Fig. 3. Sec63p does not contribute to proteasome binding to the ER. (A) Yeast 19S RP (2 pmol) were incubated in the absence or presence of 20 eq of SEC63 wild-type or sec63 T652A proteoliposomes as indicated, and binding assessed by flotation in sucrose gradients and SDS-PAGE followed by immunoblotting for the FLAG-tagged Rpn11p subunit as described for Fig. 1. The positions of proteasomes bound to membranes and of unbound proteasomes are indicated. (B) The upper panel shows a schematic drawing of the Sec61 channel and the Sec63 complex; the position of the T652A mutation in the cytosolic domain of Sec63p that disrupts the interaction with Sec62p, is indicated. Membranes from a SEC63 wild-type strain (KRY333) were solubilized in DeoxyBigCHAP and Sec63p depleted by immunoprecipitation, or lysates mock-incubated without antibodies. Depleted and mock-depleted lysates were reconstituted into proteoliposomes, and the degree of depletion of Sec63 and associated proteins assessed by quantitative immunoblotting. (Lower panel) Yeast 19S RP (2 pmol) were incubated with 15 eq of mock-depleted or Sec63p-depleted proteoliposomes as indicated, and binding assessed by flotation in sucrose gradients and SDS-PAGE followed by immunoblotting for the FLAG-tagged Rpt1p subunit.