The small GTP-binding protein, ADP-ribosylation factor 1 (ARF1) is essential for the formation of coatomer-coated vesicles from the Golgi and is also an activator of phospholipase D (PLD). Moreover, ARF1-regulated PLD is part of the signal-transduction pathway that can lead to secretion. In this study, substitution and deletion mutants of ARF1 were tested for their ability to activate PLD. These map the PLD effector region of ARF1 to the α2 helix, part of the β2-strand and the N-terminal helix and its ensuing loop. ARF mutants with an increased or decreased ability to activate PLD showed similar characteristics when tested for their ability to stimulate secretion from HL60 cells. ARF1, deleted of the N-terminal 17 amino acid residues (Ndel17), did not support PLD activity or secretion, and neither did it inhibit the activity of wild-type myristoylated ARF1 (myrARF1). In contrast, Ndel17 effectively competed with wild-type myrARF1 to prevent coatomer binding to membranes. This appears to define a structural role for Ndel17, as it can bind a high-molecular mass complex in cytosol. In addition, ethanol has no effect on recruitment of coatomer to membrane. We conclude that the function of ARF-regulated PLD is in the signal-transduction pathway leading to secretion of lysosomal granules, and not as an essential component of ARF1-mediated coatomer binding.
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July 1999
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Research Article|
June 24 1999
ADP ribosylation factor 1 mutants identify a phospholipase D effector region and reveal that phospholipase D participates in lysosomal secretion but is not sufficient for recruitment of coatomer I
David H. JONES;
David H. JONES
*Department of Physiology, Rockefeller Building, University College London, University Street, London WC1E 6JJ, U.K.
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Ben BAX;
Ben BAX
†Department of Crystallography, Birkbeck College, Malet Street, London WC1E 7HX, U.K.
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Amanda FENSOME;
Amanda FENSOME
*Department of Physiology, Rockefeller Building, University College London, University Street, London WC1E 6JJ, U.K.
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Shamshad COCKCROFT
Shamshad COCKCROFT
1
*Department of Physiology, Rockefeller Building, University College London, University Street, London WC1E 6JJ, U.K.
1To whom correspondence should be addressed (e-mail s.cockcroft@ucl.ac.uk).
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Publisher: Portland Press Ltd
Received:
February 23 1999
Revision Received:
April 01 1999
Accepted:
April 27 1999
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1999
1999
Biochem J (1999) 341 (1): 185–192.
Article history
Received:
February 23 1999
Revision Received:
April 01 1999
Accepted:
April 27 1999
Citation
David H. JONES, Ben BAX, Amanda FENSOME, Shamshad COCKCROFT; ADP ribosylation factor 1 mutants identify a phospholipase D effector region and reveal that phospholipase D participates in lysosomal secretion but is not sufficient for recruitment of coatomer I. Biochem J 1 July 1999; 341 (1): 185–192. doi: https://doi.org/10.1042/bj3410185
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