Abstract 1371 Wwm1 is a possible substrate of Rsp5 E3 ubiquitin ligase

The yeast model Saccharomyces cerevisiae was the first fully sequenced eukaryote. Despite knowing this information for almost 20 years, there are predicted genes whose functions are still unknown: genes of unknown function (GUFs). In our study, we aimed to explore the protein encoded by the YFL010C GUF: Wwm1. Wwm1 is a WW-domain-containing protein that attaches to proline-rich sequences. Deletion of WWM1 in yeast produced no phenotype, meaning this protein is not essential for its survival. However, when WWM1 was overexpressed, the cell cycle was arrested at the G1 phase, and the cell suffered clonal death. Furthermore, co-overexpression of WWM1 with the metacaspase MCA1 suppressed this phenotype, suggesting possible interactions between these two proteins. The Wwm1 protein is predicted to interact with another WW-domain-containing yeast protein, Rsp5, an essential NEDD4 family E3 ubiquitin ligase. Based on bioinformatics analysis, we found that the Wwm1 protein is a transmembrane protein with cytoplasmic, nuclear, and transmembrane regions. In attempting to characterize the function of Wwm1, we made galactose-inducible His-tagged truncation mutants of WWM1 and galactose-inducible Myc-tagged Rsp5. Yeast co-overexpressing Wwm1 and Rsp5 proteins could not grow, meaning the combined effect of overexpressing Wwm1 and Rsp5 affected yeast survival more severely than overexpressing either protein alone. In addition, full-length Wwm1 protein pulled down Rsp5, suggesting there was a direct physical interaction between these proteins, consistent with the hypothesis that Wwm1 may be a Rsp5 E3 ubiquitin ligase substrate. To test this, we have mutated the PPXY motif of Wwm1 likely to be necessary for Rsp5 recognition and begun experiments using a temperature-sensitive Rsp5 mutant strain of yeast to see if how the ubiquitination of Wwm1 is affected. Understanding the interactions of these two proteins may shed light on the physiological functions of Wwm1, particularly as they pertain to cell cycle and apoptosis. The Stauffer Charitable Trust funded this research.