Effects of Activated ADP-ribosylation Factors on Golgi Morphology Require neither Activation of Phospholipase D1 nor Recruitment of Coatomer

• Published in: The Journal of biological chemistry Vol. 275; no. 6; pp. 4022 - 4032
• Main Authors: Kuai, Jun, Boman, Annette L., Arnold, Rebecca S., Zhu, Xinjun, Kahn, Richard A.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 11.02.2000; American Society for Biochemistry and Molecular Biology
• Subjects: ADP-Ribosylation Factors - genetics; ADP-Ribosylation Factors - metabolism; Animals; Cell Line; CHO Cells; Coatomer Protein - metabolism; Cricetinae; Enzyme Activation; Fluorescent Antibody Technique; Golgi Apparatus - metabolism; Guanosine 5'-O-(3-Thiotriphosphate) - metabolism; Humans; Mutation; Myristic Acid - metabolism; Phospholipase D - metabolism; Protein Binding; Rats; Recombinant Proteins
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.275.6.4022

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.