Identification and characterization of a role for the actin cytoskeleton during sporulation in Saccharomyces cerevisiae
AuthorDavis, Dana Alan, 1969-
MetadataShow full item record
PublisherThe University of Arizona.
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AbstractThe actin cytoskeleton is essential in yeast and is composed of actin and numerous actin binding proteins. One actin binding protein, encoded by SAC6, is the yeast homolog of human fimbrin, an actin bundling protein (1). Sac6 protein is not essential for viability but is involved in many cytoskeletal functions. One common phenotype cytoskeletal mutants, including the sac6Δ, have is a defect in sporulation. Although this phenotype has been known for some time, the function of the actin cytoskeleton during sporulation is completely unknown. In order to determine the role of Sac6 protein and the actin cytoskeleton for sporulation, I accomplished the following: (1) I identified the terminal arrest point of the sac6Δ during sporulation as being immediately prior to spore wall formation, (2) By analyzing other mutants, I established that a primary function for the cytoskeleton during sporulation is for endocytosis, and (3) I identified an endocytic pathway in vegetative cells having different requirements for the actin cytoskeleton than the classical endocytic pathway. The events occurring during sporulation have been characterized. By using a number of different assays, I determined that the sac6Δ arrests late in the sporulation pathway. Different arrest points were seen depending on strain background used. However, in the SK1 background, a function for Sac6 protein in spore wall formation was identified. By examining other mutations defective for sporulation, I identified sla2Δ and chc1-521 as having sporulation defects similar to the sac6Δ. SLA2 encodes a cytoskeletal protein that has roles in endocytosis and CHC1 encodes the clathrin heavy chain that has roles in membrane trafficking, including endocytosis (2-4). Actin and Sac6 protein are also required for endocytosis (5). These data led to the model that a function of the actin cytoskeleton during sporulation is for endocytosis. An allelic series of actin mutations had previously been analyzed for ability to undergo receptor-mediated endocytosis (6). This data was compared with the sporulation ability of the actin mutations and a strong correlation was identified between these two phenotypes. I determined that endocytosis does occur throughout sporulation and that the sac6Δ has defects in endocytosis during sporulation. In order to better understand the role of endocytosis during sporulation, I analyzed the endocytosis of Ste6 protein. The half-life of this protein is known to be controlled by the endocytic machinery and it is endocytosed constitutively (7). The data obtained from this assay (although not informative with regards to sporulation) suggests that Ste6 protein has different requirements for the actin cytoskeleton than receptor-mediated endocytosis. All endocytosis appears to require the actin cytoskeleton, however this may be the first demonstration that multiple actin-dependent endocytosis pathways may exist in yeast.
Degree ProgramGraduate College
Molecular and Cellular Biology