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.

SUMMARY The Get1/2 transmembrane complex drives the insertion of tail-anchored proteins from the cytosolic chaperone Get3 into the endoplasmic reticulum membrane. Mechanistic insight into how Get1/2 coordinates this process is confounded by a lack of understanding of the basic architecture of the complex. Here we define the oligomeric state of full-length Get1/2 in reconstituted lipid bilayers by combining single-molecule and bulk fluorescence measurements with quantitative in vitro insertion analysis. We show that a single Get1/2 heterodimer is sufficient for insertion and demonstrate that the conserved cytosolic regions of Get1 and Get2 bind asymmetrically to opposing subunits of the Get3 homodimer. Taken together, our results define a simplified model for how Get1/2 and Get3 coordinate TA protein insertion.

The small molecule Retro-2 prevents ricin toxicity through a poorly-defined mechanism of action (MOA), which involves halting retrograde vesicle transport to the endoplasmic reticulum (ER). CRISPRi genetic interaction analysis revealed Retro-2 activity resembles disruption of the transmembrane domain recognition complex (TRC) pathway, which mediates post-translational ER-targeting and insertion of tail-anchored (TA) proteins, including SNAREs required for retrograde transport. Cell-based and in vitro assays show that Retro-2 blocks delivery of newly-synthesized TA-proteins to the ER-targeting factor ASNA1 (TRC40). An ASNA1 point mutant identified using CRISPR-mediated mutagenesis abolishes both the cytoprotective effect of Retro-2 against ricin and its inhibitory effect on ASNA1-mediated ER-targeting. Together, our work explains how Retro-2 prevents retrograde trafficking of toxins by inhibiting TA-protein targeting, describes a general CRISPR strategy for predicting the MOA of small molecules, and paves the way for drugging the TRC pathway to treat broad classes of viruses known to be inhibited by Retro-2.

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Demonstrating how surface chemistry and self-assembled monolayers (SAMs) control the macroscopic properties of materials is challenging as it often necessitates the use of specialized instrumentation. In this hands-on experiment, students directly measure a macroscopic property, the floatation of glass coverslips on water as a function of modifying the terminal surface groups of the glass. The glass surface is chemically modified by the self-assembly of monomolecular layers formed by two organosilanes, 3aminopropyltriethoxysilane and 1-octadecyltrimethoxysilane, which change the water contact angles. These SAMs alter the ability of the modified glass to support a mass, thus demonstrating that the bulk material property can be directly controlled by molecular surface chemistry.