protein conserved throughout eukaryotic evolution and may be directly involved in linking kinetochore function with the cell cycle-regulatory machinery. gene encoding p58 of CBF3, as a starting point. In this paper, we describe the identification of the SKP1 gene as a dosage suppressor of a ctf13-30 mutant. Genetic and biochemical analysis indicates that the 22.3 kDa Skp1 protein is a fourth subunit of CBF3 that was previously unrecognized. Unlike the other CBF3 components, Skp1p is a highly conserved protein found in multicellular eukaryotes. Furthermore, Skp1p has recently been shown to be a component of a cyclin A-cyclin-dependent kinase 2 (CDK2) complex purified from transformed human fibroblasts and to interact directly with Cdc4p and cyclin F through a novel structural motif (Bai et al., 1996 [this issue of Cell]). We conclude that Skp1p is involved in both structural and regulatory functions and may provide a direct link between the kinetochore and the cell cycle machinery. To identify further additional components of the S. cerevisiae kinetochore, we performed a screen for dosage suppressors of the temperature sensitivity caused by the ctf13-30 mutation. A 2 genomic library was transformed into a ctf13-30 mutant, and temperature-resis- clone to suppress the temperature-sensitive and chromosome missegregation phenotypes of a ctf13-30 musmall size of Skp1p, it was possible that it represented a tant. When the gene disruption was integrated into the previously unrecognized subunit of CBF3. To determine genome, dissection of a heterozygous diploid resulted whether this was the case, we made two epitope-tagged in viability segregating 2ϩ:2Ϫ (see Experimental Proceconstructs of Skp1p (see Experimental Procedures). In dures). The genomic clone was localized to the right arm the first, two tandem copies of the E1 epitope (Pluta et of chromosome IV using physical mapping methods, al., 1992), derived from the carboxy-terminal 25 amino indicating that SKP1 is a previously unidentified essenacids of avian coronavirus glycoprotein (Machamer and tial gene in S. cerevisiae. Rose, 1987) , were placed in-frame at the amino terminus The nucleotide sequence of the SKP1 genomic DNA of the SKP1 ORF under the transcriptional control of the contains a 582 bp open reading frame (ORF) that en-GAL1 promoter ( Figure 1C ). In the second construct, a codes a protein of 194 amino acids with a predicted 9 amino acid epitope derived from the HA1 protein of molecular mass of 22.3 kDa ( Figure 1A ). The restriction influenza virus (Field et al., 1988) was placed in-frame maps of genomic SKP1 and derivative clones are shown at the amino terminus of the SKP1 ORF under the control in Figure 1 . of the ADH2 promoter ( Figure 1C ). Both epitope-tagged Skp1p derivatives were able to rescue viability in a skp1-SKP1 Encodes a Fourth Subunit of CBF3 ⌬1::TRP1 deletion strain ( Figure 1B ). Whole-cell extracts were prepared from a skp1-⌬1 complex (CBF3) present in extracts of S. cerevisiae cells deletion strain carrying either epitope-tagged or unthat contains three major protein species 110, 64, and tagged copies of Skp1p. As controls, extracts were also 58 kDa in size. CBF3 binds in vitro to a 100 bp DNA probe prepared from a ctf13-⌬1 deletion strain carrying epithat spans CDEIII but lacks CDEI and CDEII (Doheny et tope-tagged Ctf13p. DNA-protein complexes were allowed to form with 32 P-labeled CDEIII sequence, and al., 1993; see Experimental Procedures). Because of the DNA-protein complexes formed with 32 Plabeled CDEIII probe and whole-cell extracts were analyzed on a nondenaturing polyacrylamide gel (Doheny et al., 1993). Antibodies were added to preformed complexes, and samples were incubated for 20 min at room temperature before gel analysis. Unbound probe was run off the bottom of the gel. (A) Lanes 1, 2, and 11 are controls. Lanes 3-6, extracts from cells containing E1-tagged or untagged Ctf13p or Skp1p proteins demonstrating an intrinsic mobility shift due to