In vitro assays of Arf1 interaction with GGA proteins

Methods Enzymol. 2005:404:316-32. doi: 10.1016/S0076-6879(05)04028-0.

Abstract

ADP-ribosylation factor 1 (Arf1) is a GTP-binding protein that regulates membrane traffic. This function of Arf1 is, at least in part, mediated by Arf1 x GTP binding to coat proteins such as coatomer, clathrin adaptor protein (AP) complexes 1 and 3, and gamma-adaptin homology-Golgi associated Arf-binding (GGA) proteins. Binding to Arf1 x GTP recruits these coat proteins to membranes, leading to the formation of transport vesicles. Whereas coatomer and the AP complexes are hetero-oligomers, GGAs are single polypeptide chains. Therefore, working with recombinant GGAs is straightforward compared to the other Arf1 effectors. Consequently, the GGAs have been used as a model for studying Arf1 interactions with effectors and as reagents to determine Arf1 x GTP levels in cells. In this chapter, we describe in vitro assays for analysis of GGA interaction with Arf1 x GTP and for determining intracellular Arf1 x GTP levels.

MeSH terms

  • ADP-Ribosylation Factor 1 / analysis
  • ADP-Ribosylation Factor 1 / antagonists & inhibitors
  • ADP-Ribosylation Factor 1 / metabolism*
  • ADP-Ribosylation Factors / analysis
  • ADP-Ribosylation Factors / metabolism*
  • Adaptor Proteins, Vesicular Transport / analysis
  • Adaptor Proteins, Vesicular Transport / metabolism*
  • Animals
  • Cattle
  • Guanosine 5'-O-(3-Thiotriphosphate) / metabolism
  • Guanosine Triphosphate / metabolism
  • Humans
  • Phosphorus Radioisotopes
  • Protein Interaction Mapping
  • Protein Structure, Tertiary
  • Recombinant Fusion Proteins / biosynthesis
  • Sulfur Radioisotopes

Substances

  • Adaptor Proteins, Vesicular Transport
  • GGA adaptor proteins
  • Phosphorus Radioisotopes
  • Recombinant Fusion Proteins
  • Sulfur Radioisotopes
  • Guanosine 5'-O-(3-Thiotriphosphate)
  • Guanosine Triphosphate
  • ADP-Ribosylation Factor 1
  • ADP-Ribosylation Factors