The 2 mu m-plasmid-encoded Rep1 and Rep2 proteins interact with each other and colocalize to the Saccharomyces cerevisiae nucleus

The efficient partitioning of the 2 mu m plasmid of Saccharomyces cerevisiae at cell division requires two plasmid-encoded proteins (Rep1p and Rep2p) and a cia-acting locus, REP3 (STB). By using protein hybrids containing fusions of the Rep proteins to green fluorescent protein (GFP), we show here that fluorescence from GFP-Rep1p or GFP-Rep2p is almost exclusively localized in the nucleus in a cir(+) strain. Nuclear localization of GFP-Rep1p and GFP Rep2p, though discernible, is less efficient in a cir(0) host. GFP-Rep2p or GFP-Rep1p is able to promote the stability of a 2 mu m circle-derived plasmid harboring REP1 or REP2, respectively, in a cir(0) background. Under these conditions, fluorescence from GFP-Rep2p or GFP-Rep1p is concentrated within the nucleus, as is the case in cir(+) cells. This characteristic nuclear accumulation is not dependent on the expression of the FLP or RAF1 gene of the 2 mu m circle. Nuclear colocalization of Rep1p and Rep2p is consistent with the hypothesis that the two proteins directly or indirectly interact to form a functional bipartite or high-order protein complex. Immunoprecipitation experiments as well as baiting assays using GST-Rep hybrid proteins suggest a direct interaction between Rep1p and Rep2p which, in principle, may be modulated by other yeast proteins. Furthermore, these assays provide evidence for Rep1p-Rep1p and Rep2p-Rep2p associations as well. The sum of these interactions may be important in controlling the effective cellular concentration of the Rep1p-Rep2p complex.