The Myristoylated Amino Terminus of ADP-ribosylation Factor 1 Is a Phospholipid- and GTP-sensitive Switch (*)
- Paul A. Randazzo(§),
- Takeshi Terui,
- Stacey Sturch,
- Henry M. Fales(1),
- Anthony G. Ferrige(2) and
- Richard A. Kahn
- From the (1)Laboratory of Biological Chemistry, Developmental Therapeutics Program, Division of Cancer Treatment, NCI and the Laboratory of Biophysical Chemistry, NHLBI, National Institutes of Health, Bethesda, Maryland 20892 and
- (2)MaxENT Solutions, Ltd., Cambridge CB75 QS1, United Kingdom
- § To whom correspondence should be addressed: NIH, Bldg. 37, Rm. 5D-02, Bethesda, MD 20892. Tel.: 301-496-3788; Fax: 301-480-2514.
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
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
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
S than for GDP, and in the presence of phospholipids, the myristoylated protein has a greater affinity for GTP
S than for GDP. Thus, N-myristoylation is a critical component in the construction of this phospholipid- and GTP-dependent switch.
Footnotes
-
↵* This work was supported by the Developmental Therapeutics Program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵1 The abbreviations used are:
- Arfs
-
ADP-ribosylation factors
- Arf1
-
human ADP-ribosylation factor 1
- GTP
S -
guanosine 5′-O-(3-thio)triphosphate
- DMPC
-
L-α-dimyristoylphosphatidylcholine
- HPLC
-
high pressure liquid chromatography.
