Functional domains of yeast plasmid-encoded Rep proteins

Abstract

Both of the Saccharomyces cerevisiae 2 microm circle-encoded Rep1 and Rep2 proteins are required for efficient distribution of the plasmid to daughter cells during cellular division. In this study two-hybrid and in vitro protein interaction assays demonstrate that the first 129 amino acids of Rep1 are sufficient for self-association and for interaction with Rep2. Deletion of the first 76 amino acids of Rep1 abolished the Rep1-Rep2 interaction but still allowed some self-association, suggesting that different but overlapping domains specify these interactions. Amino- or carboxy-terminally truncated Rep1 fusion proteins were unable to complement defective segregation of a 2 microm-based stability vector with rep1 deleted, supporting the idea of the requirement of Rep protein interaction for plasmid segregation but indicating a separate required function for the carboxy-terminal portion of Rep1. The results of in vitro baiting assays suggest that Rep2 contains two nonoverlapping domains, both of which are capable of mediating Rep2 self-association. The amino-terminal domain interacts with Rep1, while the carboxy-terminal domain was shown by Southwestern analysis to have DNA-binding activity. The overlapping Rep1 and Rep2 interaction domains in Rep1, and the ability of Rep2 to interact with Rep1, Rep2, and DNA, suggest a model in which the Rep proteins polymerize along the 2 microm circle plasmid stability locus, forming a structure that mediates plasmid segregation. In this model, competition between Rep1 and Rep2 for association with Rep1 determines the formation or disassembly of the segregation complex.

FIG. 1

Two-hybrid assay for in vivo Rep protein interaction. Activation of the lacZ reporter in the two-hybrid yeast strain CTY10/5d transformed with plasmids expressing the LexA and Gal4_AD fusion proteins, shown on the left, was measured by a permeabilized cell assay. β-Galactosidase activities represent the averages and standard deviations obtained with four independent transformants. WT, wild type.

FIG. 2

Western blot analysis was used to confirm expression of the Rep fusion proteins in the two-hybrid host. Total protein was extracted and analyzed by SDS-PAGE and Western blotting with anti-Ga14_AD antibodies and affinity-purified anti-Rep1 (a) or anti-Rep2 (b) antibodies. Protein was from strain AS2 [cir⁰] or [cir⁺] and the two-hybrid host CTY10/5d [cir⁺] cotransformed with either pLEX-REP2 expressing the LexA-Rep2 fusion protein and pGAD424 containing either full-length or truncated versions of the REP1 ORF, allowing them to be expressed as Ga14_AD fusion proteins (a), or pLEX-REP1 and pGAD424 containing either full-length or truncated versions of the REP2 ORF (b). Protein from CTY10/5d transformed with either pLEX-REP1 (a) or pLEX-REP2 (b) (lanes 7) was also included. The positions of native Rep1 and Rep2, as well as those of Rep fusion proteins, are indicated by arrowheads. Autoradiographs were scanned using Molecular Analyst (Bio-Rad) software and prepared for figures using Adobe Photoshop. WT, wild type.

FIG. 3

In vitro baiting assay demonstrating Rep protein interaction. Rep proteins and deletion derivatives were expressed as pET fusions; shown are results with full-length Rep1, Rep1Δ1–76, and Rep1Δ130–373 (top panels) and full-length Rep2, Rep2Δ1–57, and Rep2Δ59–296 (bottom panels). The pET-Rep fusion proteins were incubated with GST-Rep1, GST-Rep2, or GST bound to glutathione-agarose beads. The input (1/10 of that added to the reaction mixtures) and bound pET fusion proteins were analyzed by SDS-PAGE and Western blotting and detected with an S protein probe and chemiluminescence. The positions of the pET-Rep fusion proteins are indicated by filled arrowheads. A caret indicates the position of GST-Rep2, which cross-reacts with the S protein probe. The autoradiograph was scanned using Ofoto (Light Source) software and prepared for figures using Adobe Photoshop. WT, wild type.

FIG. 4

Southwestern assay shows Rep2 DNA-binding activity. pET-Rep fusion proteins were purified by affinity chromatography and separated by SDS-PAGE. Triplicate gels were either stained with Coomassie blue or Western blotted and incubated with a radiolabeled DNA (either a 375-bp EcoRI-BamHI fragment from the E. coli plasmid pBR322 or a 312-bp EcoRI-HindIII fragment containing the 2μm STB-proximal locus), and the signal was detected by autoradiography. The positions of the pET-Rep fusion proteins are indicated by filled arrowheads. (A higher-molecular-weight protein that copurifies with the pET-Rep2Δ59–296 fusion protein on Talon resin is observed as a slower-migrating species on the Coomassie blue-stained gel.) WT, wild type.

FIG. 5

Summary of in vivo and in vitro Rep protein interactions. Rep fusion proteins and their truncated derivatives, tested for interaction with Rep1, Rep2, or DNA by two-hybrid, baiting, and Southwestern assays, are shown as rectangles. Gray rectangles indicate interacting fusions, while white rectangles indicate no interaction observed. The degree of interaction is indicated as strongest (+++) to weakest (+). A minus indicates no interaction.