Letter | Published:

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

Nature volume 512, pages 441444 (28 August 2014) | Download Citation

Abstract

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 pathway1,2,3,4. 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, insertion8,9,10,11. 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 occur12,13. 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 transmembrane domain insertion is a non sequitur. Instead, the Get1/2 transmembrane domain helps to release substrates from Get3 by capturing their transmembrane domains, 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 endoplasmic-reticulum membrane protein targeting and insertion: a mechanism for reducing the ability of a targeting factor to shield its substrates enables substrate handover to a transmembrane-domain-docking site embedded in the endoplasmic-reticulum membrane.

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Acknowledgements

We thank O. Brandman and R. Hegde for reagents, members of the Denic laboratory for scientific advice, and J. Weissman, A. Murray, B. Stern and C. Patil for comments on the manuscript. This work was supported by the National Institutes of Health (RO1GM0999943-01) and a postdoctoral fellowship from the Sara Elizabeth O’Brien Trust Postdoctoral Fellowship Program, Bank of America, Co-Trustee (to F.W.).

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Affiliations

  1. Department of Molecular and Cellular Biology, Harvard University, Northwest Labs, Cambridge, Massachusetts 02138, USA

    • Fei Wang
    • , Charlene Chan
    • , Nicholas R. Weir
    •  & Vladimir Denic

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Contributions

F.W. performed most of the experiments described in the study. C.C. analyzed Get1 cysteine N-ethylmaleimide (NEM) accessibility. N.R.W. performed cell microscopy experiments. F.W., C.C., N.R.W. and V.D. examined the data. V.D. conceived the project, guided the experiments, and wrote the paper with F.W. and input from C.C. and N.R.W.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Vladimir Denic.

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https://doi.org/10.1038/nature13471

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