Real-time assays for the assembly-disassembly cycle of COP coats on liposomes of defined size

Methods Enzymol. 2005:404:95-107. doi: 10.1016/S0076-6879(05)04010-3.

Abstract

The assembly-disassembly cycle of COPI and COPII coats is controlled by the GTPase cycle of the small G proteins Arf1 and Sar. We describe here two spectroscopic assays that enable real-time studies of some elementary steps of coat assembly and disassembly on artificial liposomes of defined composition and curvature. A flotation assay to assess the effect of membrane curvature on protein adsorption to liposomes is also presented.

MeSH terms

  • ADP-Ribosylation Factor 1 / physiology*
  • COP-Coated Vesicles / physiology*
  • Centrifugation, Density Gradient / methods
  • Fluorescence
  • Guanine Nucleotide Exchange Factors / physiology
  • Guanosine Diphosphate / metabolism
  • Guanosine Triphosphate / metabolism
  • Light
  • Liposomes / metabolism*
  • Monomeric GTP-Binding Proteins / physiology*
  • Protein Conformation
  • Saccharomyces cerevisiae Proteins / physiology*
  • Scattering, Radiation
  • Vesicular Transport Proteins

Substances

  • Guanine Nucleotide Exchange Factors
  • Liposomes
  • Saccharomyces cerevisiae Proteins
  • Vesicular Transport Proteins
  • Guanosine Diphosphate
  • Guanosine Triphosphate
  • ADP-Ribosylation Factor 1
  • Monomeric GTP-Binding Proteins
  • SAR1 protein, S cerevisiae