The 2 microm plasmid causes cell death in Saccharomyces cerevisiae with a mutation in Ulp1 protease.
The 2 microm 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 2 microm 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 2 microm circle. Segregation of the 2 microm 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 2 microm 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.
Dobson
Melanie J
MJ
Department of Biochemistry and Molecular Biology, Faculty of Medicine, Dalhousie University, Sir Charles Tupper Medical Building, Halifax, Nova Scotia, Canada B3H 1X5. dobson@dal.ca
Pickett
Andrew J
AJ
Velmurugan
Soundarapandian
S
Pinder
Jordan B
JB
Barrett
Lori A
LA
Jayaram
Makkuni
M
Chew
Joyce S K
JS
D000483:Alleles; D002455:Cell Division; D002467:Cell Nucleus / Q000378:metabolism; D049109:Cell Proliferation; D020090:Chromosome Segregation; D003546:Cysteine Endopeptidases / Q000235:genetics*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D016195:G2 Phase; D018628:Gene Dosage; D017930:Genes, Reporter / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D010641:Phenotype; D010957:Plasmids / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000201:enzymology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D017384:Sequence Deletion / Q000235:genetics; D025841:Small Ubiquitin-Related Modifier Proteins; D020798:Two-Hybrid System Techniques
D005656:Fungal Proteins; C051237:REP protein, yeast plasmid; D012097:Repressor Proteins; C099362:SMT3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D003546:Cysteine Endopeptidases; C118083:Ulp1 protease