Accurate transfer of parental genetic material to daughter cells requires coordination of complex cellular events. The Chromosome Passenger Complex (CPC) functions in multiple stages of the cell cycle and is crucial for accurate chromosome segregation. The CPC contains a conserved serine-threonine kinase Aurora/Ipl1p and three other subunits: INCENP/Sli15p, Survivin/Bir1p, and in metazoans, Borealin/Dasra/CSC-1. These three subunits are required for CPC targeting to centromeres and central spindles. Knowledge of how this complex is regulated spatially and temporally throughout cell cycle remains incomplete. One question is how the CPC is involved in the spindle assembly checkpoint in response to lack of the spindle tension. I took an approach that combines in vivo cross-linking of the complex upon lack of the spindle tension, and mass spectrometric analysis of the purified complex, to identify associated proteins and post-translational modifications of the CPC. This analysis identified Mcl1p, a previously uncharacterized protein that is a part of the CPC. MCL1 is essential for viability and mutations in MCL1 cause chromosome missegregation and lagging chromosomes. Mcl1p co-localizes and co-purifies with the CPC, and it is essential for CPC localization, stability, integrity and function. These observations suggest that Mcl1p fills the role of Borealin/Dasra/CSC-1 and thus establish that the fundamental design of the CPC is conserved from fungi to metazoans.

Another question is how CPC function is regulated. Our previous yeast two-hybrid screening and mass spectrometry studies suggested that the CPC is modified with SUMO (small ubiquitin-like modifier). SUMO is a highly conserved polypeptide of approximately 12 kD and the SUMO gene (SMT3) is required for chromosome segregation. To investigate the role of sumoylation in the regulation of the CPC, sumoylation sites in CPC proteins were mutated. A strain containing mutations of 16 potential sumoylation sites in the CPC was examined for defects in spindle morphology, chromosome segregation, and for sensitivity to temperature and chemical stresses. No obvious phenotypes were detected for this mutant, possibly because of redundancy among the SUMO-modifications of this multi-protein complex.

Lastly, to understand how Ipl1p/Aurora kinase regulates spindle assembly prior to anaphase, the role of Ipl1p/Aurora phosphorylation of the conserved BimC kinesins (Eg5 homologs), Cin8p and Kip1p, was examined. These proteins are essential for spindle pole separation and were first tested to determine whether they are substrates of Ipl1p/Aurora. Cin8p is phosphorylated by Ipl1p in vitro and in vivo and has many Ipl1p/Aurora phosphorylation consensus motifs. Serine and Threonine residues at thirteen sites that either matched the Ipl1p/Aurora consensus motif or were identified by mass spectrometry analysis as being phosphorylated were mutated to alanines. While the cin8Δ kip1Δ double mutant is lethal, the cin8-13A kip1Δ mutant showed no growth defect at temperatures ranging from 16°C to 39°C and no sensitivity to benomyl, a microtubule-depolymerizing drug. This result suggests that Cin8p may either have cryptic phosphorylation sites for Ipl1p/Aurora or that phosphorylation of Cin8p is not essential for spindle assembly prior to anaphase.