| Faithful chromosome segregation during mitosis is vital to correct cell division. To examine the molecular mechanisms that underlie chromosome segregation, I chose to focus specifically on two proteins, Duo1p and Dam1p, which were previously identified as spindle components in the budding yeast, Saccharomyces cerevisiae. Here, I present a variety of analyses of the proteins to examine their in vivo functions and associations, and the mechanism by which they are regulated. I began by analyzing a diverse collection of duo1 and dam1 alleles to develop a deeper understanding of the functions and interactions of Duo1p and Dam1p. Duo1p and Dam1p are not required to assemble bipolar spindles, but they are required to maintain metaphase and anaphase spindle integrity. In addition, duo1 and dam1 mutants show high rates of chromosome mis-segregation, premature anaphase events while arrested in metaphase, and genetic interactions with a subset of kinetochore components consistent with a role in kinetochore function. Since Duo1p and Dam1p localize to kinetochores in chromosome spreads, this complex may serve as a link between the kinetochore and the mitotic spindle.; To further characterize the functions of Duo1p and Dam1p, I purified these proteins from yeast extracts as subunits of a ∼200 kDa complex containing Dam1p, Duo1p, and Dad1p, as well as Spc19p, Spc34p, and the previously uncharacterized proteins Dad2p, Dad3p, Dad4p and Ask1p. I also demonstrated that purified Dam1p complex binds directly to microtubules in vitro with an affinity of approximately 0.5 muM. To demonstrate that subunits of the Dam1p complex are functionally important for mitosis in vivo, I localized Spc19-GFP, Spc34-GFP, Dad2-GFP, Dad3-GFP, Dad4-GFP, and Ask1-GFP to the mitotic spindle and to kinetochores, and generated temperature sensitive mutants of DAD2 and ASK1.; Finally, I chose to examine the mechanism by which these proteins are regulated during mitosis. The Aurora kinase Ipl1p plays a crucial role in regulating kinetochore-microtubule attachments in budding yeast, but the underlying basis for this regulation is not known. To identify Ipl1p targets, I first purified 28 kinetochore proteins from yeast protein extracts. These studies identified five novel kinetochore proteins and defined two novel kinetochore sub-complexes. I then used mass spectrometry to identify 18 phosphorylation sites in 7 of these 28 proteins. Ten of these phosphorylation sites are targeted directly by Ipl1p, allowing me to identify a consensus phosphorylation site for an Aurora kinase. Systematic mutational analysis of the Ipl1p phosphorylation sites demonstrated that the essential microtubule binding protein Dam1p is a key Ipl1p target for regulating kinetochore-microtubule attachments in vivo. |
