title: Mechanism of Assembly of a Substrate Transfer Complex during Tail-anchored Protein Targeting.
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.
authors.affiliation: From the Division of Chemistry and Chemical Engineering and.
authors.forename: Harry B
authors.initials: HB
authors.lastname: Gristick
authors.affiliation: From the Division of Chemistry and Chemical Engineering and.
authors.forename: Michael E
authors.initials: ME
authors.lastname: Rome
authors.affiliation: From the Division of Chemistry and Chemical Engineering and.
authors.forename: Justin W
authors.initials: JW
authors.lastname: Chartron
authors.affiliation: From the Division of Chemistry and Chemical Engineering and.
authors.forename: Meera
authors.initials: M
authors.lastname: Rao
authors.affiliation: The Proteome Exploration Laboratory, Beckman Institute, California Institute of Technology, Pasadena, California 91125.
authors.forename: Sonja
authors.initials: S
authors.lastname: Hess
authors.affiliation: From the Division of Chemistry and Chemical Engineering and sshan@caltech.edu.
authors.forename: Shu-ou
authors.initials: SO
authors.lastname: Shan
authors.affiliation: From the Division of Chemistry and Chemical Engineering and clemons@caltech.edu.
authors.forename: William M
authors.initials: WM
authors.lastname: Clemons
mesh_terms: D000595:Amino Acid Sequence; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D055672:Static Electricity; D013379:Substrate Specificity
chemical_list: D029701:Saccharomyces cerevisiae Proteins
keywords: Saccharomyces cerevisiae; kinetics; membrane protein; protein targeting; structural biology