ArfGAP1 generates an Arf1 gradient on continuous lipid membranes displaying flat and curved regions.
ArfGAP1, which promotes GTP hydrolysis on the small G protein Arf1 on Golgi membranes, interacts preferentially with positively curved membranes through its amphipathic lipid packing sensor (ALPS) motifs. This should influence the distribution of Arf1-GTP when flat and curved regions coexist on a continuous membrane, notably during COPI vesicle budding. To test this, we pulled tubes from giant vesicles using molecular motors or optical tweezers. Arf1-GTP distributed on the giant vesicles and on the tubes, whereas ArfGAP1 bound exclusively to the tubes. Decreasing the tube radius revealed a threshold of R approximately 35 nm for the binding of ArfGAP1 ALPS motifs. Mixing catalytic amounts of ArfGAP1 with Arf1-GTP induced a smooth Arf1 gradient along the tube. This reflects that Arf1 molecules leaving the tube on GTP hydrolysis are replaced by new Arf1-GTP molecules diffusing from the giant vesicle. The characteristic length of the gradient is two orders of magnitude larger than a COPI bud, suggesting that Arf1-GTP diffusion can readily compensate for the localized loss of Arf1 during budding and contribute to the stability of the coat until fission.
Ambroggio
Ernesto
E
Laboratoire Mécanismes moléculaires du transport intracellulaire, Institut Curie, CNRS UMR 144, Paris, France.
Sorre
Benoît
B
Bassereau
Patricia
P
Goud
Bruno
B
Manneville
Jean-Baptiste
JB
Antonny
Bruno
B
D020823:ADP-Ribosylation Factor 1 / Q000032:analysis / Q000378:metabolism*; D020816:Amino Acid Motifs; D020755:Coat Protein Complex I / Q000032:analysis / Q000378:metabolism*; D004058:Diffusion; D020690:GTPase-Activating Proteins / Q000032:analysis / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D008563:Membrane Lipids / Q000032:analysis / Q000378:metabolism*; D052898:Optical Tweezers; D011485:Protein Binding; D053835:Unilamellar Liposomes / Q000032:analysis / Q000378:metabolism*
D020755:Coat Protein Complex I; D020690:GTPase-Activating Proteins; D008563:Membrane Lipids; D053835:Unilamellar Liposomes; D020823:ADP-Ribosylation Factor 1