In this study, the contributions of membrane-bound ribosomes to the regulation of endoplasmic reticulum translocon composition and Sec61␣ conformation were examined. Following solubilization of rough microsomes (RM) with digitonin, ribosomes co-sedimented in complexes containing the translocon proteins Sec61␣, ribophorin I, and TRAP␣, and endoplasmic reticulum phospholipids. Complexes of similar composition were identified in digitonin extracts of ribosome-free membranes, indicating that the

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... osome does not define the composition of the digitonin-soluble translocon. Whereas in digitonin solution a highly electrostatic ribosome-translocon junction is observed, no stable interactions between ribosomes and Sec61␣, ribophorin I, or TRAP␣ were observed following solubilization of RM with lipid-derived detergents at physiological salt concentrations. Sec61␣ was found to exist in at least two conformational states, as defined by mild proteolysis. A protease-resistant form was observed in RM and detergent-solubilized RM. Removal of peripheral proteins and ribosomes markedly enhanced the sensitivity of Sec61␣ to proteolysis, yet the readdition of inactive ribosomes to salt-washed membranes yielded only modest reductions in protease sensitivity. Addition of sublytic concentrations of detergents to salt-washed RM markedly decreased the protease sensitivity of Sec61␣, indicating that a protease-resistant conformation of Sec61␣ can be conferred in a ribosome-independent manner.