Dissection of membrane dynamics of the ARF-guanine nucleotide exchange factor GBF1. Export

@article{citeulike:4244938, abstract = {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.}, author = {Szul, T. and Garcia-Mata, R. and Brandon, E. and Shestopal, S. and Alvarez, C. and Sztul, E.}, citeulike-article-id = {4244938}, citeulike-linkout-0 = {http://dx.doi.org/10.1111/j.1600-0854.2005.00282.x}, citeulike-linkout-1 = {http://view.ncbi.nlm.nih.gov/pubmed/15813748}, citeulike-linkout-2 = {http://www.hubmed.org/display.cgi?uids=15813748}, doi = {10.1111/j.1600-0854.2005.00282.x}, issn = {1398-9219}, journal = {Traffic (Copenhagen, Denmark)}, keywords = {arf1, arno, dynamics, fluo, frap, gbf1, gef, golgi, in\_vivo, interactions, mechanism, membrane, mutant, traffic}, month = {May}, number = {5}, pages = {374--385}, posted-at = {2009-03-31 18:03:18}, priority = {2}, title = {Dissection of membrane dynamics of the ARF-guanine nucleotide exchange factor GBF1.}, url = {http://dx.doi.org/10.1111/j.1600-0854.2005.00282.x}, volume = {6}, year = {2005} }

TY - JOUR ID - citeulike:4244938 L3 - citeulike-article-id:4244938 N2 - 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. IS - 5 JF - Traffic (Copenhagen, Denmark) SN - 1398-9219 EP - 385 TI - Dissection of membrane dynamics of the ARF-guanine nucleotide exchange factor GBF1. VL - 6 SP - 374 KW - arf1 KW - arno KW - dynamics KW - fluo KW - frap KW - gbf1 KW - gef KW - golgi KW - in_vivo KW - interactions KW - mechanism KW - membrane KW - mutant KW - traffic AU - Szul, T AU - Garcia-Mata, R AU - Brandon, E AU - Shestopal, S AU - Alvarez, C AU - Sztul, E PY - 2005/05// UR - http://dx.doi.org/10.1111/j.1600-0854.2005.00282.x ER -