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Molecular and Cellular Biology, December 1998, p. 7466-7477, Vol. 18, No. 12
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

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, and Makkuni Jayaram*

Department of Microbiology and Institute of Cell and Molecular Biology, University of Texas at Austin, Austin, Texas 78712

Received 9 June 1998/Returned for modification 27 July 1998/Accepted 24 August 1998

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.

* Corresponding author. Mailing address: Department of Microbiology, University of Texas at Austin, Austin, TX 78712. Phone: (512) 471-0966. Fax: (512) 471-5546. E-mail: jayaram{at}almach.cc.utexas.edu.

Molecular and Cellular Biology, December 1998, p. 7466-7477, Vol. 18, No. 12
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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