The definition of protein–protein interactions (PPIs) in the natural cellular context is essential for properly understanding various biological processes. So far, however, most large-scale PPI analyses have not been performed in the natural cellular context. Here, we describe the construction of a Saccharomyces cerevisiae fusion library in which each endogenous gene is C-terminally tagged with the N-terminal fragment of Venus (VN) for a genome-wide bimolecular fluorescence complementation assay, a powerful technique for identifying PPIs in living cells. We illustrate the utility of the VN fusion library by systematically analyzing the interactome of the small ubiquitin-related modifier (SUMO) and provide previously unavailable information on the subcellular localization, types, and protease dependence of SUMO interactions. Our data set is highly complementary to the existing data sets and represents a useful resource for expanding the understanding of the physiological roles of SUMO. In addition, the VN fusion library provides a useful research tool that makes it feasible to systematically analyze PPIs in the natural cellular context.

Homologous recombination is exquisitely activated only during specific cell phases. In the G1 phase, homologous recombination activity is completely suppressed. According to previous reports, the activation of homologous recombination during specific cell phases depends on the kinase activity of cyclin-dependent kinase 1 (CDK1). However, the precise regulatory mechanism and target substrates of CDK1 for this regulation have not been completely determined. Here, we report that the budding yeast CDK1, Cdc28, phosphorylates the major homologous recombination regulators Rad51 and Rad52. This phosphorylation occurs in the G2/M phase by Cdc28 in combination with G2/M phase cyclins. Nonphosphorylatable mutations in Rad51 and Rad52 impair the DNA binding affinity of Rad51 and the affinity between Rad52 rings that leads to their interaction. Collectively, our data provide detailed insights into the regulatory mechanism of cell cycle–dependent homologous recombination activation in eukaryotic cells.

Rad52 is well known as a key factor in homologous recombination. Here, we report that Rad52 has functions unrelated to homologous recombination in Saccharomyces cerevisiae; it plays a role in the recruitment of Mps1 to the kinetochores and the maintenance of spindle assembly checkpoint (SAC) activity. Deletion of RAD52 causes various phenotypes related to the dysregulation of chromosome biorientation. Rad52 directly affects efficient operation of the SAC and accurate chromosome segregation. Remarkably, by using an in vitro kinase assay, we found that Rad52 is a substrate of Ipl1/Aurora and Mps1 in yeast and humans. Ipl1-dependent phosphorylation of Rad52 facilitates the kinetochore accumulation of Mps1, and Mps1-dependent phosphorylation of Rad52 is important for the accurate regulation of the SAC under spindle damage conditions. Taken together, our data provide detailed insights into the regulatory mechanism of chromosome biorientation by mitotic kinases.Rad52 | spindle assembly checkpoint | mitotic kinases | phosphorylation

The target of rapamycin complex 1 (TORC1) is mainly localized to the vacuolar membrane and regulates eukaryotic cell growth in response to nutrient availability. To obtain deeper insights into the functional roles of TORC1, we performed a genome-wide analysis of the TORC1 interactome in yeast using the bimolecular fluorescence complementation (BiFC) assay. We found that while most of the BiFC signals are observed at the vacuolar membrane, a fraction of them are detected at cytoplasmic messenger ribonucleoprotein (mRNP) granules. Moreover, mRNA-binding proteins are enriched in the TORC1 interactome, suggesting a functional relationship between TORC1 and mRNA metabolism. We show that a portion of TORC1 is consistently associated with mRNP complexes and interacts with a specific subset of mRNAs. We also demonstrate that TORC1 directly targets a translational repressor Scd6 and that the activity of Scd6 is inhibited by TORC1-dependent phosphorylation. Collectively, our data suggest that TORC1 plays a novel role in posttranscriptional regulation by controlling the activity of Scd6.

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