Velmurugan S, Ahn Y T, Yang X M, Wu X L, Jayaram M
Department of Microbiology and Institute of Cell and Molecular Biology, University of Texas at Austin, Austin, Texas 78712, USA.
Mol Cell Biol. 1998 Dec;18(12):7466-77. doi: 10.1128/MCB.18.12.7466.
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.
2 微米质粒在酿酒酵母生长群体中的稳定遗传依赖于两种质粒编码蛋白（Rep1p 和 Rep2p）以及顺式作用位点 REP3（STB）。在本研究中，我们证明 Rep1p 和 Rep2p 的短羧基末端缺失严重削弱了它们正常定位于酵母细胞核的能力。通过在缩短的 Rep 蛋白的氨基或羧基末端添加核定位序列，可以恢复核靶向及其在质粒分配中的功能作用。利用酵母体内双杂交遗传试验以及体外谷胱甘肽 S-转移酶融合捕获试验对 Rep 蛋白的缺失衍生物进行分析，结果表明 Rep1p 的氨基末端部分（约 150 个氨基酸长）负责其与 Rep2p 的相互作用。在单杂交体内试验中，我们确定 Rep1p、Rep2p 和一种宿主编码蛋白 Shf1p 能够与 STB 位点相互作用。在大肠杆菌中表达的 Shf1 蛋白在体外能与 STB 序列高度特异性结合。在缺失 SHF1 位点的酵母菌株中，2 微米环状质粒显示出相对较差的稳定性。
