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J Biol Chem. 2015 Dec 11;290(50):30006-17. doi: 10.1074/jbc.M115.677328. Epub 2015 Oct 7.

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

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From the Division of Chemistry and Chemical Engineering and.
The Proteome Exploration Laboratory, Beckman Institute, California Institute of Technology, Pasadena, California 91125.
From the Division of Chemistry and Chemical Engineering and
From the Division of Chemistry and Chemical Engineering and


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 Å 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.


Saccharomyces cerevisiae; kinetics; membrane protein; protein targeting; structural biology

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