the 60 amino acid epitope fusion in each case. Lanes 7-10, extracts from cells containing E1-tagged Ctf13p or Skp1p subsequently incubated with or without antibodies directed against the E1 epitope as indicated. Lanes 7 and 9 are controls for lanes 8 and 10. (B) Lanes 1-6, extracts containing either HAtagged or untagged Ctf13p or Skp1p subsequently incubated with or without an antibody directed against the HA epitope as indicated. Lanes 7 and 8 are from an independent transformant (repeat of lanes 5 and 6). Lane 9 is a control. complexes were resolved on a nondenaturing gel (Figure polymerase chain reaction (PCR) in vitro and gap repair in vivo (see Experimental Procedures). The four alleles 2). In the absence of anti-E1 antibody, extracts containing the E1 epitope-tagged fusions of Ctf13p and were named skp1-1, skp1-2, skp1-3, and skp1-4. The nucleotide sequence of each allele was determined (see Skp1p show a slight (but clearly observable and reproducible) intrinsic mobility shift up when compared with Experimental Procedures), and the mutations encoding changes at the protein level were found to be primarily wild-type and untagged extracts (Figure 2A, lanes 2-6) , suggesting that the E1-Skp1p fusion protein is localized in the carboxyl terminus of the protein (see Table 1 ). Chromosome missegregation was monitored visually to the complex. The magnitude of the intrinsic shift is identical for E1-tagged Ctf13p and E1-tagged Skp1p, in each skp1 temperature-sensitive strain at semi-permissive temperature using a colony color assay that indicating equal stoichiometry of these two proteins in the CBF3 complex. When E1 antibody is added to com-measures the stability of a marker chromosome fragment (Hieter et al., 1985) . By this criterion, the strains fell plexes formed using extracts from tagged and untagged Ctf13p and Skp1p strains, a supershift is observed using into two classes. Strains expressing the skp1-1, skp1-2, or skp1-3 alleles exhibited wild-type rates of chromo-the Ctf13p-tagged extract ( Figure 2A , lane 8) and the E1-tagged Skp1p extract, although the supershift with some missegregation. The strain expressing skp1-4, however, exhibited a dramatically increased rate of E1-tagged Skp1p was less pronounced (Figure 2A , lane 10). These results suggested that Skp1p is in the CBF3 chromosome missegregation comparable with that observed for the ctf13-30 mutant. We therefore decided complex, as evidenced by both the intrinsic and antibody-induced mobility shift; however, epitope accessi-to analyze further strains carrying one mutation in each class (skp1-3 and skp1-4). Our approach was to inte-bility or antibody binding stability could have compromised a supershift equivalent to that seen with epitope-grate each mutant allele as a single copy into the genome to avoid potential plasmid copy number effects. tagged Ctf13p. The addition of anti-HA antibody to complexes formed Both the skp1-3 and skp1-4 alleles contain single missense mutations (Ile-172 to Asn and Leu-146 to Ser, using extracts prepared from untagged and HA-tagged fusions of Ctf13p and Skp1p showed conclusively that respectively; Figure 1D ). Each mutant allele was integrated at the LEU2 locus in strains containing the skp1-Skp1p is present in the CBF3-CEN DNA complex. Two independent extracts containing HA-tagged Skp1p pro-⌬1 deletion mutation (see Experimental Procedures). The skp1-3 and skp1-4 mutations were found to cause duced a quantitative supershift equivalent to that seen for the HA-tagged Ctf13p ( Figure 2B, lanes 2, 6, and 8) . several allele-specific phenotypes. First, as observed with the plasmid-borne alleles, skp1-3 does not cause We conclude from these data that Skp1p is a fourth subunit of the S. cerevisiae CBF3 complex. respectively, which represent greater than 100-fold in-diploid forms colonies, whereas either homozygous mutant diploid does not. Furthermore, at permissive tem-creases relative to wild type. Second, because SKP1 was identified as a high