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Differential gradients of interaction affinities drive efficient targeting and recycling in the GET pathway

Rome, Michael E. and Chio, Un Seng and Rao, Meera and Gristick, Harry and Shan, Shu-ou (2014) Differential gradients of interaction affinities drive efficient targeting and recycling in the GET pathway. Proceedings of the National Academy of Sciences of the United States of America, 111 (46). E4929-E4935. ISSN 0027-8424. PMCID PMC4246279. doi:10.1073/pnas.1411284111.

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Efficient and accurate localization of membrane proteins requires a complex cascade of interactions between protein machineries. This requirement is exemplified in the guided entry of tail-anchored (TA) protein (GET) pathway, where the central targeting factor Get3 must sequentially interact with three distinct binding partners to ensure the delivery of TA proteins to the endoplasmic reticulum (ER) membrane. To understand the molecular principles that provide the vectorial driving force of these interactions, we developed quantitative fluorescence assays to monitor Get3–effector interactions at each stage of targeting. We show that nucleotide and substrate generate differential gradients of interaction energies that drive the ordered interaction of Get3 with successive effectors. These data also provide more molecular details on how the targeting complex is captured and disassembled by the ER receptor and reveal a previously unidentified role for Get4/5 in recycling Get3 from the ER membrane at the end of the targeting reaction. These results provide general insights into how complex protein interaction cascades are coupled to energy inputs in biological systems.

Item Type:Article
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URLURL TypeDescription Information CentralArticle
Chio, Un Seng0000-0002-5295-2690
Shan, Shu-ou0000-0002-6526-1733
Additional Information:© 2014 National Academy of Sciences. Edited by Douglas C. Rees, Howard Hughes Medical Institute, Caltech, Pasadena, CA, and approved October 2, 2014 (received for review June 20, 2014). Published online before print November 3, 2014. We thank Bil Clemons for Get1/2-CD expression constructs, purification protocols, critical discussions, and sharing unpublished structural data; Bob Keenan and Manu Hegde for Get1/2-FL expression vectors; Vlad Denic for mini-Get1/2 constructs; Peter Walter for Kar2 antibody; David Akopian for help with PLs; Dennis Woo for help with yeast insertion assays; and members of the laboratory of S.-o.S. for critical comments on the manuscript. This work was supported by National Science Foundation Graduate Research Fellowship DGE-1144469 (to M.E.R.), National Institutes of Health Training Grant 5T32GM007616- 33 (to M.R.), and the David and Lucile Packard Foundation Career Award (to S.-o.S.). Author contributions: M.E.R. and S.-o.S. designed research; M.E.R., U.S.C., and M.R. performed research; M.E.R. and H.G. contributed new reagents/analytic tools; M.E.R., U.S.C., M.R., and S.-o.S. analyzed data; and M.E.R., U.S.C., and S.-o.S. wrote the paper. The authors declare no conflict of interest. This Direct Submission article had a prearranged editor. This article contains supporting information online at 1073/pnas.1411284111/-/DCSupplemental.
Funding AgencyGrant Number
NSF Graduate Research FellowshipDGE-1144469
David and Lucile Packard FoundationUNSPECIFIED
Subject Keywords:protein targeting; tail-anchored protein; ATPase; protein interaction cascades; fluorescence
Issue or Number:46
PubMed Central ID:PMC4246279
Record Number:CaltechAUTHORS:20141105-102115424
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Official Citation:Michael E. Rome, Un Seng Chio, Meera Rao, Harry Gristick, and Shu-ou Shan Differential gradients of interaction affinities drive efficient targeting and recycling in the GET pathway PNAS 2014 111 (46) E4929-E4935; published ahead of print November 3, 2014, doi:10.1073/pnas.1411284111
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:51289
Deposited By: Tony Diaz
Deposited On:06 Nov 2014 20:47
Last Modified:10 Nov 2021 19:08

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