In budding yeast, entry into the mitotic cell cycle, or Start, requires the Cdc28 cyclin-dependent kinase (Cdk) and one of its three associated G 1 cyclins, Cln1, Cln2, or Cln3. In addition, two other G 1 cyclins, Pcl1 and Pcl2, associate with a second Cdk, Pho85, to contribute to Start. Although Pho85 is not essential for viability, Pcl1,2-Pho85 kinase complexes become essential for Start in the absence of Cln1,2-Cdc28 kinases. In addition, Pho85 interacts with a third cyclin, Pho80, to regulate acid phosphatase gene expression. Other cellular roles for Pho85 cyclin-Cdk complexes are suggested by the multiple phenotypes associated with deletion of PHO85, in addition to Start defects and deregulated acid phosphatase gene expression. Strains with pho80, pcl1, and pcl2 deletions show only a subset of the pho85 mutant phenotypes, suggesting the existence of additional Pho85 cyclins (Pcls). We used two-hybrid screening and database searching to identify seven additional cyclin-related genes that may interact with Pho85. We found that all of the new genes encode proteins that interacted with Pho85 in an affinity chromatography assay. One of these genes, CLG1, was previously suggested to encode a cyclin, based on the protein's sequence homology to Pcl1 and Pcl2. We have named the other genes PCL5, PCL6, PCL7, PCL8, PCL9, and PCL10. On the basis of sequence similarities, the PCLs can be divided into two subfamilies: the Pcl1,2-like subfamily and the Pho80-like subfamily. We found that deletion of members of the Pcl1,2 class of genes resulted in pronounced morphological abnormalities. In addition, we found that expression of one member of the Pcl1,2 subfamily, PCL9, is cell cycle regulated and is decreased in cells arrested in G 1 by pheromone treatment. Our studies suggest that Pho85 associates with multiple cyclins and that subsets of cyclins may direct Pho85 to perform distinct roles in cell growth and division.

Cyclin-dependent kinase (cdk) complexes are essential activators of cell cycle progression in all eukaryotes. In contrast to mammalian cells, in which multiple cdk's contribute to cell cycle regulation, the yeast cell cycle is largely controlled by the activity of a single cdk, CDC28. Analysis of the putative G1 cyclin PCL2 (ORFD) identified a second cyclin-cdk complex that contributes to cell cycle progression in yeast. PCL2 interacted with the cdk PHO85 in vivo and in vitro and formed a kinase complex that had G1-periodic activity. Under genetic conditions in which the Start transition was compromised, PHO85 and its associated cyclin subunits were essential for cell cycle commitment. Because PHO85 and another cyclin-like molecule, PHO80, also take part in inorganic phosphate metabolism, this cdk enzyme may integrate responses to nutritional conditions with the cell cycle.

Cellular toxicity introduced by protein misfolding threatens cell fitness and viability. Failure to eliminate these polypeptides is associated with numerous aggregation diseases. Several protein quality control mechanisms degrade non-native proteins by the ubiquitin proteasome system. Here, we use quantitative mass spectrometry to demonstrate that heat-shock triggers a large increase of ubiquitylation associated with misfolding of cytosolic proteins. We discover that the Hul5 HECT ubiquitin ligase participates in this heat-shock stress response. Hul5 is required to maintain cell fitness after heat-shock and to degrade short-lived misfolded proteins. In addition, localization of Hul5 in the cytoplasm is important for its quality control function. We identify potential Hul5 substrates in heat-shock and physiological conditions to reveal that Hul5 is required for ubiquitylation of low solubility cytosolic proteins including the Pin3 prion-like protein. These findings indicate that Hul5 is involved in a cytosolic protein quality control pathway that targets misfolded proteins for degradation.

