title: The Ribosome-Sec61 Translocon Complex Forms a Cytosolically Restricted Environment for Early Polytopic Membrane Protein Folding.
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.
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239 and.
authors.forename: Melissa A
authors.initials: MA
authors.lastname: Patterson
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239 and.
authors.forename: Anannya
authors.initials: A
authors.lastname: Bandyopadhyay
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239 and.
authors.forename: Prasanna K
authors.initials: PK
authors.lastname: Devaraneni
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239 and.
authors.forename: Josha
authors.initials: J
authors.lastname: Woodward
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239 and.
authors.forename: LeeAnn
authors.initials: L
authors.lastname: Rooney
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239 and.
authors.forename: Zhongying
authors.initials: Z
authors.lastname: Yang
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239 and the Cystic Fibrosis Foundation Therapeutics (CFFT), Cystic Fibrosis Foundation, Bethesda, Maryland 20814 skachw@ohsu.edu.
authors.forename: William R
authors.initials: WR
authors.lastname: Skach
mesh_terms: D051401:Aquaporin 4 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007425:Intracellular Membranes / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012270:Ribosomes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000069816:SEC Translocation Channels
chemical_list: C496030:AQP4 protein, human; D051401:Aquaporin 4; D008565:Membrane Proteins; D000069816:SEC Translocation Channels
keywords: aquaporin; endoplasmic reticulum (ER); membrane protein; polytopic membrane protein; protein folding; protein translocation; ribosome function; ribosome translocon complex; sec61 translocon