Global analysis of protein homomerization in Saccharomyces cerevisiae

Abstract

ABSTRACT<br/> Global analysis of protein homomerization in Saccharomyces cerevisiae.<br/> <br/> Yeonsoo Kim<br/> School of Biological Sciences<br/> The Graduate School<br/> Seoul National University<br/> In vivo analyses of protein-protein interaction (PPI) occurrence, subcellular localization, and dynamics are important issues in functional proteomic researches and various PPI analyzing methods have been developed over the past decades. Bimolecular fluorescence complementation (BiFC) assay has many advantages over other research methods in that it can provide a reliable way to detect PPI in living cells with minimal perturbation of the structure and function of target proteins and it allow the visualization of subcellular localization where PPI occurs. <br/> Previously, to facilitate the application of BiFC assay to the genome-wide analysis of PPIs, we generated a collection of yeast strains expressing full-length proteins tagged with the N-terminal fragment of Venus (VN), a yellow fluorescent protein variant, under the control of their own endogenous promoters. The VN fusion library consists of 5,911 MATa strains (representing ~95% of the yeast proteome) and was successfully exploited to screen SUMO interactome in Saccharomyces cerevisiae. In the present study, we constructed the VC (the C-terminal fragment of Venus) fusion library that consists of 5,671 MAT strains expressing C-terminally VC-tagged proteins (representing ~91% of the yeast proteome) under their native promoters. The reliability of the constructed library was then proved by using it with VN fusion library to genome-widely detect protein homomers in yeast. For a genome-wide analysis of protein homomer formation, we mated each strain of the VC fusion library with its cognate strain of the VN fusion library and performed BiFC assay. Through this analysis, we identified 186 homomer candidates, 104 of which are previously unknown homomers. Subcellular localization, highly enriched gene ontology (GO) and changes of homomeric interaction upon nitrogen starvation was further analyzed to characterize homomer population of yeast. Out of 186 protein homomers identified in this study, Pet10, a protein of unknown function which localize to lipid droplet, was further investigated for its functional relevance of homomerization and lipid accumulation. Our data set represents a useful resource for understanding the physiological roles of protein homomerization. Furthermore, thoroughly examined credibility and feasibility of the VC fusion library together with the VN fusion library in BiFC assay revealed that this system will provide a valuable platform to systematically analyze PPIs in the natural cellular context.<br/>