Reconstitution of cargo-dependent COPII coat assembly on proteoliposomes

Methods Enzymol. 2005;404:83-94. doi: 10.1016/S0076-6879(05)04009-7.

Abstract

The coat protein complex II (COPII) coat is responsible for direct capture of membrane cargo proteins and for the physical deformation of the endoplasmic reticulum (ER) membrane that drives the transport vesicle formation. The use of an in vitro reconstitution system comprising purified components is desirable for studies aimed at elucidating the role(s) of individual proteins in a specific biochemical reaction. To investigate the assembly-disassembly of COPII coats in a completely reconstituted reaction, we have developed a synthetic budding reaction involving purified coat proteins and cargo-reconstituted proteoliposomes. We describe here a fluorescence resonance energy transfer (FRET)-based method for monitoring the kinetics of COPII coat complex assembly and disassembly in cargo-reconstituted proteoliposomes. This assay allows comparison of the time course of the coat disassembly from the cargo as monitored by FRET signal with the time course of accompanying Sar1p GTP hydrolysis by tryptophan fluorescence.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • COP-Coated Vesicles / physiology*
  • Fluorescence
  • Fluorescence Resonance Energy Transfer
  • Gene Expression
  • Green Fluorescent Proteins / biosynthesis
  • Kinetics
  • Liposomes / metabolism
  • Monomeric GTP-Binding Proteins / metabolism
  • Proteolipids / physiology*
  • Qc-SNARE Proteins / biosynthesis
  • R-SNARE Proteins / biosynthesis
  • SNARE Proteins
  • Saccharomyces cerevisiae Proteins / biosynthesis
  • Saccharomyces cerevisiae Proteins / metabolism
  • Tryptophan / chemistry
  • Vesicular Transport Proteins

Substances

  • BET1 protein, S cerevisiae
  • Cyan Fluorescent Protein
  • Liposomes
  • Proteolipids
  • Qc-SNARE Proteins
  • R-SNARE Proteins
  • SNARE Proteins
  • Saccharomyces cerevisiae Proteins
  • Sec22 protein, S cerevisiae
  • Vesicular Transport Proteins
  • proteoliposomes
  • Green Fluorescent Proteins
  • Tryptophan
  • Monomeric GTP-Binding Proteins
  • SAR1 protein, S cerevisiae