copy suppressor of ctf13-30, it perature (25ЊC), the skp1-3 allele complements the chromosome segregation defect of the skp1-4 allele in the was of interest to test the reciprocal relationship, i.e., whether Ctf13p overexpression could suppress the skp1 heteroallelic diploid. Taken together these data strongly suggest two inde-temperature-sensitive alleles. Overexpression of Ctf13p rescues the temperature sensitivity of skp1-4, but not pendent functions for the Skp1 protein: a G2/M function required for kinetochore activity and chromosome seg-that of skp1-3 (data not shown). In addition, at semipermissive temperature, the chromosome missegrega-regation and a G1/S function required for cell cycle progression earlier in the cell cycle. tion phenotype caused by the skp1-4 mutation is suppressed by increased dosage of Ctf13p, even when present at one or a few extra copies on a centromere Cytological Analysis of skp1 Mutants Cytological analysis of skp1 mutants, and accompa-vector ( Figure 3 ). This suppression is kinetochore protein gene specific, since increased dosage of Ndc10p nying flow cytometric analysis of DNA content per cell, was performed in skp1 homozygous and heteroallelic has no effect, suggesting a direct interaction between the Skp1 and Ctf13 proteins. Third, following a 3 hr shift diploid mutants at permissive and nonpermissive temperatures ( Figure 4 ). As compared with wild type, skp1-to nonpermissive temperature, skp1-3 causes haploid cells to arrest with a G1 DNA content, while skp1-4 3/skp1-3 mutants exhibit typical G1 and G2 profiles of DNA content per cell in logarithmically growing cultures causes haploid cells to arrest with a G2 DNA content (data not shown). These terminal phenotypes were more at permissive temperature and arrest with a single peak of G1 content DNA after shift to nonpermissive tempera-fully characterized in diploids (see below). Fourth, skp1-3 and skp1-4 mutations exhibit interallelic comple-ture for 3 hr ( Figure 4A ). In contrast, skp1-4/skp1-4 mutants exhibit a modest accumulation of cells with a G2 mentation. Heterozygous and homozygous diploids were created from meiotic segregants of each of the DNA content during log phase growth and arrest with a single peak of G2 content DNA after shift to the nonper-integrated temperature-sensitive alleles. At nonpermissive temperature (37ЊC), the skp1-3/skp1-4 heteroallelic missive temperature. As expected from the intragenic Yeast strains carrying integrated skp1 temperature-sensitive alleles were tested for chromosome missegregation phenotypes using the colony color sectoring assay (Koshland and Hieter, 1987). Loss of the nonessential marker chromosome gives a red sector in a white colony. skp1-3 mutants exhibit a wild-type phenotype (very rarely sectored colonies) when transformed with vector only. skp1-4 mutants exhibit dramatically increased rates of chromosome loss (highly sectored colonies) when transformed with vector only. Introduction of the CTF13 gene on a low copy (CEN) vector or high copy (2) vector suppresses the chromosome missegregation phenotype of the skp1-4 mutant. Introduction of the NDC10 gene on a 2 vector has no effect. complementation data described above, the skp1-3/ budded phenotype accounting for 37% of the cell population. It appears that skp1-3 cells do not replicate their skp1-4 heteroallelic diploid exhibits DNA content profiles similar to wild type in logarithmically growing cells DNA (arrest with a G1 DNA content), but continue the budding process. This arrest phenotype is analogous at permissive temperature and after shift to the nonpermissive temperature. to that seen in a class of cdc mutants typified by cdc4. In contrast, skp1-4 cells arrest with a predominant cell Quantitation of cell and nuclear morphology following growth arrest at high temperature also revealed dra-morphology indicative of the preanaphase G2/M phase of the cell cycle. After 3 hr at the nonpermissive tempera-matic differences between the skp1-3 and