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Fig. 3. (A) Epitope-tagged mmUbc6p and hsUbc6ep are integral membrane proteins, as is the control TRAP{alpha}. Membrane preparations were extracted with buffer containing 2.5 M urea or 800 mM potassium acetate, with Na2CO3 (pH 11.6), or with 1.0% detergent. P, pellet; S, supernatant. (B) The ubiquitin-conjugating domain of both myc-tagged mmUbc6p and hsUbc6ep is orientated to the cytosol. Protease protection assay of myc-mmUbc6 and myc-hsUbc6p and the ER-lumenal control GRP78 (Santa Cruz Biotechnology, Santa Cruz, CA) and the integral ER membrane protein TRAP{alpha}. Inside-out vesicles prepared from cells expressing the epitope-tagged Ubc6 homologues were treated with proteinase K or treated with 1% Triton X-100 and proteinase K for the indicated time. Samples were subjected to western blotting using the indicated antibodies.