The microtubule organizing center (MTOC) is the key organelle for regulating the structure and function of microtubules in the cell. The long term objectives of this proposal are to identify important functional components of the yeast MTOC or spindle pole body (SPB), determine their functions, and establish the pathway of MTOC assembly. The identification of conserved MTOC components should have important implications for the treatment of cancer, the understanding of the mechanism of birth defects and the search for antifungal drugs. We have three specific aims: First, we will examine the function of the yeast centrin homolog, Cdc31p. Centrin is a highly conserved protein found in the MTOCs of plants, animals and fungi. Cdc31p is a key protein required for initiation of SPB duplication. To elucidate Cdc31p's role we will identify critical SPB proteins which interact with Cdc31p using a combination of genetic and biochemical approaches. We will identify proteins that either physically interact with Cdc31p, modify its activity in vivo or act downstream in the pathway of SPB assembly. In turn, we will determine the functions of the interacting proteins in the SPB. We will use dominant negative mutant forms of Cdc31p to help understand centrin's function in the in vitro assembly of vertebrate MTOCs. Second, we aim to determine the normal function of Dsk2p, a novel ubiquitin-related protein identified as a suppressor of a mutation in a SPB component. Is Dsk2p a component of the SPB, a modifier of an SPB component or does it act downstream in the SPB assembly pathway? Using Dsk2p specific antibodies, we will determine the localization of Dsk2p and whether it is processed like ubiquitin. We will determine whether Dsk2p binds to Cdc31p or affects the stability of Cdc31p or other SPB components. The role of the ubiquitin-dependent protein degradation pathway will be assessed. We will use a variety of genetic and physical methods to identify DSK2 related genes as well as genes and proteins that interact with Dsk2p. Finally, to determine whether the SPB is conservatively synthesized and segregated, we will examine the segregation behavior, in vivo, of tagged SPB components after pulse synthesis.
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