Role of the cytoplasmic segments of Sec61 alpha in the ribosome-binding and translocation-promoting activities of the Sec61 complex

The Sec61 complex performs a dual function in protein translocation across the RER, serving as both the high affinity ribosome receptor and the transl ocation channel. To define regions of the Sec61 complex that are involved i n ribosome binding and translocation promotion, ribosome-stripped microsome s were subjected to limited digestions using proteases with different cleav age specificities. Protein immunoblot analysis using antibodies specific fo r the NH2 and COOH terminus of Sec61 alpha was used to map the location of proteolysis cleavage sites. We observed a striking correlation between the loss of binding activity for nontranslating ribosomes and the digestion of the COOH-terminal tail or cytoplasmic loop 8 of Sec61 alpha. The proteolyze d microsomes were assayed for SRP-independent translocation activity to det ermine whether high affinity binding of the ribosome to the Sec61 complex i s a prerequisite for nascent chain transport. Microsomes that do not bind n ontranslating ribosomes at physiolagical ionic strength remain active in SR P-independent translocation, indicating that the ribosome binding and trans location promotion activities of the Sec61 complex do not strictly correlat e. Translocation-promoting activity was most severely inhibited by cleavage of cytosolic loop 6, indicating that this segment is a critical determinan t for this function of the Sec61 complex.