The distribution and translocation of the G protein ADP-ribosylation factor 1 in live cells is determined by its GTPase activity

C Vasudevan, W Han, Y Tan, Y Nie, D Li, K Shome, S C Watkins, E S Levitan, G Romero
1998 Journal of Cell Science  
ADP-ribosylation factors (ARF) are small G proteins that play key roles in vesicular transport processes. We have studied the distribution of ARF1 in live cells using chimeras of ARF1 mutants (wild type (wt) ARF1; Q71L-ARF1 (reduced GTPase); T31N (low affinity for GTP); and (Delta)Nwt (deletion of amino acids 2-18)) with green fluorescent protein (GFP). Confocal microscopy studies showed that the wt and Q71L proteins were localized in the Golgi and cytoplasm. The (Delta)Nwt and the T31N mutants
more » ... nd the T31N mutants were exclusively cytoplasmic. The behavior of the wt and Q71L proteins was studied in detail. About 15% of wt-ARF1-GFP was bound to the Golgi. Bound wt-ARF1-GFP dissociated rapidly after addition of Brefeldin A (BFA). This process did not appear to be a consequence of BFA-induced disappearance of the Golgi. Photobleaching recovery showed that essentially all the ARF-GFP was mobile, although it diffused very slowly. In contrast, about 40-50% of the Q71L mutant was found in the Golgi, and its rate of dissociation in the presence of BFA was slow and biphasic. Q71L-ARF1-GFP diffused more slowly than the wt. We conclude that ARF1 proteins exist in a dynamic equilibrium between Golgi-bound and cytosolic pools, and that the translocation of ARF in live cells requires the hydrolysis of GTP by the Golgi-bound protein.
pmid:9547306 fatcat:caxfoxxcqfd2tnoi6hn7jhq72u