title: The myristoylated amino terminus of ADP-ribosylation factor 1 is a phospholipid- and GTP-sensitive switch.
abstract: ADP-ribosylation factor 1 (Arf1) is an essential N-myristoylated 21-kDa GTP-binding protein with activities that include the regulation of membrane traffic and phospholipase D activity. Both the N terminus of the protein and the N-myristate bound to glycine 2 have previously been shown to be essential to the function of Arf in cells. We show that the bound nucleotide affects the conformation of either the N terminus or residues of Arf1 that are in direct contact with the N terminus. This was demonstrated by examining the effects of mutations in this N-terminal domain on guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) and GDP binding and dissociation kinetics. Arf1 mutants, lacking 13 or 17 residues from the N terminus or mutated at residues 3-7, had a greater affinity for GTP gamma S and a lower affinity for GDP than did the wild-type protein. As the N terminus is required for interactions with target proteins, we conclude that the N terminus of Arf1 is a GTP-sensitive effector domain. When Arf1 was acylated, the GTP-dependent conformational changes were codependent on added phospholipids. In the absence of phospholipids, myristoylated Arf1 has a lower affinity for GTP gamma S than for GDP, and in the presence of phospholipids, the myristoylated protein has a greater affinity for GTP gamma S than for GDP. Thus, N-myristoylation is a critical component in the construction of this phospholipid- and GTP-dependent switch.
authors.affiliation: Laboratory of Biological Chemistry, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: P A
authors.initials: PA
authors.lastname: Randazzo
authors.forename: T
authors.initials: T
authors.lastname: Terui
authors.forename: S
authors.initials: S
authors.lastname: Sturch
authors.forename: H M
authors.initials: HM
authors.lastname: Fales
authors.forename: A G
authors.initials: AG
authors.lastname: Ferrige
authors.forename: R A
authors.initials: RA
authors.lastname: Kahn
mesh_terms: D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D000215:Acylation; D000595:Amino Acid Sequence; D002460:Cell Line; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D019814:Myristic Acid; D009227:Myristic Acids; D010743:Phospholipids / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D009227:Myristic Acids; D010743:Phospholipids; D019814:Myristic Acid; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors