Analysis of the architecture of the heterodimeric Get1-Get2 insertase and the inhibition of tail-anchored protein biogenesis by Retro-2
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Chan, Charlene Jun-Zhi
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CitationChan, Charlene Jun-Zhi. 2019. Analysis of the architecture of the heterodimeric Get1-Get2 insertase and the inhibition of tail-anchored protein biogenesis by Retro-2. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences.
AbstractTail-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.
Citable link to this pagehttp://nrs.harvard.edu/urn-3:HUL.InstRepos:42029646
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