Purification of a membrane-associated protein complex required for protein translocation across the endoplasmic reticulum.

Abstract

The capacity of microsomal membranes to translocate nascent presecretory proteins across their lipid bilayer can be largely abolished by extracting them with high ionic strength buffers. It can be reconstituted by adding the salt extract back to the depleted membranes [Warren, G. & Doberstein, B. (1978) Nature (London) 273, 569-571]. Utilizing hydrophobic chromatography, we purified to homogeneity a protein component of the salt extract that reconstitutes the translocation activity of the extracted membranes. This component behaves as a homogeneous species upon gel filtration, ion-exchange chromatography, adsorption chromatography, and sucrose-gradient centrifugation. When examined by polyacrylamide gel electrophoresis in NaDodSO4, six polypeptides with apparent Mr of 72,000, 68,000, 54,000, 19,000, 14,000, and 9000 are observed in about equal and constant stoichiometry, suggesting that they are subunits of a complex. The sedimentation coefficient of 11S is in good agreement with the sum of the Mr of the subunits. The Mr 68,000 and 9000 subunits label intensely with N-[3H]ethylmaleimide. Thus, the reported sulfhydryl group requirement of the translocation activity in the unfractionated extract [Jackson, R. C., Walter, P. & Blobel, G. (1980) Nature (London), 286, 174-176] may be localized to either or both the Mr 68,000 and 9000 subunits of the purified complex.