Evidence for ADP-ribosylation factor (ARF) as a regulator of in vitro endosome-endosome fusion.
We have used an in vitro endosome fusion assay, recombinant ARF, synthetic peptides, and guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) to study the role of ARF during endocytosis. Previous work has shown that GTP gamma S stimulates in vitro endosome fusion in dilute cytosol (less than 0.5 mg/ml) but inhibits fusion in concentrated cytosol (greater than 1.0 mg/ml). Two peptides corresponding to the NH2-terminal 16 amino acids of human ARF1 and ARF4 blocked GTP gamma S stimulation of fusion in dilute cytosol and reversed GTP gamma S inhibition of fusion in concentrated cytosol. The addition of recombinant human ARF1 to endosomes in dilute or concentrated cytosol resulted in GTP gamma S-dependent inhibition of fusion. Only the myristoylated form of ARF inhibited fusion. The NH2-terminal ARF1 peptide reversed inhibition by recombinant ARF1. Preincubation experiments showed that endosomes could form an ARF-resistant intermediate during the fusion process. Western blot analysis revealed clathrin-coated vesicles extracted with detergent retained ARF. The results suggest that ARF is involved in both the stimulatory and inhibitory effects of GTP gamma S in dilute and concentrated cytosol, respectively. Furthermore, myristoylation, the NH2-terminal domain, and binding to GTP appear to be critical for ARF activity during an early prefusion step required for endocytosis.
Lenhard
J M
JM
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110.
Kahn
R A
RA
Stahl
P D
PD
D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism*; D007700:Kinetics; D008561:Membrane Fusion; D051379:Mice; D008969:Molecular Sequence Data; D015388:Organelles / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D011994:Recombinant Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors