FIG. 1.

Purification of Rev1-Rev7 complex. (A) Rev1 copurifies with the Rev7 protein. N-terminal GST-Rev1 fusion protein was overexpressed in a wild-type yeast strain and was purified first on glutathione-Sepharose beads and then on an S-Sepharose column. (i) The GST-Rev1-containing protein sample was analyzed on a 10% denaturing polyacrylamide gel and stained with Coomassie blue. The positions of Rev7 present in the purified Rev1 sample (lane 1) and of purified Rev7 alone (lane 2) are indicated. The positions of molecular weight standards are indicated. (ii) Western blot analysis with anti-Rev7 antibodies to purified GST Rev1-Rev7 (lane 1) and to purified Rev7 (lane 2). (B) In vitro reconstitution of the Rev1-Rev7 complex. Purified Rev7 (9 μg) was added to the GST-Rev1-containing yeast extract (100 μg). After incubation, the reaction mixture was bound to glutathione-Sepharose beads; this was followed by washings and elution of the bound proteins with PreScission protease (2 units), which cleaves the GST tag. Fractions enriched for the Rev1-Rev7 complex were pooled, concentrated, and analyzed on an SDS-12% polyacrylamide gel and developed by Coomassie blue staining. Lane 2, Rev1 (200 ng); lane 3, Rev1-Rev7 complex (200 ng); lane 4, Rev7 (200 ng). *, unidentified but possibly heat shock proteins.