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Molecular and Cellular Biology, May 2005, p. 4299-4310, Vol. 25, No. 10
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.10.4299-4310.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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

Melanie J. Dobson,1* Andrew J. Pickett,1 Soundarapandian Velmurugan,2 Jordan B. Pinder,1 Lori A. Barrett,1 Makkuni Jayaram,2 and Joyce S. K. Chew1

Department of Biochemistry and Molecular Biology, Faculty of Medicine, Dalhousie University, Sir Charles Tupper Medical Building, Halifax, Nova Scotia, Canada B3H 1X5,1 Department of Microbiology, University of Texas at Austin, Austin, Texas 787122

Received 7 October 2004/ Returned for modification 20 November 2004/ Accepted 13 February 2005

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.

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, Faculty of Medicine, Dalhousie University, Sir Charles Tupper Medical Building, Halifax, Nova Scotia, Canada B3H 1X5. Phone: (902) 494-7182. Fax: (902) 494-1355. E-mail: dobson{at}dal.ca.

Molecular and Cellular Biology, May 2005, p. 4299-4310, Vol. 25, No. 10
0022-538X/05/$08.00+0     doi:10.1128/MCB.25.10.4299-4310.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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