The 2 micrometer plasmid stability system: analyses of the interactions among plasmid- and host-encoded components.
The stable inheritance of the 2 micrometer 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 2 micrometer circle-derived plasmid shows relatively poor stability.
Velmurugan
S
S
Department of Microbiology and Institute of Cell and Molecular Biology, University of Texas at Austin, Austin, Texas 78712, USA.
Ahn
Y T
YT
Yang
X M
XM
Wu
X L
XL
Jayaram
M
M
D051696:Activating Transcription Factors; D002467:Cell Nucleus / Q000235:genetics; D017362:Cyclic AMP Response Element-Binding Protein / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000235:genetics*; D005455:Fluorescent Antibody Technique; D005656:Fungal Proteins / Q000235:genetics*; D005784:Gene Amplification / Q000235:genetics; D015966:Gene Expression Regulation, Fungal / Q000235:genetics; D005809:Genes, Regulator; D000074081:MutS Homolog 3 Protein; D019913:Nuclear Localization Signals / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D011506:Proteins / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D017384:Sequence Deletion / Q000235:genetics; D015534:Trans-Activators / Q000235:genetics*; D014157:Transcription Factors
D051696:Activating Transcription Factors; C410226:CST6 protein, S cerevisiae; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C089762:Msh3 protein, mouse; D000074081:MutS Homolog 3 Protein; D019913:Nuclear Localization Signals; D011506:Proteins; C051237:REP protein, yeast plasmid; C490689:REP2 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors