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Published December 11, 2015 | public
Journal Article Open

Mechanism of assembly of a substrate-transfer complex during tail-anchored protein targeting


Tail-anchored (TA) proteins, defined as having a single transmembrane helix at their C terminus, are post-translationally targeted to the endoplasmic reticulum membrane by the guided entry of TA proteins (GET) pathway. In yeast, the handover of TA substrates is mediated by the heterotetrameric Get4/Get5 complex (Get4/5), which tethers the co-chaperone Sgt2 to the targeting factor, the Get3 ATPase. Binding of Get4/5 to Get3 is critical for efficient TA targeting; however, questions remain about the formation of the Get3·Get4/5 complex. Here we report crystal structures of a Get3·Get4/5 complex from Saccharomyces cerevisiae at 2.8 and 6.0 Å that reveal a novel interface between Get3 and Get4 dominated by electrostatic interactions. Kinetic and mutational analyses strongly suggest that these structures represent an on-pathway intermediate that rapidly assembles and then rearranges to the final Get3·Get4/5 complex. Furthermore, we provide evidence that the Get3·Get4/5 complex is dominated by a single Get4/5 heterotetramer bound to one monomer of a Get3 dimer, uncovering an intriguing asymmetry in the Get4/5 heterotetramer upon Get3 binding. Ultrafast diffusion-limited electrostatically driven Get3·Get4/5 association enables Get4/5 to rapidly sample and capture Get3 at different stages of the GET pathway.

Additional Information

© 2015 The American Society for Biochemistry and Molecular Biology, Inc. Received July 7, 2015; Revision received October 2, 2015; First Published on October 7, 2015. This work was supported by National Science Foundation Graduate Research Fellowship DGE-1144469 (to M. E. R.) and National Institutes of Health Training Grant 5T32GM007616-33 (to H. B. G. and M. R.) and Research Grants R01GM097572 (to W. M. C.) and R01GM107368 (to S. S.). The Proteome Exploration Laboratory was supported by Gordon and Betty Moore Foundation Grant GBMF775 and the Beckman Institute (to S. H.). The atomic coordinates and structure factors (codes 5BW8 and 5BWK) have been deposited in the Protein Data Bank (http://wwpdb.org/). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. We thank Graeme Card, Ana Gonzalez, and Michael Soltis for help with data collection at Stanford Synchrotron Radiation Lightsource Beamline 12-2. We are grateful to Gordon and Betty Moore for support of the Molecular Observatory at Caltech. Operations at Stanford Synchrotron Radiation Lightsource are supported by the United States Department of Energy and National Institutes of Health. Author Contributions: H. B. G., M. E. R., S. S., and W. M. C. designed the study and wrote the paper. H. B. G. determined the 2.8-Å crystal structure and characterized the intermediate complex. M. E. R. performed experiments to define stoichiometry and kinetic analysis. J. W. C. determined the structure of the 6.0-Å complex. M. R. helped conceptually with the study. S. H. assisted in the mass spectrometry analysis. All authors reviewed the results and approved the final version of the manuscript.

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Published - J._Biol._Chem.-2015-Gristick-30006-17.pdf


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August 20, 2023
August 20, 2023