Yeast 2 µm plasmid copy number is elevated by a mutation in the nuclear gene <i>UBC4</i>

Abstract

The copy number of the Saccharomyces cerevisiae endogenous 2 microm plasmid is under strict control to ensure efficient propagation to the daughter cell without significantly reducing the growth rate of the mother or the daughter cell. A recessive mutation has been identified that resulted in an elevated but stable 2 microm plasmid copy number, which could be complemented by a genomic DNA clone containing the UBC4 gene, encoding an E2 ubiquitin-conjugating enzyme. A ubc4::URA3 deletion resulted in the same elevated 2 microm plasmid copy number. An analysis of the endogenous 2 microm transcripts revealed that the steady-state abundance of REP1, REP2, FLP and RAF were all increased 4-5-fold in the mutant. Analysis of the mutant ubc4 allele identified a single base pair mutation within the UBC4 coding region, which would generate a glutamic acid to lysine amino acid substitution within a region of conserved tertiary structure located within the first alpha-helix of Ubc4p. These investigations represent the first molecular characterization of a mutation within a Saccharomyces cerevisiae nuclear gene shown to affect 2 microm steady-state plasmid copy number and implicate the ubiquitin-dependent proteolytic pathway in host control of 2 microm plasmid copy number.