title: High yield production of myristoylated Arf6 small GTPase by recombinant N-myristoyl transferase.
abstract: Small GTP-binding proteins of the Arf family (Arf GTPases) interact with multiple cellular partners and with membranes to regulate intracellular traffic and organelle structure. Understanding the underlying molecular mechanisms requires in vitro biochemical assays to test for regulations and functions. Such assays should use proteins in their cellular form, which carry a myristoyl lipid attached in N-terminus. N-myristoylation of recombinant Arf GTPases can be achieved by co-expression in E. coli with a eukaryotic N-myristoyl transferase. However, purifying myristoylated Arf GTPases is difficult and has a poor overall yield. Here we show that human Arf6 can be N-myristoylated in vitro by recombinant N-myristoyl transferases from different eukaryotic species. The catalytic efficiency depended strongly on the guanine nucleotide state and was highest for Arf6-GTP. Large-scale production of highly pure N-myristoylated Arf6 could be achieved, which was fully functional for liposome-binding and EFA6-stimulated nucleotide exchange assays. This establishes in vitro myristoylation as a novel and simple method that could be used to produce other myristoylated Arf and Arf-like GTPases for biochemical assays.
authors: Laboratoire d'Enzymologie et Biochimie Structurales, Centre de Recherche de Gif (CNRS), Gif-sur-Yvette, France.
authors: Dominique
authors: D
authors: Padovani
authors: Mahel
authors: M
authors: Zeghouf
authors: José A
authors: JA
authors: Traverso
authors: Carmela
authors: C
authors: Giglione
authors: Jacqueline
authors: J
authors: Cherfils
mesh_terms: D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000217:Acyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D008081:Liposomes / Q000378:metabolism; D008958:Models, Molecular; D019814:Myristic Acid / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
chemical_list: D000089982:ADP-Ribosylation Factor 6; D008081:Liposomes; D011994:Recombinant Proteins; D019814:Myristic Acid; D006160:Guanosine Triphosphate; D000217:Acyltransferases; C050480:glycylpeptide N-tetradecanoyltransferase; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human
keywords: Arf; Arf-like; myristoylation; small GTPases