The mechanism of tail-anchored protein insertion into the ER membrane

Mol Cell. 2011 Sep 2;43(5):738-50. doi: 10.1016/j.molcel.2011.07.020. Epub 2011 Aug 11.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Vesicular Transport / chemistry
  • Adaptor Proteins, Vesicular Transport / genetics
  • Adaptor Proteins, Vesicular Transport / metabolism
  • Adenosine Triphosphatases / chemistry
  • Adenosine Triphosphatases / genetics
  • Adenosine Triphosphatases / metabolism
  • Endoplasmic Reticulum / metabolism*
  • Guanine Nucleotide Exchange Factors / chemistry
  • Guanine Nucleotide Exchange Factors / genetics
  • Guanine Nucleotide Exchange Factors / metabolism
  • Liposomes
  • Membrane Proteins / chemistry*
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Protein Transport
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / chemistry
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism

Substances

  • Adaptor Proteins, Vesicular Transport
  • GET1 protein, S cerevisiae
  • Get2 protein, S cerevisiae
  • Guanine Nucleotide Exchange Factors
  • Liposomes
  • Membrane Proteins
  • Saccharomyces cerevisiae Proteins
  • Adenosine Triphosphatases
  • Get3 protein, S cerevisiae