Proteasome 19S particle binding to wildtype, sec61Y345H and sec61∆L7 proteoliposomes. Microsomes from wildtype or sec61 mutant yeast were prepared, stripped of ribosomes, solubilized, and total protein reconstituted into proteoliposomes. Membranes were incubated with purified 19S particles in the presence of 5 mM ATP, and samples analyzed by flotation in 1.8 M sucrose for 1 h at 200,000 g. Fractions were collected from the top and analyzed by SDS-PAGE and immunoblotting for Sec61p and the 19S subunit Rpn12p. Rpn12p in each fraction was quantified. Note that 19S particles in the absence of membranes (green) remain at the bottom of the gradient. YTX69 is the standard wildtype yeast strain used in the lab for proteasome and ribosome binding experiments; SEC61, sec61Y345H, and sec61∆L7 were all in the KRY461 background.
Tretter et al. BMC Cell Biology 2013 14:56 doi:10.1186/1471-2121-14-56