The 2μm plasmid stability system: Analyses of the interactions among plasmid- and host-encoded components

Soundarapandian Velmurugan, Yong Tae Ahn, Xian Mei Yang, Xu Li Wu, Makkuni Jayaram

Research output: Contribution to journalArticlepeer-review

23 Scopus citations

Abstract

The stable inheritance of the 21μm 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 21μm circle-derived plasmid shows relatively poor stability.

Original languageEnglish (US)
Pages (from-to)7466-7477
Number of pages12
JournalMolecular and cellular biology
Volume18
Issue number12
DOIs
StatePublished - Dec 1998
Externally publishedYes

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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