The 2μm Plasmid Stability System: Analyses of the Interactions among Plasmid- and Host-Encoded Components

ABSTRACT

The stable inheritance of the 2μm plasmid in a growing population of Saccharomyces cerevisiae is dependent on two plasmid-encoded proteins (Rep1p and Rep2p), together with the cis-acting locus REP3 (STB). In this study we demonstrate that short carboxy-terminal deletions of Rep1p and Rep2p severely diminish their normal capacity to localize to the yeast nucleus. The nuclear targeting, as well as their functional role in plasmid partitioning, can be restored by the addition of a nuclear localization sequence to the amino or the carboxy terminus of the shortened Rep proteins. Analyses of deletion derivatives of the Rep proteins by using the in vivo dihybrid genetic test in yeast, as well as by glutathione S-transferase fusion trapping assays in vitro demonstrate that the amino-terminal portion of Rep1p (ca. 150 amino acids long) is responsible for its interactions with Rep2p. In a monohybrid in vivo assay, we have identified Rep1p, Rep2p, and a host-encoded protein, Shf1p, as being capable of interacting with the STB locus. The Shf1 protein expressed in Escherichia coli can bind with high specificity to the STB sequence in vitro. In a yeast strain deleted for the SHF1 locus, a 2μm circle-derived plasmid shows relatively poor stability.

ACKNOWLEDGMENT

This work was supported by a grant from the Council for Tobacco Research.

ADDENDUM IN PROOF

Scott-Drew and Murray (S. Scott-Drew and J. A. Murray, J. Cell Sci. 111:1779–1789, 1998) have recently demonstrated that Rep1p and Rep2p form a complex. By confocal microscopy they have further demonstrated that the proteins occupy specific sites in the nucleus and that these sites are distributed to both mother and daughter cells during cell division.