Liposomes in the Study of GDP⧸GTP Cycle of Arf and Related Small G Proteins

The liposomes of submicrometer size and defined composition provide a minimal system by which the membrane confinement of proteins through specific and nonspecific interactions with lipids can be mimicked in the test tube. Several steps of the ADP-ribosylation factor/SAR1A (Arf/Sar) cycle have been reconstituted using liposomes and purified proteins. By a combination of biochemical and morphological approaches, the guanosine-5’-triphosphate (GTP)-dependent translocation of Arf and Sar1 to membrane lipids, the subsequent recruitment of coat complexes, and the deformation of membranes induced by these polymeric structures can be analyzed. Additionally, several spectroscopic methods have been developed to monitor some reactions in real time. Tryptophan fluorescence allows the monitoring of the conversion of Arf or Sar1 between guanosine diphosphate (GDP)- and GTP-bound forms. Fluorescence resonance energy transfer (FRET) between tryptophan residues and a fluorescent lipid analog, such as diphenyl hexatriene-phosphatidylcholine (DPH-PC), allows monitoring of the translocation of Arf1 on liposomes. Light scattering allows monitoring of the assembly of protein coats on liposomes. These methods together with a liposome sedimentation assay are described in this chapter. The emphasis is on Arf and Sar, although several experiments with Rac, a member of the Rho family, are also presented.