ADP-ribosylation factor and phosphatidic acid levels in Golgi membranes during budding of coatomer-coated vesicles

ADP-ribosylation factor and phosphatidic acid levels in Golgi membranes during budding of coatomer-coated vesicles

Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021

Contributed by James E. Rothman

Abstract

The finding that ADP-ribosylation factor (ARF) can activate phospholipase D has led to debate as to whether ARF recruits coat proteins through direct binding or indirectly by catalytically increasing phosphatidic acid production. Here we test critical aspects of these hypotheses. We find that Golgi membrane phosphatidic acid levels do not rise—in fact they decline—during cell-free budding reactions. We confirm that the level of membrane-bound ARF can be substantially reduced without compromising coat assembly [Ktistakis, N. T., Brown, H. A., Waters, M. G., Sternweis, P. C. & Roth, M. G. (1996) J. Cell Biol. 134, 295–306], but find that under all conditions, ARF is present on the Golgi membrane in molar excess over bound coatomer. These results do not support the possibility that the activation of coat assembly by ARF is purely catalytic, and they are consistent with ARF forming direct interactions with coatomer. We suggest that ARF, like many other G proteins, is a multifunctional protein with roles in trafficking and phospholipid signaling.

Footnotes

↵ * To whom reprint requests should be addressed. e-mail: mark-stamnes{at}uiowa.edu.
↵ † Present address: Department of Physiology and Biophysics, University of Iowa School of Medicine, Iowa City, IA 52242.
↵ ‡ Present address: Imperial Cancer Research Fund, Lincoln’s Inn Fields, London WC2 3PX, United Kingdom.
↵ § Present address: The Bone and Mineral Centre, University College London, London WC1E 6JJ, United Kingdom.
ABBREVIATIONS:

ARF,

ADP ribosylation factor;

COPI,

coat protomer I;

PLD,

phospholipase D;

PA,

phosphatidic acid;

PC,

phosphatidylcholine