Accurate chromosome segregation requires the execution and coordination of many processes during mitosis, including DNA replication, sister chromatid cohesion, and attachment of chromosomes to spindle microtubules via the kinetochore complex. Additional pathways are likely involved because faithful chromosome segregation also requires proteins that are not physically associated with the chromosome. Using kinetochore mutants as a starting point, we have identified genes with roles in chromosome stability by performing genome-wide screens employing synthetic genetic array methodology. Two genetic approaches (a series of synthetic lethal and synthetic dosage lethal screens) isolated 211 nonessential deletion mutants that were unable to tolerate defects in kinetochore function. Although synthetic lethality and synthetic dosage lethality are thought to be based upon similar genetic principles, we found that the majority of interactions associated with these two screens were nonoverlapping. To functionally characterize genes isolated in our screens, a secondary screen was performed to assess defects in chromosome segregation. Genes identified in the secondary screen were enriched for genes with known roles in chromosome segregation. We also uncovered genes with diverse functions, such as RCS1, which encodes an iron transcription factor. RCS1 was one of a small group of genes identified in all three screens, and we used genetic and cell biological assays to confirm that it is required for chromosome stability. Our study shows that systematic genetic screens are a powerful means to discover roles for uncharacterized genes and genes with alternative functions in chromosome maintenance that may not be discovered by using proteomics approaches.chromosome stability ͉ synthetic genetic array ͉ kinetochore

The budding yeast kinetochore is composed of an inner and outer protein complex, which binds to centromere (CEN) DNA and attaches to microtubules. We performed a genetic synthetic dosage lethality screen to identify novel kinetochore proteins in a collection of chromosome transmission fidelity mutants. Our screen identified several new kinetochore-related proteins including YLR381Wp/Ctf3p, which is a member of a conserved family of centromere-binding proteins. High fidelity chromosome transmission, which is necessary for eukaryotic cell survival, requires coordination of many events including DNA replication, sister chromatid cohesion, and kinetochore function. The kinetochore, which is composed of centromere (CEN) DNA and associated proteins, mediates attachment of chromosomes to the spindle. The kinetochore also provides a site for centromeric cohesion and generates signals to arrest cell cycle progression if metaphase has not been achieved properly (for review, see Kitagawa and Hieter 2001). Shortly after spindle pole body (SPB) duplication, sister centromeres separate transiently and oscillate along the spindle axis until anaphase when permanent sister separation occurs (Goshima and Yanagida 2000; He et al. 2000;Tanaka et al. 2000;Pearson et al. 2001). The mechanism by which kinetochores assemble, attach to microtubules, and migrate toward SPBs in anaphase is not well understood.In the budding yeast Saccharomyces cerevisiae, the minimal required CEN DNA element (CDE) consists of two palindromic sequences, CDEI and CDEIII, flanking an A-T rich CDEII sequence. CDEIII, which is essential, is bound by the inner kinetochore complex CBF3 (for reviews, see Clarke 1998; Ortiz and Lechner 2000; Pidoux and Allshire 2000). CBF3 is composed of four essential proteins: Ndc10p/Ctf14p/Cep2p, Cep3p, Ctf13p, and Skp1p. Skp1p and its interacting partner Sgt1p have roles at the kinetochore in G 2 and in SCF-mediated degradation in G 1 (Bai et al. 1996;Connelly and Hieter 1996;Kitagawa et al. 1999). CEN DNA is thought to be wrapped around a specialized nucleosome containing a conserved histone H3-like protein, Cse4p (Cnp1 in fission yeast and CENP-A in higher eukaryotes), in place of a core histone H3 (for review, see Sullivan 2001). In addition to its conserved C-terminal histone fold domain, Cse4p contains a unique and essential N-terminal domain that interacts with members of the yeast outer kinetochore (Keith et al. 1999;Ortiz et al. 1999;Chen et al. 2000). The outer kinetochore protein complex, composed of Mcm21p, Okp1p, and Ctf19p, interacts with

Kinetochore proteins contribute to the fidelity of chromosome transmission by mediating the attachment of a specialized chromosomal region, the centromere, to the mitotic spindle during mitosis. In budding yeast, a subset of kinetochore proteins, referred to as the outer kinetochore, provides a link between centromere DNA-binding proteins of the inner kinetochore and microtubule-binding proteins. Using a combination of chromatin immunoprecipitation, in vivo localization, and protein coimmunoprecipitation, we have established that yeast Chl4p and Iml3p are outer kinetochore proteins that localize to the kinetochore in a Ctf19p-dependent manner. Chl4p interacts with the outer kinetochore proteins Ctf19p and Ctf3p, and Iml3p interacts with Chl4p and Ctf19p. In addition, Chl4p is required for the Ctf19p-Ctf3p and Ctf19p-Iml3p interactions, indicating that Chl4p is an important structural component of the outer kinetochore. These physical interaction dependencies provide insights into the molecular architecture and centromere DNA loading requirements of the outer kinetochore complex.

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