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 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 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.
Vasudevan
C
C
Department of Pharmacology, University of Pittsburgh, Pittsburgh, PA 15261, USA.
Han
W
W
Tan
Y
Y
Nie
Y
Y
Li
D
D
Shome
K
K
Watkins
S C
SC
Levitan
E S
ES
Romero
G
G
D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001692:Biological Transport; D020126:Brefeldin A; D003517:Cyclopentanes / Q000494:pharmacology; D003600:Cytosol / Q000737:chemistry*; D005347:Fibroblasts / Q000737:chemistry / Q000648:ultrastructure; D005470:Fluorometry; D020558:GTP Phosphohydrolases / Q000032:analysis / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D006056:Golgi Apparatus / Q000737:chemistry / Q000187:drug effects; D049452:Green Fluorescent Proteins; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis; D018942:Macrolides; D010777:Photochemistry; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000032:analysis; D013329:Structure-Activity Relationship
D000900:Anti-Bacterial Agents; D003517:Cyclopentanes; D008164:Luminescent Proteins; D018942:Macrolides; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D020126:Brefeldin A; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors