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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.

Author information

1
From the Division of Chemistry and Chemical Engineering and.
2
The Proteome Exploration Laboratory, Beckman Institute, California Institute of Technology, Pasadena, California 91125.
3
From the Division of Chemistry and Chemical Engineering and sshan@caltech.edu.
4
From the Division of Chemistry and Chemical Engineering and clemons@caltech.edu.

Abstract

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.

KEYWORDS:

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

PMID:
26451041
PMCID:
PMC4705998
DOI:
10.1074/jbc.M115.677328
[Indexed for MEDLINE]
Free PMC Article

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