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The structural need for signalling sequence function across the endoplasmic reticulum (ER) is well understood but the mechanical basis for signal sequence function after the targeting reaction is unclear. An investigation into the role of secretory nascent chains in regulation of the ribosome-membrane junctions in the mammalian ER is presented. The study shows that formation of a protease-resistant ribosome-membrane function is stage- and precursor-specific and that the hydrophobic core of the signal sequence accesses the cystolic domain of the membrane.

Chemical cross-linking of completed translocation reaction between purified sec61p ribosome receptor and ribosomes of nuclease-treated reticulocytes was utilized to establish the identification of a novel stage of ribosome/nascent chain association with endoplasmic reticulum. Results have shown that the binding event occurs autonomously from interactions with sec61p and subsequently followed by physical interaction of the ribosome with sec61p.

An analysis of chemical cross-linking shows that ribosome-specific changes during the interaction between nascent chains and Sec61p is accompanied by translocation across the ER membrane. During translocation nascent chains are crosslinked efficiently to Sec61p through its signal sequence and mature portion. At early stages of translocation, ribosomal interaction with Sec61p mediates the nascent chain-Sec61p interactions.

The functions of the signal recognition particle (SR) and the signal sequence receptor (SSR) in protein translocation were studied. Extracts of canine pancreas rough microsome membranes were depleted of either SR or SSR and observed for activity. SR depletion resulted in loss of elongation arrest release, nascent chain targeting, protein translocation and membrane protein integration. Ribosomal binding was not affected. SSR depletiondid not show any effect on protein translocation activities, indicating that either it is not required for translocation or it is a functionally redundant component.