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© The Rockefeller University Press, 0021-9525/2000//553 $5.00
The Journal of Cell Biology, Volume 149, Number 3, , 2000 553-566

Original Article

Partitioning of the 2-µm Circle Plasmid of Saccharomyces cerevisiae

: Functional Coordination with Chromosome Segregation and Plasmid-Encoded Rep Protein Distribution



Soundarapandian Velmurugana, Xian-Mei Yanga, Clarence S.-M. Chana, Melanie Dobsonb, and Makkuni Jayarama

a Section of Molecular Genetics and Microbiology, Institute for Cell and Molecular Biology, University of Texas at Austin, Austin, Texas 78712
b Department of Biochemistry, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7
Section of Molecular Genetics and Microbiology, Institute for Cell and Molecular Biology, University of Texas at Austin, Austin, TX 78712.(512) 471-5546(512) 471-0966

jayaram{at}icmb.utexas.edu

The efficient partitioning of the 2-µm plasmid of Saccharomyces cerevisiae at cell division is dependent on two plasmid-encoded proteins (Rep1p and Rep2p), together with the cis-acting locus REP3 (STB). In addition, host encoded factors are likely to contribute to plasmid segregation. Direct observation of a 2-µm–derived plasmid in live yeast cells indicates that the multiple plasmid copies are located in the nucleus, predominantly in clusters with characteristic shapes. Comparison to a single-tagged chromosome or to a yeast centromeric plasmid shows that the segregation kinetics of the 2-µm plasmid and the chromosome are quite similar during the yeast cell cycle. Immunofluorescence analysis reveals that the plasmid is colocalized with the Rep1 and Rep2 proteins within the yeast nucleus. Furthermore, the Rep proteins (and therefore the plasmid) tend to concentrate near the poles of the yeast mitotic spindle. Depolymerization of the spindle results in partial dispersion of the Rep proteins in the nucleus concomitant with a loosening in the association between plasmid molecules. In an ipl1-2 yeast strain, shifted to the nonpermissive temperature, the chromosomes and plasmid almost always missegregate in tandem. Our results suggest that, after DNA replication, plasmid distribution to the daughter cells occurs in the form of specific DNA-protein aggregates. They further indicate that the plasmid partitioning mechanism may exploit at least some of the components of the cellular machinery required for chromosomal segregation.

Key Words: ipl1-2 mutation • tubulin • plasmid cohesion • mitotic spindle • BRN1

© 2000 The Rockefeller University Press

Abbreviations used in this paper: [ciro], strains devoid of endogenous 2-µm circles; [cir+], strains containing 2-µm circles; DAPI, 4', 6'-diamidino-2-phenylindole; GFP, green fluorescent protein.


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