Analysis of the architecture of the heterodimeric Get1-Get2 insertase and the inhibition of tail-anchored protein biogenesis by Retro-2

Abstract

Tail-anchored (TA) proteins, including SNAREs involved in vesicle transport, are post-translationally targeted to the endoplasmic reticulum membrane by the guided entry of tail-anchored proteins (GET) pathway in yeast and the transmembrane domain (TMD) recognition complex (TRC) pathway in mammals. The conserved targeting factor Get3 (in yeast; ASNA1 in mammals) binds the carboxyl-terminal TMD of TA proteins and forms a targeting complex, which engages its integral heterodimeric receptor at the ER membrane for TMD release and insertion into the lipid bilayer. I first addressed the structure and stoichiometry of the yeast Get1-Get2 complex. In vivo site-specific crosslinking and accessibility assays revealed an aqueous interface between the transmembrane regions of Get1 and Get2. To distinguish between competing models for how the Get1-Get2 heterodimer interacts with the Get3-TA targeting complex and mediates TMD insertion, I reconstituted a single-chain version of Get1-Get2 complex into proteoliposomes and nanodiscs. In vitro assays, alongside fluorescence tools, showed a single copy of the Get1-Get2 heterodimer is sufficient to recruit the Get3-TA targeting complex and insert the TMD into the ER membrane. Next, I investigated the mechanism of action of the small molecule Retro-2, which protects cells from toxins and pathogens that exploit vesicular trafficking. Genetic profiling analysis revealed that Retro-2 treatment resembles inhibition of the TRC pathway. Using dual-colour self-cleaving fluorescent reporters, I showed the compound inhibited TA protein biogenesis in mammalian cells. In vitro assays further revealed the mechanism of action in which Retro-2 blocks the transfer of newly-synthesized TA proteins from the upstream chaperone SGTA to ASNA1. These data propose a model in which Retro-2 directly inhibits ASNA1-mediated TA protein targeting, leading to the depletion of the SNAREs that mediate vesicular transport, and thus ultimately preventing the entry or replication of various pathogens.