ArfGAP1 generates an Arf1 gradient on continuous lipid membranes displaying flat and curved regions

EMBO J. 2010 Jan 20;29(2):292-303. doi: 10.1038/emboj.2009.341. Epub 2009 Nov 19.

Abstract

ArfGAP1, which promotes GTP hydrolysis on the small G protein Arf1 on Golgi membranes, interacts preferentially with positively curved membranes through its amphipathic lipid packing sensor (ALPS) motifs. This should influence the distribution of Arf1-GTP when flat and curved regions coexist on a continuous membrane, notably during COPI vesicle budding. To test this, we pulled tubes from giant vesicles using molecular motors or optical tweezers. Arf1-GTP distributed on the giant vesicles and on the tubes, whereas ArfGAP1 bound exclusively to the tubes. Decreasing the tube radius revealed a threshold of R approximately 35 nm for the binding of ArfGAP1 ALPS motifs. Mixing catalytic amounts of ArfGAP1 with Arf1-GTP induced a smooth Arf1 gradient along the tube. This reflects that Arf1 molecules leaving the tube on GTP hydrolysis are replaced by new Arf1-GTP molecules diffusing from the giant vesicle. The characteristic length of the gradient is two orders of magnitude larger than a COPI bud, suggesting that Arf1-GTP diffusion can readily compensate for the localized loss of Arf1 during budding and contribute to the stability of the coat until fission.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ADP-Ribosylation Factor 1 / analysis
  • ADP-Ribosylation Factor 1 / metabolism*
  • Amino Acid Motifs
  • Coat Protein Complex I / analysis
  • Coat Protein Complex I / metabolism*
  • Diffusion
  • GTPase-Activating Proteins / analysis
  • GTPase-Activating Proteins / metabolism*
  • Golgi Apparatus / metabolism
  • Membrane Lipids / analysis
  • Membrane Lipids / metabolism*
  • Optical Tweezers
  • Protein Binding
  • Unilamellar Liposomes / analysis
  • Unilamellar Liposomes / metabolism*

Substances

  • Coat Protein Complex I
  • GTPase-Activating Proteins
  • Membrane Lipids
  • Unilamellar Liposomes
  • ADP-Ribosylation Factor 1