FIGURE 8.
Ecm29 and Hul5 are jointly recruited to rpt6-Δ1 proteasomes. A, proteasomes (2 μg) purified via Rpn11-TEV-protein A (43) were analyzed by 4–12% SDS-PAGE and immunoblotting with indicated antibodies. Ubp6 is a major proteasome-associated protein. Rpt4 is an ATPase subunit of the RP. B, whole cell extracts (20 μg) were subjected to 4–12% SDS-PAGE and immunoblotting (IB). These strains contain 3× HA epitope at the C terminus of Hul5, expressed from its chromosomal locus. C, genetic interactions among hul5Δ, ecm29Δ, and rpt6-Δ1. 5-fold serial dilutions of yeast strains were spotted on indicated plates and grown for 2–3 days at 30 °C. Concentrations of test compounds were 4 mm H2O2, 0.4 μg/ml 4-NQO, and 1 μg/ml canavanine.