skp1-4 mutants. After 3 hr at nonpermissive temperature, skp1-3 ture, 48% of cells were large budded with an undivided nucleus positioned at or near the neck between mother cells are predominantly unbudded with a single DNA mass. A novel class of cells with abnormally elongated and daughter cells. The majority of cells with this morphology were characterized by DNA at or spanning the buds (21%) is also seen ( Figures 4B and 5 ). After 6 hr at nonpermissive temperature, 57% of skp1-3 cells neck and a short mitotic spindle. Cells with a short spindle spanning the neck accounted for 16% of the cell exhibit novel cell morphologies, including a multiple SKP1/SKP1 wild-type, skp1-3/skp1-3 and skp1-4/skp1-4 homozygous mutants, and skp1-3/skp1-4 heteroallelic diploids were grown to logarithmic phase at 25ЊC and shifted to 37ЊC for 3 and 6 hr, and nuclear and bud morphology were scored. The criteria used for each morphologic class scored are shown schematically above the columns. The numbers shown represent percentages of the total cells scored (far right column). Asterisk, in this population of cells, 67% had the nucleus at the neck and 33% had the nucleus spanning the neck (n ϭ 203). We also tested whether cells grown at nonpermissive were aligned and a consensus generated (Figure 6) . A remarkable conservation of this protein has been pre-temperature could recover when shifted back to the permissive temperature. About 60% of skp1-3 cells were served over the kingdoms, with the highest similarity toward the carboxyl terminus of this protein family. The able to recover from the G1 arrest after 6 hr at 37ЊC. In contrast, only 10% of skp1-4 cells were able to recover temperature-sensitive mutations in the skp1-3 and skp1-4 alleles are in completely conserved amino acids. from the G2/M arrest (data not shown). S. cerevisiae Skp1p has an apparent 28 amino acid insertion when aligned with the other homologs (resi- To date the components of the CBF3 complex in S. dues 37-64). It was possible that these additional residues were important for the specific functions of Skp1p cerevisiae have not shown highly significant amino acid sequence similarities to other proteins currently present in yeast. An in-frame deletion of the 28 amino acids was constructed (skp1-⌬2; see Figure 1E ) and shown in the public databases. However, when a six-way conceptual translation of the database of expressed se-to rescue a null mutation of SKP1. We conclude that the S. cerevisiae protein is not a functionally distinct quence tags (dbEST) (Boguski et al., 1994) was searched with the Skp1p amino acid sequence, significant member of the protein family. matches were found to ORFs encoded in ESTs from Arabidopsis thaliana, Caenorhabditis elegans, and Discussion Homo sapiens. Clones corresponding to the EST sequences were obtained in each case, and the complete CBF3, a multisubunit protein complex that binds the essential CDEIII sequence in centromere DNA, was pre-sequences of the Skp1p homologs in these three organisms were determined. The human Skp1p homolog se-viously shown to contain three major subunits (58, 64, and 110 kDa in size). We describe the identification of quence is identical to the sequence of p19 , which is a component of a cyclin A-CDK2 a fourth intrinsic subunit of CBF3, Skp1p, which was isolated by screening for dosage suppressors of a complex purified from transformed human fibroblasts. Coincidentally, the gene encoding human p19 was also known kinetochore protein mutant. The discovery of Skp1p is of broad significance for several reasons. In named SKP1 (for S phase kinase-associated protein). In addition, a search of the nonredundant protein database addition to its essential structural role at the kinetochore, analysis of skp1 mutants suggest a role in cell (NRDB) revealed three sequences with high similarity to Skp1p: OCP-II (Chen et al., 1995), FP21 (West et al., cycle progression at both G2/M and G1/S. Skp1p represents an intrinsic kinetochore protein