Welcome to the new version of CaltechAUTHORS. Login is currently restricted to library staff. If you notice any issues, please email coda@library.caltech.edu
Published November 6, 2014 | Supplemental Material + Published
Journal Article Open

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


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.

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 We thank Bil Clemons for Get1/2-CD expression constructs, purification protocols, critical discussions, and sharing unpublished structural data; Bob Keenan and Manu Hegde for Get1/2-FL expression vectors; Vlad Denic for mini-Get1/2 constructs; Peter Walter for Kar2 antibody; David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 www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1411284111/-/DCSupplemental.

Attached Files

Published - E4929.full.pdf

Supplemental Material - pnas.201411284SI.pdf


Files (2.4 MB)
Name Size Download all
837.7 kB Preview Download
1.6 MB Preview Download

Additional details

August 22, 2023
October 18, 2023