Having used biochemical approaches to identify components of the Saccharomyces cerevisiae microtubule cytoskeleton, we will now determine how these components function during mitosis, meiosis, and nuclear fusion. In addition, our recently completed genetic selections for genes that enhance the function of a beta-tubulin mutant promise to provide unique insights into mechanisms used to modulate microtubule stability in vivo. The principles learned from our studies will apply to all eukaryotic cells because the properties of microtubules are highly conserved. These specific questions will be addressed: What are the in vivo functions of MAP27, MAP38 and MAP50? The in vivo functions of three biochemically identified yeast MAPs, MAP27, MAP38 and MAP50, will be genetically elucidated. MAP38 and MAP50 have been shown by immunofluorescence to associate with both intra-nuclear and cytoplasmic microtubules in vivo. Therefore, determining the functions of these two proteins will be given the highest priority. MAP27 has GTP- binding activity suggesting that it might regulate or insure the proper assembly of microtubules. The genes that encode MAP27, MAP38 and MAP50 have been isolated. Using in vitro mutagenesis and gene replacement, we will construct mutant alleles of these genes. The in vivo roles of each MAP will be elucidated using four well-established and highly sensitive in vivo assays of microtubule function in yeast. Once the genetic analysis of the MAP functions is completed, the biochemical activities that underlie their in vivo roles will be determined. What are the in vivo functions of genes identified by suppression of a benomyl-dependent beta-tubulin mutation? We have already identified genes that can either be mutated or overexpressed to suppress the tub2- 150 beta-tublin mutant. These genes are likely to encode novel proteins that regulate microtubule assembly in vivo. The same sensitive genetic tests for in vivo microtubule function referred to for the MAP27, MAP38, and MAP50 genes (above) will be used to elucidate the roles of the suppressor genes. Double mutant analysis will determine the order in which each of the proteins acts in processes such as mitotic spindle assembly, and which subsets of proteins function together to regulate microtubule assembly during the cell cycle. In total, these studies will provide novel insights into the mechanisms by which accessory proteins interact with tubulin in living cells to mediate such functions as chromosome segregation, nuclear migration and nuclear fusion.
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