Ribosome-independent regulation of translocon composition and Sec61α conformation

In this study, the contributions of membrane-bound ribosomes to the regulation of endoplasmic reticulum translocon composition and Sec61 alpha conformation were examined. Following solubilization of rough microsomes (RM) with digitonin, ribosomes co-sedimented in complexes containing the translocon proteins Sec61 alpha, ribophorin I, and TRAP alpha, and endoplasmic reticulum phospholipids. Complexes of similar composition were identified in digitonin extracts of ribosome-free membranes, indicating that the ribosome 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 alpha, ribophorin I, or TRAP alpha were observed following solubilization of RM with lipid derived detergents at physiological salt concentrations. Sec61 alpha 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 alpha 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 alpha, indicating that a protease-resistant conformation of Sec61 alpha can be conferred in a ribosome-independent manner.