title: Differential gradients of interaction affinities drive efficient targeting and recycling in the GET pathway.
abstract: Efficient and accurate localization of membrane proteins requires a complex cascade of interactions between protein machineries. This requirement is exemplified in the guided entry of tail-anchored (TA) protein (GET) pathway, where the central targeting factor Get3 must sequentially interact with three distinct binding partners to ensure the delivery of TA proteins to the endoplasmic reticulum (ER) membrane. To understand the molecular principles that provide the vectorial driving force of these interactions, we developed quantitative fluorescence assays to monitor Get3-effector interactions at each stage of targeting. We show that nucleotide and substrate generate differential gradients of interaction energies that drive the ordered interaction of Get3 with successive effectors. These data also provide more molecular details on how the targeting complex is captured and disassembled by the ER receptor and reveal a previously unidentified role for Get4/5 in recycling Get3 from the ER membrane at the end of the targeting reaction. These results provide general insights into how complex protein interaction cascades are coupled to energy inputs in biological systems.
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125.
authors.forename: Michael E
authors.initials: ME
authors.lastname: Rome
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125.
authors.forename: Un Seng
authors.initials: US
authors.lastname: Chio
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125.
authors.forename: Meera
authors.initials: M
authors.lastname: Rao
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125.
authors.forename: Harry
authors.initials: H
authors.lastname: Gristick
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125 sshan@caltech.edu.
authors.forename: Shu-ou
authors.initials: SO
authors.lastname: Shan
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D025941:Protein Interaction Mapping; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D025801:Ubiquitin / Q000378:metabolism
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; D002352:Carrier Proteins; C503510:GET1 protein, S cerevisiae; C548889:GET4 protein, S cerevisiae; C515526:Get2 protein, S cerevisiae; D020662:Guanine Nucleotide Exchange Factors; C510068:Mdy2 protein, S cerevisiae; D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C529048:Get3 protein, S cerevisiae; D003545:Cysteine
keywords: ATPase; fluorescence; protein interaction cascades; protein targeting; tail-anchored protein