The 2μm Plasmid Causes Cell Death in Saccharomyces cerevisiae with a Mutation in Ulp1 Protease

Abstract

The 2μm circle plasmid confers no phenotype in wild-type Saccharomyces cerevisiae but in a nib1 mutant, an elevated plasmid copy number is associated with cell death. Complementation was used to identify nib1 as a mutant allele of the ULP1 gene that encodes a protease required for removal of a ubiquitin-like protein, Smt3/SUMO, from protein substrates. The nib1 mutation replaces conserved tryptophan 490 with leucine in the protease domain of Ulp1. Complete deletion of ULP1 is lethal, even in a strain that lacks the 2μm circle. Partial deletion of ULP1, like the nib1 mutation, results in clonal variations in plasmid copy number. In addition, a subset of these mutant cells produces lineages in which all cells have reduced proliferative capacity, and this phenotype is dependent upon the presence of the 2μm circle. Segregation of the 2μm circle requires two plasmid-encoded proteins, Rep1 and Rep2, which were found to colocalize with Ulp1 protein in the nucleus and interact with Smt3 in a two-hybrid assay. These associations and the observation of missegregation of a fluorescently tagged 2μm circle reporter plasmid in a subset of ulp1 mutant cells suggest that Smt3 modification plays a role in both plasmid copy number control and segregation.

ACKNOWLEDGMENTS

We thank C. Holm for the gift of yeast strains CH568 and CH569, Marc Gartenberg for the plasmid pBIS-GALkFLP-(TRP1), Barbara Goettgens (Core Facility, Institute for Cell and Molecular Biology, University of Texas, Austin) for assistance with confocal microscopy, and J. Benjamin and Y. Zhang for technical assistance. We are grateful to Erica Johnson for communicating results prior to publication and for helpful comments on the manuscript.

This work was supported by an NSERC grant to M.J.D. and grants from the National Institutes of Health and the Robert Welch Foundation to M.J.