that is highly con-1995), and an unknown ORF (Lu et al., 1995) . OCP-II is a protein found in the organ of Corti in the guinea pig served in evolutionarily diverse eukaryotes, including yeast and mammals. Yeast Skp1p (suppressor of kineto-inner ear, and its role in auditory function is unknown. FP21 is a glycoprotein of unknown function in Dictyo-chore protein) is the homolog of human SKP1 (S phase kinase-associated protein), which is a subunit of a cyclin stelium and appears to have an unusual glycosylation Several lines of evidence indicate that Skp1p is a 22.3 species. Recently, a mouse gene encoding a Skp1p homolog was cloned and related sequences were kDa subunit of the CBF3 complex. First, increased expression of SKP1 suppresses the temperature-sensitive mapped to two locations in the mouse genome, one corresponding to the cloned gene and the other to a lethality and chromosome missegregation phenotype caused by ctf13-30, a missense mutation in the 58 kDa cross-hybridizing sequence (Chen et al., 1995) . It is possible that one of these represents a pseudogene, al-subunit of CBF3. Second, SKP1 is an essential gene, and the temperature-sensitive mutation skp1-4 causes though this point needs clarification. In C. elegans, however, SKP1-related ESTs were derived from two phenotypes similar to those observed in a ctf13 mutant, including chromosome missegregation at semi-permis-nonidentical mRNA sequences (only one of which was sequenced to completion and reported here). The hu-sive temperature and a terminal phenotype at nonpermissive temperature indicative of a defect in the G2/M man genome may therefore contain a single functional SKP1 gene or, alternatively, two functional genes, one phase of the cell cycle. Finally, antibodies recognizing an epitope-tagged Skp1 protein decrease the electro-of which is expressed at low levels and not represented in the cDNA libraries used for EST sequencing. Skp1p phoretic mobility of a CBF3-CEN DNA-protein complex formed in vitro. homologs present in the public databases include OCP-II (Cavia porcellus), FP21 (Dictyostelium discoideum), Within the CBF3 complex, the stoichiometry of Skp1p is apparently equal to Ctf13p. CBF3 has an apparent and an uncharacterized ORF from the chlorella virus (PBCV-1) genome; however, very little is known about native molecular mass of 240 kDa, consistent with the original hypothesis that it is a heterotrimeric complex the function of these three proteins in their respective organisms. The human Skp1p homolog protein se-containing one molecule each of the 58, 64, and 110 kDa subunits. The intrinsic mobility shift data (Figure quence is identical to a sequence reported by for p19 SKP1 , a protein that copurifies with a 2A) indicate that Skp1p and Ctf13p are present at an equimolar ratio. Addition of a 60 amino acid segment p9-p45 SKP2 -cyclin A-CDK2 complex derived from transformed fibroblasts. Although addition of p45 or p45 and to either Skp1p or Ctf13p results in an identical mobility change for the CBF3-CEN DNA complex in the absence p19 to cyclin A-CDK2 complexes had no effect on kinase activity in vitro, microinjection of anti-p45 antibody into of added anti-epitope antibody. Exact comigration of these complexes has been confirmed in extended elec-cultured cells prevented entry into S phase. Microinjection of anti-p19 antibody had no effect, so there is cur-trophoresis runs (data not shown). This "intrinsic" shift is not subject to variation of epitope accessibility to rently no direct evidence that p19 SKP1 modulates CDK activity either in vitro or in vivo. The G1/S arrest observed antibody binding, as might be encountered in a standard supershift assay (as in Figure 2B ). CBF3 therefore con-in S. cerevisiae skp1-3 mutants reported here supports the hypothesis that p19 is an essential element of the tains a minimum of four subunits, which are likely to be in an equimolar ratio. The DNase I footprint of CBF3 cyclin A-CDK2 S phase kinase. exhibits asymmetry around