THE DISTRIBUTION AND TRANSLOCATION OF THE G-PROTEIN ADP-RIBOSYLATION FACTOR-1 IN LIVE CELLS IS DETERMINED BY ITS GTPASE ACTIVITY

ADP-ribosylation factors (ARF) are small G proteins that play key role s in vesicular transport processes. We have studied the distribution o f ARF1 in live cells using chimeras of ARF1 mutants (wild type (wt) AR F1; Q71L-ARF1 (reduced GTPase); T31N (low affinity for GTP); and Delta (N)wt (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(N)wt and the T31 N mutants were exclusively cytoplasmic. The behavior of the wt and Q71 L proteins was studied in detail. About 15% of wt-ARF1-GFP was bound t o the Golgi. Bound wt-ARF1-GFP dissociated rapidly after addition of B refeldin A (BFA), This process did not appear to be a consequence of B FA-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 a nd biphasic, Q71L-ARF1-GFP diffused more slowly than the wt. We conclu de that ARF1 proteins exist in a dynamic equilibrium between Golgi-bou nd and cytosolic pools, and that the translocation of ARF in live cell s requires the hydrolysis of GTP by the Golgi-bound protein.