THE 2-MU-M PLASMID STABILITY SYSTEM - ANALYSES OF THE INTERACTIONS AMONG PLASMID-ENCODED AND HOST-ENCODED COMPONENTS

The stable inheritance of the 2 mu m plasmid in a growing population o f Saccharomyces cerevisiae is dependent on two plasmid encoded protein s (Rep1p and Rep2p), together with the cis-acting locus REP3 (STB). In thisstudy we demonstrate that short carboxy-terminal deletions of Rep 1p and Rep2p severely diminish their normal capacity to localize to th e yeast nucleus. The nuclear targeting, as well as their functional ro le in plasmid partitioning, can be restored by the addition of a nucle ar localization sequence to the amino or the carboxy terminus of the s hortened Rep proteins. Analyses of deletion derivatives of the Rep pro teins by using the in vivo dihybrid genetic test in yeast, as well as by glutathione S-transferase fusion trapping assays in vitro demonstra te that the amino-terminal portion of Rep1p (ca, 150 amino acids long) is responsible for its interactions with Rep2p. In a monohybrid in vi vo assay, we have identified Rep1p, Rep2p, and a host-encoded protein, Shf1p, as being capable of interacting,vith the STB locus. The Shf1 p rotein expressed in Escherichia coli can bind with high specificity to the STB sequence in vitro. In a yeast strain deleted for the SHF1 loc us, a 2 mu m circle-derived plasmid shows relatively poor stability.