the center of the dyad and encompasses 56 bp of DNA (Jehn et al., 1991) . It is Skp1p Is Required for Cell Cycle Progression possible that multiple CBF3 complexes bind to a single at Both G1/S and G2/M CDEIII sequence, especially given the dyad symmetry The distinct sets of phenotypes caused by different temwithin the 25 bp CDEIII consensus sequence. cdc28 mutant alleles (see Nasmyth, 1993) . The G2/M These biochemical data strongly support the conclusion accumulation and chromosome missegregation phenothat Skp1p is an essential component of CBF3. Furthertypes in skp1-4 mutants are similar to those observed more, as these authors point out, the copurification of in ctf13 and cep3/cbf3c mutants, both of which carry Skp1p (Cbf3d) with Ctf13p (Cbf3c) through a series of mutations in CBF3 components. The G2/M delay in cell chromatographic steps provides evidence that these cycle progression could be due to a physical impediproteins directly interact. Our genetic suppression data ment to chromosome separation or, alternatively, to acprovide in vivo evidence for protein-protein interaction: tivation of a cell cycle checkpoint. The identification of increased expression of SKP1 suppresses a ctf13 mutamad and bub mutants that bypass a cell cycle arrest tion and, reciprocally, increased expression of CTF13 induced by defects in spindle assembly provides strong suppresses the skp1-4 mutation. evidence for a surveillance mechanism that monitors spindle integrity in yeast (Hoyt et al., 1991; Li and Murray, Skp1p Is an Evolutionarily Conserved 1991). Two groups have recently shown that these same Kinetochore Component functions are required for the G2/M delay observed in The identification and sequence analysis of homologs of the S. cerevisiae SKP1 gene from A. thaliana, C. elegans, response to kinetochore defects caused by mutations in ctf13 or CEN DNA (Wang and Burke, 1995; Pangilinan tached kinetochores but is extinguished when bipolar attachment is achieved may be a component or target and Spencer, 1996). It will therefore be of interest to determine the effects of mad and bub mutants on the of a kinetochore checkpoint (Campbell and Gorbsky, 1995). As suggested previously (Li and Nicklas, 1995; skp1-4-mediated G2/M delay. The allele-specific G1/S arrest observed in skp1-3 mu-Gorbsky, 1995), eventual achievement of bipolar attachtants appears to be distinct from the G1 arrest observed ment could, through mechanical tension on the kinetoin cdc28 mutants. The skp1-3-mediated arrest is similar chore complex, induce a conformational change in a to those exhibited by mutants later in G1. In particular, key regulatory protein and thereby regulate the activity the multiple budded phenotype is strikingly similar to of an associated kinase. Though speculative, it is possi-cdc4, cdc34, and cdc53 mutants, which arrest at the ble that Skp1p may function at the kinetochore to signal G1/S boundary. As noted above, the homology of Skp1p completion of metaphase. For example, Skp1p could to human p19, which may associate with a cyclin facilitate docking of a cyclin-CDK complex (or proteins A-CDK2 complex in mammalian cells, is consistent with involved in regulating a kinase) at the kinetochore. This a regulatory role for entry into S phase. In addition, Bai specific localization of a regulatory kinase (or other proet al. (1996) provide strong evidence that Skp1p plays tein complex) may act as a determinant in its activity a critical role in the transition from G1 to S phase in upon a kinetochore-localized substrate. Perhaps the yeast and mammals. Combined genetic and biochemiregulatory capacity of Skp1p has been recruited to the cal experiments show that Skp1p directly interacts with kinetochore by its direct incorporation within the CBF3-Cdc4p and cyclin F through a novel structural motif. CEN DNA complex. The ctf13-30 allele (YPH972) has been previously described (Doheny Skp1p is an intrinsic subunit of the kinetochore complex are described in Table 2 a These