Analysis of Arf GTP-binding protein function in cells.
This unit describes techniques and approaches that can be used to study the functions of the ADP-ribosylation factor (Arf) GTP-binding proteins in cells. There are six mammalian Arfs and many more Arf-like proteins (Arls), and these proteins are conserved in eukaryotes from yeast to humans. Like all GTPases, Arfs cycle between GDP-bound, inactive and GTP-bound active conformations, facilitated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) that catalyze GTP binding and hydrolysis, respectively. This unit describes approaches that can be taken to examine the localization and function of Arf and Arl proteins in cells. A simple protocol for measuring activation (GTP-binding) of specific Arf proteins in cells using a pull-down assay is also described. Approaches that can be taken to assess function of GEFs and GAPs in cells is described.
Cohen
Lee Ann
LA
Laboratory of Cell Biology, NHLBI, National Institutes of Health, Bethesda, Maryland, USA.
Donaldson
Julie G
JG
D020727:ADP-Ribosylation Factors / Q000378:metabolism*; D000818:Animals; D018929:Cell Culture Techniques; D006801:Humans; D008967:Molecular Biology / Q000379:methods*; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport
D020727:ADP-Ribosylation Factors; D020559:Monomeric GTP-Binding Proteins