The Get1/2 transmembrane complex is an endoplasmic-reticulum membrane protein insertase

• Published in: Nature (London) 2014-08, Vol.512 (7515), p.441-444
• Main Authors: Wang, Fei, Chan, Charlene, Weir, Nicholas R., Denic, Vladimir
• Format: Article
• Language: English
• Subjects: 14; 14/19; 631/337; 631/45/612/1237; 631/80/2023; 82; 82/1; 82/29; 82/80; 82/83; Adaptor Proteins, Vesicular Transport - chemistry; Adaptor Proteins, Vesicular Transport - genetics; Adaptor Proteins, Vesicular Transport - metabolism; Adenosine Triphosphatases - metabolism; Binding Sites; Endoplasmic Reticulum - chemistry; Endoplasmic Reticulum - enzymology; Endoplasmic Reticulum - metabolism; Guanine Nucleotide Exchange Factors - metabolism; Humanities and Social Sciences; Intracellular Membranes - chemistry; Intracellular Membranes - enzymology; Intracellular Membranes - metabolism; letter; Lipid Bilayers - chemistry; Lipid Bilayers - metabolism; Membrane Proteins - chemistry; Membrane Proteins - genetics; Membrane Proteins - metabolism; multidisciplinary; Multiprotein Complexes - chemistry; Multiprotein Complexes - metabolism; Mutant Proteins - chemistry; Mutant Proteins - genetics; Mutant Proteins - metabolism; Mutation; Protein Binding; Protein Structure, Tertiary - genetics; Protein Transport - genetics; Saccharomyces cerevisiae - cytology; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Science
• ISSN: 0028-0836; 1476-4687
• DOI: 10.1038/nature13471

The receptor for the cytoplasmic factor that targets tail-anchored proteins to the endoplasmic reticulum is an enzyme that enables a facilitated insertion path into the lipid bilayer. Protein insertion in the endoplasmic reticulum. Tail-anchored proteins, of which there are hundreds in a cell, are inserted into the membrane of the cellular organelle, the endoplasmic reticulum, by a protein targeting pathway. It has been suggested that the final step in this pathway may be a spontaneous mechanism, with the hydrophobic transmembrane domain of the tail-anchored protein interacting with the lipid bilayer to insert itself into the membrane. Here Vladimir Denic and colleagues reveal a facilitated insertion mechanism or insertase. The authors find that the transmembrane domain of the protein to be inserted — initially shielded from the cytoplasm by a targeting factor called Get3 — is made accessible to a membrane-embedded binding site within the Get1/2 protein complex. 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 1 , 2 , 3 , 4 . 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) 5 , 6 , 7 . 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 8 , 9 , 10 , 11 . 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 12 , 13 . 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 transmembr