Author

Daobing Wang

Date of Graduation

Spring 2010

Degree

Master of Science in Biology

Department

Biology

Committee Chair

Kyoungtae Kim

Keywords

endocytosis, vps1, rvs167, lifespan, dissociation, interaction

Subject Categories

Biology

Abstract

The yeast dynamin-related protein Vps1 shares high homology with dynamin in its N-terminal GTPase and C-terminal GED domains. To test the significance of two conserved domains in early endocytosis, an endocytic maker, Abp1, was fused with GFP in three GTPase mutants (vps1K42E, vps1S43N, and vps1G315D) and a GED-truncated mutant. First, the lifespan of Abp1-GFP at the endocytic sites was significantly increased in GED-truncated vps1 mutant when compared to that of Wild Type (WT) cells. In particular, the lifespan of Abp1 in the mutant was essentially identical to that of a mutant lacking Vps1, suggesting the significance of the GED for the proper assembly or maturation of endocytic components at endocytic sites. Second, Abp1-GFP in WT moved inward in a rapid/directed fashion and dissociated from the post-internalized patch within 5-7 s, whereas Abp1-GFP in the GED mutant exhibited not only random motion as is the case in vps1Δ, but also a slow dissociation from the patch. Consistent with Abp1-GFP disassembly, I found that the disassembly rate of the endocytic adaptor protein Sla1 was significantly decreased, and conclude that the defects in disassembly might attribute to the decreased efficiency of Prk1 kinase and Sjl2 phosphatase, as well as the fragmented actin cables. In addition, I observed that both the full-length of VPS1 and the C-terminal half of the gene lacking the GED domain interact genetically at a non-permissive temperature with the RVS167gene, an essential player for endocytic scission. The physiological significance of the genetic interaction between VSP1 and RVS167 was confirmed by the significantly delayed maturation of Abp1-GFP at a non-permissive temperature. The investigation of the presumed physical interaction between Vps1 and Rvs167 will provide insights into the scission event of endocytosis.

Copyright

© Daobing Wang

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