Abstract
Transmembrane topology of polytopic membrane proteins (PMPs) is established in the endoplasmic reticulum (ER) by the ribosome Sec61-translocon complex (RTC) through iterative cycles of translocation initiation and termination. It remains unknown, however, whether tertiary folding of transmembrane domains begins after the nascent polypeptide integrates into the lipid bilayer or within a proteinaceous environment proximal to translocon components. To address this question, we used cysteine scanning mutagenesis to monitor aqueous accessibility of stalled translation intermediates to determine when, during biogenesis, hydrophilic peptide loops of the aquaporin-4 (AQP4) water channel are delivered to cytosolic and lumenal compartments. Results showed that following ribosome docking on the ER membrane, the nascent polypeptide was shielded from the cytosol as it emerged from the ribosome exit tunnel. Extracellular loops followed a well defined path through the ribosome, the ribosome translocon junction, the Sec61-translocon pore, and into the ER lumen coincident with chain elongation. In contrast, intracellular loops (ICLs) and C-terminalresidues exited the ribosome into a cytosolically shielded environment and remained inaccessible to both cytosolic and lumenal compartments until translation was terminated. Shielding of ICL1 and ICL2, but not the C terminus, became resistant to maneuvers that disrupt electrostatic ribosome interactions. Thus, the early folding landscape of polytopic proteins is shaped by a spatially restricted environment localized within the assembled ribosome translocon complex.
Highlights
Mechanisms that guide membrane protein folding in the endoplasmic reticulum membrane remain unresolved
We showed that transmembrane segment (TM) 1, 3, and 5 encode signal anchor (SA) activity that initiates polypeptide translocation into the endoplasmic reticulum (ER) lumen, whereas TMs 2, 4, and 6 encode stop transfer (ST) activity that cotranslationally terminates translocation and redirects the growing polypeptide into the
Defining Peptide Accessibility by Cysteine Modification—In the current study, native AQP4 Cys residues were substituted with Ala and single Cys residues were systematically introduced at the N and C termini and within each intracellular loops (ICLs) and Extracellular loops (ECLs) (Fig. 1, A and B)
Summary
Mechanisms that guide membrane protein folding in the endoplasmic reticulum membrane remain unresolved. Transmembrane topology of polytopic membrane proteins (PMPs) is established in the endoplasmic reticulum (ER) by the ribosome Sec61-translocon complex (RTC) through iterative cycles of translocation initiation and termination It remains unknown, whether tertiary folding of transmembrane domains begins after the nascent polypeptide integrates into the lipid bilayer or within a proteinaceous environment proximal to translocon components. Intracellular loops (ICLs) accumulate beneath the ribosome and gain access to cytosol only after the nascent chain is released from the ribosome These data indicate that the RTC provides a cytosolically restricted environment that simultaneously accommodates multiple hydrophilic peptide regions during early stages of biogenesis and folding
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