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Figure 2


Fig. 2. Sec63p and Kar2p co-precipitate with ER-associated proteasomes. (A) Yeast microsomes were isolated from KRY665 in which the Rpt1p subunit of the 19S RP base was tagged with protein A. Membranes were solubilized in DeoxyBigCHAP, and protein-A-tagged Rpt1p and associated proteins isolated by batch absorption to IgG-Sepharose. Protein A-Rpt1p and associated proteins were eluted from washed beads with 0.5 M acetic acid, pH 3.4 (HAc-eluate). Equivalent amounts of microsomes, lysate, material not bound to IgG-Sepharose (supernatant) and 50x HAc-eluate were separated by SDS-PAGE and individual proteins detected by immunoblotting with polyclonal antisera. The asterisk marks a band non-specifically labelled by the Rpt5 antibody. (B) Yeast microsomes were isolated from RJD1171 in which Rpt1p is FLAG tagged. Microsomes were solubilized as above and Rpt1p and associated proteins isolated by adsorption to anti-FLAG agarose. Proteins were eluted with 200 µg/ml FLAG peptide for 1 hour at 4°C. Equivalent amounts of microsomes, lysate, material not bound to anti-FLAG-agarose (supernatant) and 100x of the FLAG eluate were analyzed by SDS-PAGE and immunoblotting as above. The asterisk marks a band non-specifically labelled by the Hrd1 antibody. Note that data in the top and bottom panels are from two separate experiments.





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