A genetic synthetic dosage lethality (SDL) screen using CTF13 encoding a known kinetochore protein as the overexpressed reference gene identified two chromosome transmission fidelity (ctf) mutants, YCTF58 and YCTF26. These mutant strains carry independent alleles of a novel gene, which we have designated CTF19. In light of its potential role in kinetochore function, we have cloned and characterized the CTF19 gene in detail. CTF19 encodes a nonessential 369–amino acid protein. ctf19 mutant strains display a severe chromosome missegregation phenotype, are hypersensitive to benomyl, and accumulate at G2/M in cycling cells. CTF19 genetically interacts with kinetochore structural mutants and mitotic checkpoint mutants. In addition, ctf19 mutants show a defect in the ability of centromeres on minichromosomes to bind microtubules in an in vitro assay. In vivo cross-linking and chromatin immunoprecipitation demonstrates that Ctf19p specifically interacts with CEN DNA. Furthermore, Ctf19-HAp localizes to the nuclear face of the spindle pole body and genetically interacts with a spindle-associated protein. We propose that Ctf19p is part of a macromolecular kinetochore complex, which may func- tion as a link between the kinetochore and the mitotic spindle.

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