FIG. 1.

Doa1 binds Ub and polyubiquitin chains. (A) Direct yeast two-hybrid interaction assay between Doa1 and Ub chains. Yeast EGY48 was transformed with plasmids expressing Doa1-LexA (DNA binding domain), Ub1-5 (transcriptional activation domain), and a lacZ reporter plasmid, patched onto HC Gal-X-Gal medium, and incubated for 2 days at 30°C. The number of linked Ub monomers, 0 to 5, is indicated. (B) Doa1 binds Ub in vitro. Cell lysate (2 mg) derived from strain Doa1-myc (FR521) was incubated in the presence of ubiquitin (200 μg) or BSA (200 μg) for 2 h at 4°C. Ubiquitin agarose was then added, followed by incubation at 4°C for 3 h. Bound proteins were separated by SDS-PAGE and subjected to anti-myc Western blotting. IP, immunoprecipitation.