LOCALIZATION AND INTERACTION OF THE PROTEIN-COMPONENTS OF THE YEAST 2-MU CIRCLE PLASMID PARTITIONING SYSTEM SUGGEST A MECHANISM FOR PLASMIDINHERITANCE

Replicating plasmids are highly unstable in yeast, because they are re tained in mother cells. The 2 mu circle plasmid overcomes this materna l inheritance bias by using a partitioning system that involves the pl asmid encoded proteins Rep1p and Rep2p, and the cis-acting locus STB. It is thus widely exploited as a cloning vehicle in yeast. However, li ttle is known about the cellular or molecular mechanisms by which effe ctive partitioning is achieved, and models of both free diffusion and plasmid localisation have been proposed. Here we show that Rep1p and R ep2p proteins interact to form homo- and hetero-complexes in vitro. In vivo, Rep1p and Rep2p are shown to be nuclear proteins, exhibiting su b-nuclear concentration in distinct foci, The number of foci appears c onstant regardless of plasmid copy number and cell ploidy level, Befor e cell division, the number of foci increases, and we observe approxim ately equal allocation of foci to mother and daughter cell nuclei. We show that whereas Rep2p expressed alone is found exclusively in the nu cleus, Rep1p requires the presence of Rep2p for effective nuclear loca lisation. High levels of 2 mu plasmid induce a multiple-budded elongat ed cell phenotype, which we show can be phenocopied by overexpression of both REP1 and REP2 together but not alone. Taken together, these re sults suggest that Rep1p and Rep2p interact in vivo, and occupy define d nuclear sites that are allocated to both mother and daughter nuclei during division. We propose a model for 2 mu m plasmid partitioning ba sed on these results, involving the association of plasmid DNA with sp ecific, segregated subnuclear sites.