Abstract #89B

GTP binding of Bud4 is necessary for proper bud-site selection and for the integrity of septins during cytokinesis. Pil Jung Kang, Hay-Oak Park. Department of Molecular Genetics, Ohio State Univ, Columbus, OH.
   Cells of the budding yeast S. cerevisiae exhibit two distinct budding patterns that reflect the genetic programming of cell polarization. Haploid a and a cells bud in the axial pattern whereas diploid a/a cells bud in the bipolar pattern. It is believed that these different patterns occur in response to a cell type-specific cortical marker. Despite our current knowledge of proteins involved in budding, how polarity is established toward a spatially defined site is largely unknown. To gain insight into the mechanism by which the cell type-specific budding pattern is established, we have begun a molecular analysis of the axial-specific proteins including Bud3, Bud4, and Axl1. Bud3 and Bud4 are thought to assemble at the mother-bud neck by interacting with septins, and mark a site for budding and for the assembly of new septins in the subsequent division cycle (Chant et al., 1995; Sanders & Herskowitz 1996). Here we report that Bud4 is a GTP-binding protein whose intrinsic GTPase activity is extremely low. We found that GTP hydrolysis by Bud4 could be stimulated by yeast cell extract, suggesting the presence of a GTPase activating protein for Bud4. The GTP-binding motif of Bud4 was necessary for proper localization of Axl1 and for the integrity of septins during cytokinesis. In wild-type cells, Cdc3, a constituent of the septin ring, assembles as a single ring before bud emergence, and remains at the mother-bud neck until the M phase. Cdc3 develops into double rings during cytokinesis, which split to mother and daughter cells after cytokinesis (Kim et al., 1991). We found that in the bud4 mutant lacking the GTP-binding motif, Cdc3-GFP initially assembled in a single ring as in the wild type. Cdc3-GFP, however, dispersed and never formed double rings during cytokinesis nor was it inherited as a discrete ring by both mother and daughter cells after cell separation. Taken together, these data suggest that the GTP-binding property of Bud4 is likely to contribute to the assembly of the axial landmark and inheritance of the spatial information.

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