Effects of activated ADP-ribosylation factors on Golgi morphology require neither activation of phospholipase D1 nor recruitment of coatomer.
Nine mutations in the switch I and switch II regions of human ADP-ribosylation factor 3 (ARF3) were isolated from loss-of-interaction screens, using two-hybrid assays with three different effectors. We then analyzed the ability of the recombinant proteins to (i) bind guanine nucleotides, (ii) activate phospholipase D1 (PLD1), (iii) recruit coatomer (COP-I) to Golgi-enriched membranes, and (iv) expand and vesiculate Golgi in intact cells. Correlations of activities in these assays were used as a means of testing specific hypotheses of ARF action, including the role of PLD1 activation in COP-I recruitment, the role of COP-I in Golgi vesiculation caused by expression of the dominant activating mutant [Q71L]ARF3, and the need for PLD1 activation in Golgi vesiculation. Because we were able to find at least one example of a protein that has lost each of these activities with retention of the others, we conclude that activation of PLD1, recruitment of COP-I to Golgi, and vesiculation of Golgi in cells are functionally separable processes. The ability of certain mutants of ARF3 to alter Golgi morphology without changes in PLD1 activity or COP-I binding is interpreted as evidence for at least one additional, currently unidentified, effector for ARF action at the Golgi.
Kuai
J
J
Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322-3050, USA.
Boman
A L
AL
Arnold
R S
RS
Zhu
X
X
Kahn
R A
RA
D020727:ADP-Ribosylation Factors / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D020756:Coatomer Protein / Q000378:metabolism*; D006224:Cricetinae; D004789:Enzyme Activation; D005455:Fluorescent Antibody Technique; D006056:Golgi Apparatus / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D009154:Mutation; D019814:Myristic Acid / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins
D020756:Coatomer Protein; D011994:Recombinant Proteins; D019814:Myristic Acid; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D010739:Phospholipase D; C106801:phospholipase D1; C116687:ARF3 protein, human; C492129:Arf3 protein, rat; D020727:ADP-Ribosylation Factors