strains are mitotic segregants that have lost the marker chromosome fragment (and therefore form red colonies). of a 1.8 kb BglII fragment containing the TRP1 gene was accom- The plasmid containing the GAL1 promotor-E1 epitope fused plished by blunt-end ligation into the NsiI site. One-step gene reto SKP1, p415GEU1SKP, was constructed from the plasmid placement was accomplished by excision of a NotI-XhoI fragment p415GEU1, a derivative of p414GEU1 (Doheny et al., 1993), which and transformation into YPH985 selecting for Trp ϩ transformants. replaced the TRP1 gene with the LEU2 gene using PvuI-PvuI sites The integration of the TRP1 marker into the genome was confirmed in the vector sequence (Sikorski and Hieter, 1989) . A PCR strategy by Southern blot analysis of restricted genomic DNA. Trp ϩ diploids was used to place the E1 epitope in-frame with the second codon were sporulated, and 20 tetrads dissected resulted in 2ϩ:2Ϫ segreof the SKP1 ORF using an engineered XhoI cloning site and a downgation for viability. All viable spores were Trp Ϫ , demonstrating that stream genomic EcoRI site (see Figure 1A ). To avoid possible PCR SKP1 was an essential gene. errors, wild-type genomic sequence from NsiI to PvuII replaced the SKP1 was physically mapped to chromosome IV by hybridization PCR-generated sequence. When transformed into the skp1 deletion of a radioactive 32 P-labeled (Feinberg and Vogelstein, 1984) EcoRIstrain, p415GEU1SKP rescued growth on galactose-containing, but NotI (polylinker) fragment isolated from 3B to filters containing overnot dextrose-containing, media. The untagged construct was prelapping and cosmid clones of the S. cerevisiae genome (L. Riles pared by cloning the XhoI-PvuII fragment from p415GEU1SKP into and M. Olson). This placed SKP1 on the right arm of chromosome p415GEU2 (J. Kroll, unpublished data) at the XhoI-SmaI sites in the IV, between gcn2 and trp4. polylinker. When transformed into the skp1 deletion strain, The skp1-⌬1::TRP1 allele is a deletion of the entire SKP1 ORF p415GEU2SKP rescued growth on both galactose-and dextrosebetween the PstI and Eco57I sites and replacement with vector and containing media, presumably because of low level expression un-TRP1 sequences using one-step gene replacement ( Figure 1B) . This der glucose-repressing conditions. deletion extends from 100 bp upstream of the start of SKP1 to 20 The HA epitope was fused to SKP1 in pAD5 (Neiman et al., 1993) , bp upstream of the stop codon. The integration vector, pRS304 which is a 2-LEU2 plasmid that carries an ADH1 promoter, an ⌬SKP1, was constructed by inserting the ‫3.1ف‬ kb PstI (polylinker)-ATG, and the Lerner HA epitope (Field et al., 1988) followed by a PstI fragment and the 1.16 kb Eco57I-ClaI fragment from the original SalI cloning site. A 1.3 kb XhoI-SacI fragment from p415GEU1SKP library clone 3B into the PstI site and the ClaI-KpnI (blunt) site of was cloned into the SalI-SacI site in the polylinker of pAD5, resulting pRS304 (Sikorski and Hieter, 1989), respectively. pRS304⌬SKP1 in pAD5SKP. This HA-tagged construct was able to rescue viability was linearized with EcoRI and transformed into YPH1096 selecting in the skp1 deletion strain. for Trp ϩ transformants. Transformants were streaked on 5-fluoroor-Extracts were prepared for biochemical analysis of CBF3-CEN otic acid (FOA) (Boeke et al., 1987) to determine whether the integra-DNA complexes according to the methods described in Doheny et tion took place in the genome or on the Ura ϩ plasmid, pRS316SKP. al. (1993) . The DNA fragment containing CDEIII used in the gel mobil-Transformants that failed to grow on FOA were selected, and Southity shift assay was the same as reported previously (Doheny et al., ern blot analysis of genomic DNA was performed to confirm the 1993) except it was labeled by a different method. The plasmid integration and removal of the SKP1 ORF. YPH1099 is the skp1 null pSF262a (a gift from P. Sorger) allows excision of the CDEIII region with