Investigation of Post-Translational Modification and Function of the Yeast Plasmid Partitioning Proteins Rep1 and Rep2

Abstract

The 2-micron circle of Saccharomyces cerevisiae is one of a small number of similar DNA plasmids found only in budding yeast. To understand how this cryptic parasite persists, despite conferring no advantage to the host, I investigated the plasmid-encoded Rep1 and Rep2 proteins. Interaction of Rep1 and Rep2 with each other and with the plasmid STB locus is required for equal partitioning of plasmid copies at mitosis. The Rep proteins also repress expression of Flp, the recombinase that mediates plasmid copy-number amplification. In this study, absence of Rep1 and Rep2, or over-expression of the plasmid-encoded Raf antirepressor, increased expression of a longer, novel FLP transcript. Translation of this mRNA may explain elevated Flp activity at low plasmid copy number. Raf competed for Rep2 selfassociation and interaction with Rep1, suggesting the mechanism of Raf anti-repression. Deletion analysis identified a target site for Rep protein repression of FLP that is also repeated in the STB locus, suggesting this as the sequence required for Rep protein association with both regions of the plasmid. Distinct roles for Rep1 and Rep2 were identified; Rep1 was found to depend on Rep2 for post-translational stability, with Rep2 dependent on Rep1 for stable association with STB. Lysine-to-arginine substitutions in Rep1 and Rep2 impaired their association with the host covalent-modifier protein SUMO, suggesting these were sites of sumoylation. The substitutions did not affect interaction of the Rep proteins with each other or their stability but did perturb plasmid inheritance, suggesting that Rep protein sumoylation contributes to their plasmid partitioning function. When Rep1 was mutant, both Rep proteins lost their normal localization to the nuclear foci where 2-micron plasmids cluster, and were impaired for association with STB, supporting this as the cause of defective plasmid inheritance. The potential sumoylation-dependent association of the Rep proteins with the 2-micron plasmid partitioning locus suggests the plasmid has acquired a strategy common to eukaryotic viral and host genomes that depend on sumoylation of their segregation proteins for faithful inheritance. Collectively, my results shed light on how the 2-micron plasmid maintains the delicate balance of persisting without harming its host.