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Crystal structure of ATP-bound Get3–Get4–Get5 complex reveals regulation of Get3 by Get4

Gristick, Harry B. and Rao, Meera and Chartron, Justin W. and Rome, Michael E. and Shan, Shu-ou and Clemons, William M., Jr. (2014) Crystal structure of ATP-bound Get3–Get4–Get5 complex reveals regulation of Get3 by Get4. Nature Structural & Molecular Biology, 21 (5). pp. 437-442. ISSN 1545-9985. PMCID PMC4386898. doi:10.1038/nsmb.2813.

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[img] Image (JPEG) (Supplementary Figure 1: Representative ITC isotherms for nucleotide-dependent complex formation.) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 2: Surface properties of Get3 and Get4) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 3: Stereo views of the Get3D-Get4–5N interface) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 4: TA targeting assay) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 5: Tetramers of Get3) - Supplemental Material
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Correct localization of membrane proteins is essential to all cells. Chaperone cascades coordinate the capture and handover of substrate proteins from the ribosomes to the target membranes, yet the mechanistic and structural details of these processes remain unclear. Here we investigate the conserved GET pathway, in which the Get4–Get5 complex mediates the handover of tail-anchor (TA) substrates from the cochaperone Sgt2 to the Get3 ATPase, the central targeting factor. We present a crystal structure of a yeast Get3–Get4–Get5 complex in an ATP-bound state and show how Get4 primes Get3 by promoting the optimal configuration for substrate capture. Structure-guided biochemical analyses demonstrate that Get4-mediated regulation of ATP hydrolysis by Get3 is essential to efficient TA-protein targeting. Analogous regulation of other chaperones or targeting factors could provide a general mechanism for ensuring effective substrate capture during protein biogenesis.

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Shan, Shu-ou0000-0002-6526-1733
Clemons, William M., Jr.0000-0002-0021-889X
Additional Information:© 2014 Macmillan Publishers Limited. Received: 28 August 2013; Accepted: 24 March 2014; Published online: 13 April 2014. We are grateful to V. Denic (Harvard University) for providing the Δget3 Δget5 yeast strain. We thank G. Card, A. Gonzalez and M. Soltis for help with data collection at the Stanford Synchrotron Radiation Lightsource (SSRL) beamline 12-2. We are grateful to G. Moore and B. Moore for support of the Molecular Observatory at the California Institute of Technology. Operations at the SSRL are supported by the US Department of Energy and US National Institutes of Health (NIH). This work was supported by career awards from the David and Lucile Packard Foundation and the Henry Dreyfus Foundation (S.S.), US National Science Foundation Graduate Research Fellowship DGE-1144469 (M.E.R.), NIH training grant 5T32GM007616-33 (H.B.G. and M.R.) and NIH research grant R01GM097572 (W.M.C. Jr.). Author Contributions: H.B.G. performed all experiments except those noted. M.R. performed Get4 inhibition studies, and M.E.R. performed translocation experiments, both supervised by S.S. J.W.C. initiated the project and aided in refinement. S.S. contributed to experimental design and interpretation. W.M.C. Jr. conceived and supervised the project. H.B.G. and W.M.C. Jr. wrote the manuscript. All authors discussed the results and implications and commented on the manuscript.
Funding AgencyGrant Number
David and Lucile Packard FoundationUNSPECIFIED
Camille and Henry Dreyfus FoundationUNSPECIFIED
NSF Graduate Research FellowshipDGE-1144469
NIH Predoctoral Fellowship5T32GM007616-33
Issue or Number:5
PubMed Central ID:PMC4386898
Record Number:CaltechAUTHORS:20140423-093035077
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:45135
Deposited By: Jason Perez
Deposited On:23 Apr 2014 17:44
Last Modified:10 Nov 2021 17:00

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