ArfGAP1 dynamics and its role in COPI coat assembly on Golgi membranes of living cells.
Secretory protein trafficking relies on the COPI coat, which by assembling into a lattice on Golgi membranes concentrates cargo at specific sites and deforms the membranes at these sites into coated buds and carriers. The GTPase-activating protein (GAP) responsible for catalyzing Arf1 GTP hydrolysis is an important part of this system, but the mechanism whereby ArfGAP is recruited to the coat, its stability within the coat, and its role in maintenance of the coat are unclear. Here, we use FRAP to monitor the membrane turnover of GFP-tagged versions of ArfGAP1, Arf1, and coatomer in living cells. ArfGAP1 underwent fast cytosol/Golgi exchange with approximately 40% of the exchange dependent on engagement of ArfGAP1 with coatomer and Arf1, and affected by secretory cargo load. Permanent activation of Arf1 resulted in ArfGAP1 being trapped on the Golgi in a coatomer-dependent manner. These data suggest that ArfGAP1, coatomer and Arf1 play interdependent roles in the assembly-disassembly cycle of the COPI coat in vivo.
Liu
Wei
W
Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
Duden
Rainer
R
Phair
Robert D
RD
Lippincott-Schwartz
Jennifer
J
D020823:ADP-Ribosylation Factor 1 / Q000378:metabolism; D020727:ADP-Ribosylation Factors / Q000378:metabolism*; D000818:Animals; D022181:COP-Coated Vesicles / Q000378:metabolism*; D019556:COS Cells; D002522:Chlorocebus aethiops; D020755:Coat Protein Complex I / Q000378:metabolism*; D020756:Coatomer Protein / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism*; D008954:Models, Biological; D021381:Protein Transport / Q000502:physiology
D020755:Coat Protein Complex I; D020756:Coatomer Protein; D020690:GTPase-Activating Proteins; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors