Resumen:
rfGAP1, 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 ma