Mitotic chromosome segregation is accomplished by the spindle, a motile microtubule-based structure. In the simple eukaryote S. cerevisiae, all of the microtubule-based motor proteins of the kinesin and dynein families are now known (total of six and one, respectively). Most act within the spindle, but the roles performed by each are not completely understood. A major goal of the proposed experiments is to determine these roles and the nature of the complex interactions exhibited by these motors. We are focusing upon two uncharacterized kinesin-related motors, Rrc805p and Kip2p, that we have linked to the essential process of mitotic spindle positioning (or """"""""nuclear migration""""""""). Surprisingly, these two motors and the dynein nuclear migration motor can all be eliminated without causing lethality. This indicates a previously unappreciated nuclear migration role for at least one of the four remaining kinesin-related motors (or the presence of novel microtubule-based motors - see below). We have also revealed a deleterious action of the Kip2p motor in the absence of dynein and Rrc8O5p. The causes of these related effects will be revealed by the construction of multiple motor mutant strains and observing the in vivo behavior of spindles and chromosomes. We will also perform a series of experiments designed to determine the molecular mechanism of action of the most important spindle assembly and pole-separating motor, Cin8p. This motor belongs to the BimC family of kinesin-related proteins that is conserved throughout the eukaryotes. The structure and function of wild- type and mutant Cin8p motor complexes will be examined by in vivo and in vitro assays. We have determined that the tail domain of Cin8p is required for multimerization, proper localization and normal regulation. These properties will be explored further. We will also describe the mechanisms of cell cycle regulation that act upon Cin8p. Finally, we will investigate the mitotic role performed by MONl, the largest gene in yeast, encoding a protein with features similar to molecular motors.
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