Reconstitution of coat protein complex II (COPII) vesicle formation from cargo-reconstituted proteoliposomes reveals the potential role of GTP hydrolysis by Sar1p in protein sorting

J Biol Chem. 2004 Jan 9;279(2):1330-5. doi: 10.1074/jbc.C300457200. Epub 2003 Nov 19.

Abstract

Secretory proteins are transported from the endoplasmic reticulum (ER) in vesicles coated with coat protein complex II (COPII). To investigate the molecular mechanism of protein sorting into COPII vesicles, we have developed an in vitro budding reaction comprising purified coat proteins and cargo reconstituted proteolipsomes. Emp47p, a type-I membrane protein, is specifically required for the transport of an integral membrane protein, Emp46p, from the ER. Recombinant Emp46/47p proteins and the ER resident protein Ufe1p were reconstituted into liposomes whose composition resembles yeast ER membranes. When the proteoliposomes were mixed with COPII proteins and GMP-PNP, Emp46/47p, but not Ufe1p, were concentrated into COPII vesicles. We also show here that reconstituted Emp47p accelerates the GTP hydrolysis by Sar1p as stimulated by its GTPase-activating protein, Sec23/24p, both of which are components of the COPII coat. Furthermore, this GTP hydrolysis decreases the error of cargo sorting. We suggest that GTP hydrolysis by Sar1p promotes exclusion of improper proteins from COPII vesicles.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • COP-Coated Vesicles / chemistry*
  • Electrophoresis, Polyacrylamide Gel
  • Endoplasmic Reticulum / metabolism
  • Escherichia coli / metabolism
  • GTPase-Activating Proteins
  • Guanosine Triphosphate / chemistry
  • Guanosine Triphosphate / metabolism
  • Hydrolysis
  • Light
  • Liposomes / metabolism
  • Membrane Proteins / chemistry
  • Membrane Proteins / metabolism
  • Microscopy, Electron
  • Plasmids / metabolism
  • Proteins / chemistry
  • Proteolipids / metabolism
  • Qa-SNARE Proteins
  • Recombinant Proteins / metabolism
  • Saccharomyces cerevisiae Proteins / chemistry
  • Saccharomyces cerevisiae Proteins / metabolism
  • Scattering, Radiation
  • Time Factors
  • Tryptophan / chemistry
  • Vesicular Transport Proteins

Substances

  • EMP46 protein, S cerevisiae
  • EMP47 protein, S cerevisiae
  • GTPase-Activating Proteins
  • Liposomes
  • Membrane Proteins
  • Proteins
  • Proteolipids
  • Qa-SNARE Proteins
  • Recombinant Proteins
  • SEC23 protein, S cerevisiae
  • SEC24 protein, S cerevisiae
  • Saccharomyces cerevisiae Proteins
  • UFE1 protein, S cerevisiae
  • Vesicular Transport Proteins
  • proteoliposomes
  • Guanosine Triphosphate
  • Tryptophan