During the yeast cell cycle, the distribution of actin changes in a highly specific way that reflects the changes in localization of growth that occur during this time. However, little is known of how the alterations in actin localization and growth are mediated or regulated. Actin-binding proteins are likely important, as mutants lacking these proteins are defective in these processes. However, despite the fact that many of these proteins have been well characterized biochemically, little is known of how the the activities of the various actin-biinding proteins in vitro affect actin organization and growth in vivo. The P.I. is focusing on the actin-filament cross-linking protein fimbrin (Sac6), which is the only actin-filament cross-linking protein known in yeast. Cross-linking of actin filaments is likely essential for many of the functions of actin, as individual filaments are unable to withstand strong compressional forces. However, little is known of either the roles of regulation of cross-linking in yeast or in other cells. In this grant, the P.I. proposes to identify the roles and possible regulation of actin-filament corss-linking by Sac6. In particular, the P.I. will first examine the molecular architecture of actin-filament cross-linking. The P.I. will ask whether the two actin binding domains of Sac6 bind to the same or different sites on actin. Second, the P.I. will determine whether Sac6 is regulated or has roles at specific stages of the cell cycle. Third, the P.I. will use genetic screens to identify proteins that reveal the regulation or roles of Sac6 in the cell. These screen will involve looking for suppression of the lethality caused by over-expression of Sac6, or expression of the Sac6-19 mutant, which binds actin extra-tightly.
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