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J. Cell Biol.,
Volume 142, Number 2, July 27, 1998 391-402
Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health
(NIH), Bethesda, Maryland 20892
Small GTP-binding proteins such as ADP-
ribosylation factor 1 (ARF1) and Sar1p regulate the
membrane association of coat proteins involved in intracellular membrane trafficking. ARF1 controls the
clathrin coat adaptor AP-1 and the nonclathrin coat
COPI, whereas Sar1p controls the nonclathrin coat
COPII. In this study, we demonstrate that membrane
association of the recently described AP-3 adaptor is
regulated by ARF1. Association of AP-3 with membranes in vitro was enhanced by GTP
S and inhibited
by brefeldin A (BFA), an inhibitor of ARF1 guanine
nucleotide exchange. In addition, recombinant myristoylated ARF1 promoted association of AP-3 with membranes. The role of ARF1 in vivo was examined by
assessing AP-3 subcellular localization when the intracellular level of ARF1-GTP was altered through
overexpression of dominant ARF1 mutants or ARF1-
GTPase-activating protein (GAP). Lowering ARF1-GTP levels resulted in redistribution of AP-3 from
punctate membrane-bound structures to the cytosol as
seen by immunofluorescence microscopy. In contrast,
increasing ARF1-GTP levels prevented redistribution of AP-3 to the cytosol induced by BFA or energy depletion. Similar experiments with mutants of ARF5 and
ARF6 showed that these other ARF family members
had little or no effect on AP-3. Taken together, our results indicate that membrane recruitment of AP-3 is
promoted by ARF1-GTP. This finding suggests that
ARF1 is not a regulator of specific coat proteins, but
rather is a ubiquitous molecular switch that acts as a
transducer of diverse signals influencing coat assembly.
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