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

Abstract

Summary Hundreds of tail-anchored (TA) proteins, including SNAREs involved in vesicle fusion, are inserted post-translationally into the endoplasmic reticulum (ER) membrane by a dedicated protein targeting pathway1–4. Prior to insertion, the C-terminal transmembrane domains (TMDs) of TA proteins are shielded in the cytosol by the conserved targeting factor Get3 (in yeast; TRC40 in mammals)5–7. The Get3 ER 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 insertion8–11. Because TA 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 ER lipid bilayer and permit spontaneous insertion to occur12,13. In this study, we used cell reporters and biochemical reconstitution to define mutations in the Get1/2 transmembrane domain that disrupted TA 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 prefer to stay bound to Get3 despite their proximity to the lipid bilayer; as a consequence, spontaneous TMD insertion is non sequitur. Instead, the Get1/2 transmembrane domain helps release substrates from Get3 by capturing their TMDs 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 ER membrane protein targeting and insertion: a mechanism for reducing the ability of a targeting factor to shield its substrates enables substrate hand over to a TMD-docking site embedded in the ER membrane.

Loss of GET pathway orthologs in Arabidopsis thaliana causes root hair growth defects and affects SNARE abundance

“…2 A-D). Because both ScGET1 and HsWRB also localize to the ER membrane, this lends further support for At4g16444 being the A. thaliana GET1 ortholog (20,35). Additionally, direct in planta interaction analysis using coimmunoprecipitation mass spectrometry (CoIP-MS) of AtGET3a-GFP-expressing lines identified At4g16444 with high confidence consistently in two biological replicates among the interactors (Dataset S1).…”

“…This complex binds to the TMD and delivers the TA protein to the cytosolic ATPase GET3 (17,18). GET3 arranges as zinc-coordinating homodimer and shuttles the client protein to the endoplasmic reticulum (ER) membrane receptors GET1 and GET2, which finalize insertion of the TA protein (15,19,20).…”

Mice lacking WRB reveal differential biogenesis requirements of tail-anchored proteins in vivo

“…This receptor is thought to work as an insertase for TA proteins at the ER membrane11. Many aspects of the molecular mechanism of TRC40/Get3-mediated insertion of TA proteins have been dissected.…”

Hydrophobic handoff for direct delivery of peroxisome tail-anchored proteins

“…Hydrophobicity in this region could also cooperate with alpha-d to prompt substrate release. Interestingly, insertion of ER TA proteins was recently shown to require membrane-embedded substrate TMD-binding sites formed by the TMDs of the Get1/Get2 receptor, which act after ATP hydrolysis and GET3 conformational changes 24 . Thus, despite their many differences, a requirement for intervention at the level of the bilayer after substrate delivery to the membraneassociated receptor may be a common feature of ER and peroxisome insertion machineries.…”

“…A final step in the insertion process requires Get1/Get2 TMDs, which act as membrane embedded binding sites that capture the substrate TMD, and thereby facilitate its release from Get3 (ref. 24).…”