Assembly and function of the Saccharomyces cerevisiae Ctf19 kinetochore subcomplex

Nkp1p and Nkp2p are part of the eleven-member CTF19 kinetochore subcomplex of S. cerevisiae. Of the nine nonessential CTF19 members, Nkp1p and Nkp2p are the least well-characterized. Relative to other complex members, they have been found to make minimal contributions to some CTF19 complex functions (e.g., chromosome segregation and spindle disassembly), and their participation in other CTF19 complex functions (e.g., cohesin localization and kinetochore function) has not been tested. Furthermore, unlike the rest of the complex members, the assembly of Nkp1p and Nkp2p to the CEN has not been studied.;We used chromatin immunoprecipitation (ChIP) to systematically determine assembly dependency relationships between CTF19 complex members. We found that Nkp1p and Nkp2p are strongly interdependent for CEN association. We also found two additional groups of interdependent proteins in the complex: Ctf19p-Mcm21p and Ctf3p-Mcm16p-Mcm22p. We conclude that Nkp1p-Nkp2p comprises a distinct branch of CTF19 complex assembly at the CEN.;Functional characterization of Nkp1p and Nkp2p revealed further evidence that these proteins are functionally distinct from the other CTF19 members: Nkp1p and Nkp2p were not required for normal growth in the presence of benomyl or for proper cohesin localization. We also found that Nkp1p and Nkp2p are functionally distinct from one another, despite their complete interdependence for CEN association: Nkp2p is required for high-temperature growth, whereas Nkp1p is not. Furthermore, we found that Nkp1p performs a role in kinetochore function that Nkp2p does not.;We also report characterization of cse4-N48, a nonsense allele of the yeast centromeric histone H3 variant. cse4-N48 expression causes increased cellular resistance to benomyl, as well as spindle morphology defects. We found that the benomyl resistance effect depends on each nonessential CTF19 complex member. Furthermore, ChIP experiments indicated that cse4-N48 expression disrupts CEN association of CTF 19 complex members, suggesting that cse4-N48 induces its effect by disrupting the CTF19 complex.;Finally, we report the identification by mass spectrometry of candidate sites of phosphorylation on SAC proteins. We mutated several candidate sites and found that one bub3 phospho-mimic allele and three mad3 phospho-mimic alleles displayed increased sensitivity to benomyl, indicating that phosphorylation on these residues may negatively regulate SAC function.