BIOCHEMICAL FRACTIONATION AND ASSEMBLY OF THE MEMBRANE-COMPONENTS THAT MEDIATE NASCENT CHAIN TARGETING AND TRANSLOCATION

Fractionation of a microsomal detergent extract with ammonium sulfate allows separation of the signal recognition particle receptor (SR-alpha), which is required for targeting of the nascent chain, from other microsomal proteins, such as signal peptidase, whose activity is displayed during subsequent translocation. The reconstituted SR-alpha-enriched fraction is functional in assays of precursor targeting and elongation arrest release but lacks translocation activity. This defect can be complemented by addition, prior to reconstitution, of a separate protein subfraction. In addition, protein components necessary for translocation can be reversibly depleted from the complementary fraction, under conditions where precursor targeting is retained, by sulfhydryl-directed chromatography. Thus, precursor binding and translocation can be biochemically uncoupled, indicating that they are sequential reactions mediated by distinct components.