title: The mechanism of tail-anchored protein insertion into the ER membrane.
abstract: Tail-anchored (TA) proteins access the secretory pathway via posttranslational insertion of their C-terminal transmembrane domain into the endoplasmic reticulum (ER). Get3 is an ATPase that delivers TA proteins to the ER by interacting with the Get1-Get2 transmembrane complex, but how Get3's nucleotide cycle drives TA protein insertion remains unclear. Here, we establish that nucleotide binding to Get3 promotes Get3-TA protein complex formation by recruiting Get3 to a chaperone that hands over TA proteins to Get3. Biochemical reconstitution and mutagenesis reveal that the Get1-Get2 complex comprises the minimal TA protein insertion machinery with functionally critical cytosolic regions. By engineering a soluble heterodimer of Get1-Get2 cytosolic domains, we uncover the mechanism of TA protein release from Get3: Get2 tethers Get3-TA protein complexes into proximity with the ATPase-dependent, substrate-releasing activity of Get1. Lastly, we show that ATP enhances Get3 dissociation from the membrane, thus freeing Get1-Get2 for new rounds of substrate insertion.
authors.affiliation: Northwest Labs, Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.
authors.forename: Fei
authors.initials: F
authors.lastname: Wang
authors.forename: Andrew
authors.initials: A
authors.lastname: Whynot
authors.forename: Matthew
authors.initials: M
authors.lastname: Tung
authors.forename: Vladimir
authors.initials: V
authors.lastname: Denic
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008081:Liposomes; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; C503510:GET1 protein, S cerevisiae; C515526:Get2 protein, S cerevisiae; D020662:Guanine Nucleotide Exchange Factors; D008081:Liposomes; D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins; D000251:Adenosine Triphosphatases; C529048:Get3 protein, S cerevisiae