Model for eukaryotic tail-anchored protein binding based on the structure of Get3

Abstract

The Get3 ATPase directs the delivery of tail-anchored (TA) proteins to the endoplasmic reticulum (ER). TA-proteins are characterized by having a single transmembrane helix (TM) at their extreme C terminus and include many essential proteins, such as SNAREs, apoptosis factors, and protein translocation components. These proteins cannot follow the SRP-dependent co-translational pathway that typifies most integral membrane proteins; instead, post-translationally, these proteins are recognized and bound by Get3 then delivered to the ER in the ATP dependent Get pathway. To elucidate a molecular mechanism for TA protein binding by Get3 we have determined three crystal structures in apo and ADP forms from Saccharomyces cerevisae (ScGet3-apo) and Aspergillus fumigatus (AfGet3-apo and AfGet3-ADP). Using structural information, we generated mutants to confirm important interfaces and essential residues. These results point to a model of how Get3 couples ATP hydrolysis to the binding and release of TA-proteins.

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©2009 by the National Academy of Sciences. Communicated by Douglas C. Rees, California Institute of Technology, Pasadena, CA, July 8, 2009 (received for review June 24, 2009). Published online before print August 14, 2009, doi: 10.1073/pnas.0907522106 We thank S. Shan and A. Palazzo for discussion and critical comments on the manuscript and Gordon and Betty Moore for support of the Molecular Observatory at Caltech. All data collection was performed at beamline 12-2 at Stanford Synchrotron Radiation Lightsource and we thank G. Card and M. Soltis for beamline assistance. Operations at SSRL are supported by the U.S. Department of Energy and National Institutes of Health. W.M.C. is supported by the Searle Scholar program and a Burroughs-Wellcome Fund Career Award for the Biological Sciences. Author contributions: C.J.M.S. and W.M.C. designed research; C.J.M.S. performed research; C.J.M.S. and M.Z. contributed new reagents/analytic tools; C.J.M.S., J.W.C., and W.M.C. analyzed data; and C.J.M.S., J.W.C., and W.M.C. wrote the paper. The authors declare no conflict of interest. Data deposition: The atomic coordinates and structure factors have been deposited in the RCSB Protein Data Bank, www.pdb.org (PDB ID codes 31BG for AfGet3-ADP and 31DQ for ScGet3). This article contains supporting information online at www.pnas.org/cgi/content/full/0907522106/DCSupplemental.

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Supplemental Material - 0907522106SI.pdf

Published - Suloway2009p5850P_Natl_Acad_Sci_Usa.pdf

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