Dissection of membrane dynamics of the ARF-guanine nucleotide exchange factor GBF1.
ADP-ribosylation factor (ARF)-facilitated recruitment of COP I to membranes is required for secretory traffic. The guanine nucleotide exchange factor GBF1 activates ARF and regulates ARF/COP I dynamics at the endoplasmic reticulum (ER)-Golgi interface. Like ARF and coatomer, GBF1 peripherally associates with membranes. ADP-ribosylation factor and coatomer have been shown to rapidly cycle between membranes and cytosol, but the membrane dynamics of GBF1 are unknown. Here, we used fluorescence recovery after photobleaching to characterize the behavior of GFP-tagged GBF1. We report that GBF1 rapidly cycles between membranes and the cytosol (t1/2 is approximately 17 +/- 1 seconds). GBF1 cycles faster than GFP-tagged ARF, suggesting that in each round of association/dissociation, GBF1 catalyzes a single event of ARF activation, and that the activated ARF remains on membrane after GBF1 dissociation. Using three different approaches [expression of an inactive (E794K) GBF1 mutant, expression of the ARF1 (T31N) mutant with decreased affinity for GTP and Brefeldin A treatment], we show that GBF1 is stabilized on membranes when in a complex with ARF-GDP. GBF1 dissociation from ARF and membranes is triggered by its catalytic activity, i.e. the displacement of GDP and the subsequent binding of GTP to ARF. Our findings imply that continuous cycles of recruitment and dissociation of GBF1 to membranes are required for sustained ARF activation and COP I recruitment that underlies ER-Golgi traffic.
Szul
Tomasz
T
Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL 35924, USA.
Garcia-Mata
Rafael
R
Brandon
Elizabeth
E
Shestopal
Svetlana
S
Alvarez
Cecilia
C
Sztul
Elizabeth
E
D020727:ADP-Ribosylation Factors / Q000235:genetics / Q000378:metabolism*; D020126:Brefeldin A / Q000494:pharmacology; D020755:Coat Protein Complex I / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D036681:Fluorescence Recovery After Photobleaching; D006056:Golgi Apparatus / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008954:Models, Biological; D009154:Mutation; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology
D020755:Coat Protein Complex I; C120445:GBF1 protein, human; D020662:Guanine Nucleotide Exchange Factors; D011500:Protein Synthesis Inhibitors; D049452:Green Fluorescent Proteins; D020126:Brefeldin A; D020727:ADP-Ribosylation Factors