Regulation of ribosome–membrane junction at early stages of presecretory protein translocation across the endoplasmic reticulum

A series of fusion protein constructs were de-signed to investigate the contribution of secretory na-scent chains to regulation of the ribosome–membrane junction in the mammalian endoplasmic reticulum. As a component of these studies, the membrane topology of the signal sequence was determined at stages of pro-tein translocation immediately after targeting and be-fore signal sequence cleavage. Truncated translation products were used to delimit the analysis to defined stages of translocation. In a study of secretory protein precursors, formation of a protease-resistant ribosome–membrane junction, currently thought to define the pathway of the translo-cating nascent chain, was observed to be precursor- and stage-dependent. Analysis of the binding of early inter-mediates indicated that the nascent chain was bound to the membrane independent of the ribosome, and that the binding was predominately electrostatic. The mem-brane topology of the signal sequence was determined as a function of the stage of translocation, and was found to be identical for all assayed intermediates. Un-expectedly, the hydrophobic core of the signal se-quence was observed to be accessible to the cytosolic face of the membrane at stages of translocation imme-diately after targeting as well as stages before signal se-quence cleavage. Removal of the ribosome from bound intermediates did not disrupt subsequent translocation, suggesting that the active state of the protein-conduct-ing channel is maintained in the absence of the bound ribosome. A model describing a potential mode of reg-ulation of the ribosome–membrane junction by the na-scent chain is presented