Date of Award

2008

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology and Molecular Genetics

First Advisor

Johnson, Douglas

Abstract

The S. cerevisisae Rho GTPase Cdc42p localizes around the entire plasma membrane, but is only activated at sites of polarized growth in a cell cycle-dependent manner. This spatial and temporal control ensures that Cdc42p is inactive within the cytoplasm and outside of the polarized cortical domain, and is active at sites of polarized growth. This dissertation addresses spatial control of Cdc42p by examining complex formation with its sole guanine nucleotide dissociation inhibitor (GDI) Rdi1p and its sole guanine nucleotide exchange factor (GEF) Cdc24p. Rdi1p is not essential; however, when overexpressed, it is lethal, suggesting that it does have an important negative regulatory function. To study complex formation and localization, the technique of bimolecular fluorescence complementation (BiFC) was used to study in vivo Rdi1p-Cdc42p complex formation and localization, and how mutations to Rdi1p’s two functional domains, the regulatory arm and the geranylgeranyl binding pocket, affected the complex. Rdi1p and Cdc42p interacted diffusely throughout the cytoplasm of cells at all stages of the cell cycle and their interaction was enhanced at incipient bud sites, the tips and sides of small- and large-budded cells and at the mother-bud neck region. Mutant Rdi1p was still able to interact with Cdc42p; however, the complex was absent from the cytoplasm. The Cdc42p Switch II domain mutation has the same phenotype as the Rdi1p mutations and, surprisingly, Cdc42p without a geranylgeranyl modification can still interact with Rdi1p in the cytoplasm, although with decreased affinity. These results are consistent with Rdi1p extracting Cdc42p from sites of polarized growth, which is dependent on both of its functional domains, and sequestering Cdc42p in the cytoplasm, where it is inactive. Cdc42p is also activated at incipient bud sites, tips of small buds and the mother-bud neck by the precise targeting of Cdc24p. This research also sought to identify protein(s) and regulatory mechanisms that function in targeting and maintenance of Cdc24p at polarized growth sites. The GFP-tagged targeting and anchoring domain of Cdc24p (aa 647-854) was used to screen haploid deletion mutants for localization to polarized growth sites. boi2Δ, ent2Δ, hua1Δ, and rsc2Δ mutants did not target GFP-Cdc24-647-854p to sites of polarized growth; therefore, Boi2p, Ent2p, Hua1p and Rsc2p have direct or indirect roles in targeting and maintenance of Cdc24p to sites of polarized growth. skg6Δ tos2Δ mutants abnormally localized GFP-Cdc24-647-854p pre-anaphase to the mother-bud neck. The same abnormal localization of GFP-Cdc24-647-854p was seen when potential CDK phosphorylation sites were mutated. This indicates that Tos2p, and its homolog Skg6p, and phosphorylation of the Cdc24p targeting and anchoring domain, are important for the maintenance of Cdc24p at sites of polarized growth. This dissertation research contributes to our understanding of how Cdc42p regulators affect its localization and activity by studying the spatial and temporal localization of the Cdc42p-Rdi1p complex and by identifying protein(s) involved in targeting and maintenance of Cdc24p at sites of polarized growth. i

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