The membrane cytoskeletons of mammalian cells and budding yeast have so far been studied intensively but by almost completely distinct methodologies. We propose hare to initiate a comparison of the molecular components and physical properties of the membrane cytoskeletons of yeast and mammals. We hope that general principles may be distilled when common features are found. (1) Physical properties of yeast with mutations in membrane cytoskeleton proteins. Mutants isolated in a wide variety of cytoskeletal proteins in budding yeast show osmosensitivity and apparent membrane fragility. These observations suggest that the yeast membrane actin cytoskeletal proteins in budding yeast show osmosensitivity and apparent membrane fragility. These observations suggest that the yeast membrane actin cytoskeleton, like the erythrocyte membrane cytoskeleton, plays a critical role in providing structural integrity to the plasma membrane. We will use the ~sensitive force technique: and the ~micropipette technique~ to test the physical properties of the yeast cell cortex in cells with and without cell walls. These studies will provide a deeper understanding of how physical al properties of the cell cortex are determined. (2) Identification of mammalian homologues of yeast membrane cytoskeleton proteins. Genetic analysis of the yeast membrane cytoskeleton has identified both homologues of actin-binding proteins found in other cells ( capping protein, cofilin and fimbrin) and novel proteins (Sla2p, Abp1p, Rvs167, Srv2 and S1a1p). Finding mammalian homologues of the novel proteins will provide an opportunity to apply knowledge from genetic analysis in yeast to mammalian cells. The prospects for finding such homologues are good. A mammalian Srv2 homologue has already been found, an Sla2 homologue exists in C. Elegans, and Sla1 homologue exists in S. pombe, and an Abp1 homolog exists in S. exiguus. (3). Erythrocyte cytoskeleton homologues in yeast. Band 4.1, spectrin and ankyrin have been extensively studies in erythrocytes using biochemistry and molecular-genetics. Finding proteins in yeast with either extensive sequence homology, or homology to a functional domain, would allow classical genetic analysis of their functions. We will query the yeast genome database (presently, 70% accessible, 90% complete) using particularly conserved domains of these cytoskeletal proteins. We will disrupt the genes for any homologues identified in yeast for analysis as described in Aim (1), and will raise antisera to immunolocalize the proteins in vivo.
Showing the most recent 10 out of 311 publications
