The Ribosome-Sec61 Translocon Complex Forms a Cytosolically Restricted Environment for Early Polytopic Membrane Protein Folding

摘要: Transmembrane topology of polytopic membrane proteins (PMPs) is established in the endoplasmic reticulum (ER) by ribosome Sec61-translocon complex (RTC) through iterative cycles translocation initiation and termination. It remains unknown, however, whether tertiary folding transmembrane domains begins after nascent polypeptide integrates into lipid bilayer or within a proteinaceous environment proximal to translocon components. To address this question, we used cysteine scanning mutagenesis monitor aqueous accessibility stalled translation intermediates determine when, during biogenesis, hydrophilic peptide loops aquaporin-4 (AQP4) water channel are delivered cytosolic lumenal compartments. Results showed that following docking on ER membrane, was shielded from cytosol as it emerged exit tunnel. Extracellular followed well defined path ribosome, junction, pore, lumen coincident with chain elongation. In contrast, intracellular (ICLs) C-terminalresidues exited cytosolically remained inaccessible both compartments until terminated. Shielding ICL1 ICL2, but not C terminus, became resistant maneuvers disrupt electrostatic interactions. Thus, early landscape shaped spatially restricted localized assembled complex.