Mechanism of Assembly of a Substrate Transfer Complex during Tail-anchored Protein Targeting

被引:11
作者
Gristick, Harry B. [1 ]
Rome, Michael E. [1 ]
Chartron, Justin W. [1 ]
Rao, Meera [1 ]
Hess, Sonja [2 ]
Shan, Shu-ou [1 ]
Clemons, William M., Jr. [1 ]
机构
[1] CALTECH, Beckman Inst, Div Chem & Chem Engn, Pasadena, CA 91125 USA
[2] CALTECH, Beckman Inst, Proteome Explorat Lab, Pasadena, CA 91125 USA
基金
美国国家卫生研究院; 美国国家科学基金会;
关键词
ENDOPLASMIC-RETICULUM; MEMBRANE INSERTION; STRUCTURAL BASIS; GET3; RECOGNITION; ELECTROSTATICS; BINDING; SYSTEM;
D O I
10.1074/jbc.M115.677328
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 angstrom 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.
引用
收藏
页码:30006 / 30017
页数:12
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