GENETICS OF CENTRIN AND THE SPINDLE POLE BODY IN YEAST

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Microtubule organizing centers (MTOC, called the spindle pole body or SPB in yeast) are the key organelles for regulating the structure and function of microtubules in the cell. Microtubules emanating from the MTOC form the spindle apparatus for chromosome segregation and interact with cortical sites to orient the nucleus and spindle within the cell. Several proteins of the SPB and the protein complexes that orient the spindle are highly conserved in eukaryotes. For example, Cdc31p is an SPB protein required for the earliest step in SPB duplication. Cdc31p is the yeast homologue of centrin, a highly conserved calmodulin-like MTOC protein. Kar1p is an integral membrane protein at the SPB that is required to localize Cdc31p. However, Cdc31p has additional functions in the cell; in addition to a role in SPB duplication, Cdc31p associates with a cytoplasmic kinase, Kic1p, and is required for cell morphology and integrity. Cytoplasmic dynein and components of the dynactin complex form part of one pathway for SPB/nuclear orientation. Act5p is the yeast homologue of the mammalian actin-related protein, Arp1, the most abundant subunit of the dynactin complex. Little is known about how these components interact with each other and with other proteins to duplicate the SPB and orient it in the cell. Our long-range objectives are to identify components of the SPB, understand their functions in duplication and orientation and how these processes are regulated and integrated in the cell cycle. We will use a variety of genetic and molecular techniques to test four specific hypotheses about SPB duplication and orientation. First, we identified the PKC1 pathway as a potential key regulator that coordinates SPB duplication with the cell cycle. We will test the hypothesis that the PKC1 pathway acts by regulating the activity of Cdc31 p. Second, we have identified two KAR1 interacting genes, NEM1 and SPOT that encode nuclear envelope proteins. We will test the hypothesis that Nem1p and Spo7p interact with Kar1p or Cdc31p for assembly of the SPB half- bridge. Third, we propose that specific regions of CDC31 mediate its different functions. We will test this by the phenotypic and biochemical characterization of a large number of CDC31 mutations. Fourth, we have begun a genetic analysis to identify the remaining components and regulators of the dynactin complex. A variety of ACT5 and suppressor mutants will be used to understand the how the components of the dynactin complex interact to help orient the SPB/nucleus. This research should provide fundamental knowledge about the basic structure and function of the MTOC and is relevant to significant areas of human health, including the mechanisms of birth defects and cancer. The MTOC proteins identified here may be important targets for anti-mitotic drugs for the treatment of cancer and fungal infections.