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The mechanism of membrane-associated steps in tail-anchored protein insertion

Abstract

Tail-anchored (TA) membrane proteins destined for the endoplasmic reticulum are chaperoned by cytosolic targeting factors that deliver them to a membrane receptor for insertion. Although a basic framework for TA protein recognition is now emerging, the decisive targeting and membrane insertion steps are not understood. Here we reconstitute the TA protein insertion cycle with purified components, present crystal structures of key complexes between these components and perform mutational analyses based on the structures. We show that a committed targeting complex, formed by a TA protein bound to the chaperone ATPase Get3, is initially recruited to the membrane through an interaction with Get2. Once the targeting complex has been recruited, Get1 interacts with Get3 to drive TA protein release in an ATPase-dependent reaction. After releasing its TA protein cargo, the now-vacant Get3 recycles back to the cytosol concomitant with ATP binding. This work provides a detailed structural and mechanistic framework for the minimal TA protein insertion cycle.

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Figure 1: Reconstitution of TA protein insertion with purified components.
Figure 2: Get2 fragment complex with ADP·AlF 4 -bound Get3.
Figure 3: Get1 fragment complex with Get3.
Figure 4: Mutational analysis of the function of Get1, Get2 and Get3.
Figure 5: ATP-dependent recycling of empty Get3 from Get1.
Figure 6: Model for TA protein insertion.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors for S. cerevisiae Get3 in complex with Get1(21–104) and for 2+-ADP·AlF4 2-bound S. cerevisiae Get3 in complex with Get2(1–38) have been deposited in the Protein Data Bank under accession codes 3ZS8 and 3ZS9, respectively.

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Acknowledgements

Data were collected at beamline 21-IDG at the Advanced Photon Source (APS), Argonne National Laboratory, and we thank the beamline staff for support. We thank T. Dever for yeast strains, T. Rapoport for the Sec61α antibody, M. Downing for technical assistance, members of the Hegde, Keenan and E. Perozo labs and D. Freymann for advice, and A. Shiau and S. Shao for discussions and comments on the manuscript. Use of the APS, an Office of Science User Facility operated for the US Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported by the US DOE under contract no. DE-AC02-06CH11357. This work was supported by the Intramural Research Program of the NIH (to R.S.H.), the Camille and Henry Dreyfus Postdoctoral Program in Environmental Chemistry (to R.J.K. and E.B.), an Edward Mallinckrodt, Jr. Foundation Grant (to R.J.K.) and NIH Grant R01 GM086487 (to R.J.K.).

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Authors and Affiliations

Authors

Contributions

A.M., M.D. and E.B. produced, purified and characterized recombinant Get1, Get2 (full length and fragments) and Get3. M.M. and R.S.H. performed the reconstitution experiments, including the substrate release and membrane insertion assays. A.M., M.D. and R.J.K. carried out crystallization and structure determination as well as the interaction analyses. R.S.H. and R.J.K. designed the project. M.M., R.S.H. and R.J.K. wrote the paper. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Ramanujan S. Hegde or Robert J. Keenan.

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The authors declare no competing financial interests.

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This file contains Supplementary Figures 1-20 with legends, Supplementary Table 1, a Supplementary Discussion, Supplementary Notes and Supplementary References. (PDF 16767 kb)

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Mariappan, M., Mateja, A., Dobosz, M. et al. The mechanism of membrane-associated steps in tail-anchored protein insertion. Nature 477, 61–66 (2011). https://doi.org/10.1038/nature10362

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