MOLECULAR BIOLOGY OF MICROTUBULE INTERACTING PROTEINS

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DESCRIPTION: The long-term objective of the research proposed is to identify and characterize proteins that have essential functions in the assembly and activities of microtubules. Most of these proteins and their functions have not been described but they likely have roles in determining the many essential cellular activities of microtubules. The investigator's earlier work supported by this grant led to the discovery of one such protein known as gamma tubulin. Gamma tubulin is related to but distinct from the alpha and beta tubulins which interact to form the heterodimer known as tubulin. Since the discovery of gamma tubulin, data from several labs have shown that gamma tubulin is ubiquitous among eukaryotes and it is found primarily in the microtubule organizing centers (MTOCs) of fungal and animal cells. This has led to the hypothesis that gamma tubulin is essential for microtubule assembly from MTOCs but questions about the function of gamma tubulin, its role in regulating microtubule assembly, its interactions with microtubules, and its interactions with other components of MTOCs remain to be answered. The first aim of the proposed work is to isolate and characterize conditionally-lethal gamma tubulin mutations in the filamentous fungus Aspergillus nidulans. Such mutants have been difficult to isolate by conventional means so the "alanine scanning" approach is proposed. These conditional mutants should be useful tools for clarification of gamma tubulin function and for pseudoreversion analysis for identification of gamma tubulin interacting proteins. Mutants will be examined to determine the effects of mutations on nuclear division, mitosis, mitotic spindle formation, the cytoplasmic microtubule network, and localization and stability of gamma tubulin. The second aim is to identify and characterize proteins that interact with gamma tubulin. Two approaches will be taken. The first approach will involve a yeast two-hybrid screen while the second will use pseudoreversion analysis similar to that used in discovery of gamma tubulin. Genes identified by either of these complementary approaches will be characterized further to determine the potential roles for their products in microtubule assembly and function. The final aim of the proposal is to investigate a novel tubulin identified in the C. elegans genome project. This putative gene product was originally identified as a gamma tubulin but closer analysis shows that it shares only 40-44% identity with gamma tubulins and less with other tubulins. This is sufficiently different to propose that this represents a new tubulin family. This will be investigated by first determining whether C. elegans has a conventional gamma tubulin. If identified, the investigator then intents to search for homologs of this novel tubulin in other organisms and begin to dissect its function.