STAGE-SPECIFIC AND RIBOSOME-SPECIFIC ALTERATIONS IN NASCENT CHAIN-SEC61P INTERACTIONS ACCOMPANY TRANSLOCATION ACROSS THE ER-MEMBRANE

Near-neighbor interactions between trans! locating nascent chains and Sec61p were investigated by chemical cross-linking. At stages of trans location before signal sequence cleavage, nascent chains could be cros s-linked to Sec61p at high (60-80%) efficiencies. Cross-linking occurr ed through the signal sequence and the mature portion of wild-type and signal cleavage mutant nascent chains. At later stages of translocati on, as represented through truncated translocation intermediates, cros s-linking to Sec61p was markedly reduced. Dissociation of the ribosome into its large and small subunits after assembly of the precursor int o the translocon, but before cross-linking, resulted in a dramatic red uction in subsequent crosslinking yield, indicating that at early stag es of translocation, nascent chain-Sec61p interactions are in part med iated through interactions of the ribosome with components of the ER m embrane, such as Sec61p. Dissociation of the ribosome was, however, wi thout effect on subsequent translocation. These results are discussed with respect to a model in which Sec61p performs a function essential for the initiation of protein translocation.