title: The Get1/2 transmembrane complex is an endoplasmic-reticulum membrane protein insertase.
abstract: Hundreds of tail-anchored proteins, including soluble N-ethylmaleimide-sensitive factor attachment receptors (SNAREs) involved in vesicle fusion, are inserted post-translationally into the endoplasmic reticulum membrane by a dedicated protein-targeting pathway. Before insertion, the carboxy-terminal transmembrane domains of tail-anchored proteins are shielded in the cytosol by the conserved targeting factor Get3 (in yeast; TRC40 in mammals). The Get3 endoplasmic-reticulum receptor comprises the cytosolic domains of the Get1/2 (WRB/CAML) transmembrane complex, which interact individually with the targeting factor to drive a conformational change that enables substrate release and, as a consequence, insertion. Because tail-anchored protein insertion is not associated with significant translocation of hydrophilic protein sequences across the membrane, it remains possible that Get1/2 cytosolic domains are sufficient to place Get3 in proximity with the endoplasmic-reticulum lipid bilayer and permit spontaneous insertion to occur. Here we use cell reporters and biochemical reconstitution to define mutations in the Get1/2 transmembrane domain that disrupt tail-anchored protein insertion without interfering with Get1/2 cytosolic domain function. These mutations reveal a novel Get1/2 insertase function, in the absence of which substrates stay bound to Get3 despite their proximity to the lipid bilayer; as a consequence, the notion of spontaneous transmembrane domain insertion is a non sequitur. Instead, the Get1/2 transmembrane domain helps to release substrates from Get3 by capturing their transmembrane domains, and these transmembrane interactions define a bona fide pre-integrated intermediate along a facilitated route for tail-anchor entry into the lipid bilayer. Our work sheds light on the fundamental point of convergence between co-translational and post-translational endoplasmic-reticulum membrane protein targeting and insertion: a mechanism for reducing the ability of a targeting factor to shield its substrates enables substrate handover to a transmembrane-domain-docking site embedded in the endoplasmic-reticulum membrane.
authors.affiliation: Department of Molecular and Cellular Biology, Harvard University, Northwest Labs, Cambridge, Massachusetts 02138, USA.
authors.forename: Fei
authors.initials: F
authors.lastname: Wang
authors.affiliation: Department of Molecular and Cellular Biology, Harvard University, Northwest Labs, Cambridge, Massachusetts 02138, USA.
authors.forename: Charlene
authors.initials: C
authors.lastname: Chan
authors.affiliation: Department of Molecular and Cellular Biology, Harvard University, Northwest Labs, Cambridge, Massachusetts 02138, USA.
authors.forename: Nicholas R
authors.initials: NR
authors.lastname: Weir
authors.affiliation: Department of Molecular and Cellular Biology, Harvard University, Northwest Labs, Cambridge, Massachusetts 02138, USA.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Denic
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D001665:Binding Sites; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D007425:Intracellular Membranes / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism*; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism*; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000235:genetics; D021381:Protein Transport / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; C503510:GET1 protein, S cerevisiae; C515526:Get2 protein, S cerevisiae; D020662:Guanine Nucleotide Exchange Factors; D008051:Lipid Bilayers; D008565:Membrane Proteins; D046912:Multiprotein Complexes; D050505:Mutant Proteins; D029701:Saccharomyces cerevisiae Proteins; D000251:Adenosine Triphosphatases; C529048:Get3 protein, S cerevisiae