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The structural basis of tail-anchored membrane protein recognition by Get3

Nature volume 461, pages 361–366 (2009)Cite this article

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Abstract

Targeting of newly synthesized membrane proteins to the endoplasmic reticulum is an essential cellular process. Most membrane proteins are recognized and targeted co-translationally by the signal recognition particle. However, nearly 5% of membrane proteins are ‘tail-anchored’ by a single carboxy-terminal transmembrane domain that cannot access the co-translational pathway. Instead, tail-anchored proteins are targeted post-translationally by a conserved ATPase termed Get3. The mechanistic basis for tail-anchored protein recognition or targeting by Get3 is not known. Here we present crystal structures of yeast Get3 in ‘open’ (nucleotide-free) and ‘closed’ (ADP·AlF4--bound) dimer states. In the closed state, the dimer interface of Get3 contains an enormous hydrophobic groove implicated by mutational analyses in tail-anchored protein binding. In the open state, Get3 undergoes a striking rearrangement that disrupts the groove and shields its hydrophobic surfaces. These data provide a molecular mechanism for nucleotide-regulated binding and release of tail-anchored proteins during their membrane targeting by Get3.

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Figure 1: Get3 is a dynamic, metal-stabilized homodimer.
Figure 2: A composite hydrophobic groove at the closed dimer interface.
Figure 3: Functional analysis of the hydrophobic groove.
Figure 4: The Get3 nucleotide sensor.
Figure 5: Model for TA protein targeting.

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Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors for the S. pombe Get3 and Mg2+ ADP·AlF4--bound S. cerevisiae Get3 crystal structures are deposited in the Protein Data Bank under accession codes 2WOO and 2WOJ.

Change history

  • 17 September 2009

    Author affiliations were added for A.S. and M.D. on 17 September 2009.

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Acknowledgements

Data were collected at beamlines 21-IDG and 23-IDD at the Advanced Photon Source (APS), Argonne National Laboratory, and we thank the beamline staff for support. Use of the APS was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract no. DE-AC02-06CH11357. We thank B. Glick and R. Strack for reagents and advice, X. Li for assay characterization, and A. Shiau and T. Steck for comments on the manuscript. This work was supported by a grant from the Edward Mallinckrodt, Jr. Foundation (to R.J.K.) and by the Intramural Research Program of the National Institutes of Health (to R.S.H.).

Author Contributions A.M., A.S. and M.E.D. carried out cloning, protein purification and crystallization. A.M. performed the mutagenesis. M.M. and R.S.H. performed the TA substrate-binding assay. M.D. carried out the ATPase assay. M.E.D. performed the yeast genetic complementation assay. A.M., A.S. and R.J.K. carried out data collection. A.M. and R.J.K. carried out structure determination and model building. R.J.K. supervised the work and wrote the manuscript. All authors discussed the results and commented on the manuscript.

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Authors and Affiliations

  1. Department of Biochemistry & Molecular Biology, The University of Chicago, Gordon Center for Integrative Science, Room W238, 929 East 57th Street, Chicago, Illinois 60637, USA,

    Agnieszka Mateja, Anna Szlachcic, Maureen E. Downing, Malgorzata Dobosz & Robert J. Keenan

  2. Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Room 101, Building 18T, 18 Library Drive, Bethesda, Maryland 20892, USA ,

    Malaiyalam Mariappan & Ramanujan S. Hegde

  3. Department of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, Tamka 2, 50-137 Wroclaw, Poland,

    Anna Szlachcic

  4. Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Krakow, Poland ,

    Malgorzata Dobosz

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Correspondence to Robert J. Keenan.

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Mateja, A., Szlachcic, A., Downing, M. et al. The structural basis of tail-anchored membrane protein recognition by Get3. Nature 461, 361–366 (2009). https://doi.org/10.1038/nature08319

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