HindIII-EcoRI, resulting in a 100 bp fragment that was labeled strain generated in this way. Mutational analysis of centromere DNA from chromosome Genetics USA 74, of yeast chromosomes: a colony color assay that measures nondis-5463-5467. junction and chromosome loss S. cerevisiae genes yeast host strains designed for efficient manipulation of DNA in required for cell cycle arrest in response to loss of microtubule Saccharomyces cerevisiae Fluoresence of intact yeast cells treated with alkali cations Centromere DNA mutations inof the Saccharomyces cerevisiae centromere CDEIII sequence: reduce a mitotic delay in Saccharomyces cerevisiae Isolation and charactercontains a cyclin-CDK complexing homolog, as identified by in vitro ization of a gene (CBF2) specifying an essential protein component reconstitution Visual assay for chromosome codes a centromere protein of Saccharomyces cerevisiae Checkpoint genes required to segregation, constitutes a putative DNA binding subunit of the Sacdelay cell division in response to nocodazole respond to impaired charomyces cerevisiae kinetochore complex, CBF3. EMBO J. 13, kinetochore function in the yeast Saccharomyces cerevisiae Aberrantly segregating cencomplex, CBF3, is a major component of the budding yeast centrotromeres activate the spindle assembly checkpoint in budding mere Mitotic forces control a cell-cycle glycoprotein from Dictyostelium Analysis of 45 kb of DNA located at the left end of the chlorella S phase kinase A specific transmembrane GenBank Accession Numbers domain of a coronavirus E1 glycoprotein is required for its retention in the Golgi region A rapid method for localized mutagenesis of yeast genes Control of the yeast cell cycle by the Cdc28 protein kinase Functional homology of protein kinases required for sexual differentiation in Schizosaccharomyces pombe and Saccharomyces cerevisiae suggests a conserved signal transduction module in eukaryotic organisms An efficient method to generate phosphatase insensitive 3Ј labelled DNA probes using Taq polymerase Abnormal kinetochore structure activates the spindle assembly checkpoint in budding yeast Identification of a subdomain of CENP-B that is necessary and sufficient for localization to the human centromere The centromere: hub of chromosomal activities Anaphase onset in vertebrate somatic cells is controlled by a checkpoint that monitors sister kinetochore attachment to the spindle Methods in Yeast using Taq polymerase according to the method described pre-communicating results prior to publication. We thank F. Spencer, R. Skibbens, and J. Lamb for comments on the manuscript; M. viously (Niedenthal and Hegemann, 1993) .Basrai for contributions to experimental design; G. Merkulov for construction of the E1-tagged SKP1 expression vector; S. Tugend-Generation and Integration of Temperature-Sensitive Alleles Synthetic oligonucleotides were synthesized corresponding to se-reich for help in protein sequence alignments; and members of the Hieter lab for helpful discussions and ideas. This work was sup-quences approximately 250 bp on either side of the polylinker of pRS316. They served as primers for a PCR using pRS316SKP as ported by a National Institutes of Health grant (CA16519) to P. H. a template following conditions for mutagenesis as described by Muhlrad et al. (1992) . Approximately 10 ng of template was amplified Received April 10, 1996; revised June 11, 1996. under standard PCR conditions (Perkin Elmer) except the concentration of MgCl2 was increased to 3 mM and the dNTP pool was References reduced to 60 M. Approximately 500 ng of mutagenized PCR product was cotransformed into YPH1099 with 500 ng of XhoI-linearized Bai, C., Sen, P., Hofmann, K., Ma, L., Goebl, M., Harper, J.W., and pRS315 and 5 g of herring sperm DNA (Sigma). Transformants were Elledge, S.J. (1996) . SKP1 connects cell cycle regulators to the replica plated twice to medium containing FOA to ensure eviction of ubiquitin proteolysis machinery through a novel motif, the F-box.