The ADP ribosylation factor (ARF) GTP binding proteins are believed to mediate cytoskeletal remodeling and vesicular trafficking along the secretory pathway. Here we show that ARF6 is specifically associated with dense-core secretory granules in neuroendocrine PC12 cells. Stimulation with a secretagogue triggers the recruitment of secretory granules to the cell periphery and the concomitant activation of ARF6 by the plasma membrane-associated guanine nucleotide exchange factor, ARF nucleotide binding site opener (ARNO). Expression of the constitutively inactive ARF6(T27N) mutant inhibits secretagogue-dependent exocytosis from PC12 cells. Using a mutant of ARF6 specifically impaired for PLD1 stimulation, we find that ARF6 is functionally linked to phospholipase D (PLD)1 in the exocytotic machinery. Finally, we show that ARNO, ARF6, and PLD1 colocalize at sites of exocytosis, and we demonstrate direct interaction between ARF6 and PLD1 in stimulated cells. Together, these results provide the first direct evidence that ARF6 plays a role in calcium-regulated exocytosis in neuroendocrine cells, and suggest that ARF6-stimulated PLD1 activation at the plasma membrane and consequent changes in membrane phospholipid composition are critical for formation of the exocytotic fusion pore.

Small GTPases of the ADP-ribosylation factor (ARF) family play a key role in membrane trafficking by regulating coated vesicle formation, and guanine nucleotide exchange is essential for the ARF function. Brefeldin A blocks the ARF-triggered coat assembly by inhibiting the guanine nucleotide exchange on ARFs and causes disintegration of the Golgi complex and tubulation of endosomal membranes. BIG2 is one of brefeldin A-inhibited guanine nucleotide exchange factors for the ARF GTPases and is associated mainly with the trans-Golgi network. In the present study, we have revealed that another population of BIG2 is associated with the recycling endosome and found that expression of a catalytically inactive BIG2 mutant, E738K, selectively induces membrane tubules from this compartment. We also have shown that BIG2 has an exchange activity toward class I ARFs (ARF1 and ARF3) in vivo and inactivation of either ARF exaggerates the BIG2(E738K)-induced tubulation of endosomal membranes. These observations together indicate that BIG2 is implicated in the structural integrity of the recycling endosome through activating class I ARFs.

Activation of ADP-ribosylation factors (ARFs), ϳ20-kDa guanine nucleotide-binding proteins that play an important role in intracellular vesicular trafficking, depends on guanine nucleotide-exchange proteins (GEPs), which accelerate replacement of bound GDP with GTP. Two major families of ARF GEPs are known: ϳ200-kDa molecules that are inhibited by brefeldin A (BFA), a fungal metabolite that blocks protein secretion and causes apparent disintegration of Golgi structure, and ϳ50-kDa GEPs that are insensitive to BFA. We describe here two human brain cDNAs that encode BFA-inhibited GEPs. One is a ϳ209-kDa protein 99.5% identical in deduced amino acid sequence (1,849 residues) to a BFAinhibited ARF GEP (p200) from bovine brain. The other smaller protein, which is ϳ74% identical (1,785 amino acids), represents a previously unknown gene. We propose that the former, p200, be named BIG1 for (brefeldin A-inhibited GEP1) and the second, which encodes a ϳ202-kDa protein, BIG2. A protein containing sequences found in BIG2 had been purified earlier from bovine brain. Human tissues contained a 7.5-kilobase BIG1 mRNA and a 9.4-kilobase BIG2 transcript. The BIG1 and BIG2 genes were localized, respectively, to chromosomes 8 and 20. BIG2, synthesized as a His 6 fusion protein in Sf9 cells, accelerated guanosine 5-3-O-(thio)triphosphate binding by recombinant ARF1, ARF5, and ARF6. It activated native ARF (mixture of ARF1 and ARF3) more effectively than it did any of the nonmyristoylated recombinant ARFs. BIG2 activity was inhibited by BFA in a concentration-dependent manner but not by B17, a structural analog without effects on Golgi function. Although several clones for ϳ50-kDa BFA-insensitive ARF GEPs are known, these new clones for the ϳ200-kDa BIG1 and BIG2 should facilitate characterization of this rather different family of proteins as well as the elucidation of mechanisms of regulation of BFAsensitive ARF function in Golgi transport.

The ARF (ADP ribosylation factor) family of 20-kDa GTPbinding proteins, originally named because of the ability to stimulate cholera toxin-catalyzed ADP ribosylation of G s␣ (1), are now known as critical components of diverse intracellular vesicular trafficking pathways (2). ARF function depends on its alternation between inactive GDP-and active GTP-bound conformations. As ARF has no detectable GTPase activity and exchanges bound nucleotide very slowly at physiological concentrations of Mg 2ϩ , its cycling between active and inactive forms is controlled by GTPase-activating proteins (GAP) and guanine nucleotide-exchange proteins (GEP). Protease-sensitive ARF GEP activity was found in Golgi membranes and was inhibited by the fungal metabolite brefeldin A (BFA) that blocks vesicular transport (3, 4). A cytosolic ARF GEP was also inhibited by BFA, but after purification from bovine brain and rat spleen, the GEP was no longer BFA sensitive (5, 6).Available data are consistent with the possibilities that ARF GEP is not itself a target of BFA or that there are BFAinsensitive as well as BFA-sensitive forms of ARF GEP. We undertook to purify a BFA-sensitive GEP from bovine brain cytosol. As reported here, after Ϸ12,000-fold overall purification, a BFA sensitive-GEP was obtained, which behaved on gel filtration as a complex of Ϸ670 kDa. A component protein of Ϸ200 kDa was separated by SDS͞PAGE and exhibited BFA-sensitive GEP activity after elution from the gel and renaturation. Amino acid sequences of peptides from this protein were very similar to those of Sec7 from Saccharomyces cerevisiae (7), consistent with the view that the BFA-sensitive 200-kDa ARF GEP is a mammalian counterpart of Sec7. MATERIALS AND METHODSMaterials. DEAE-Sephacel was purchased from Pharmacia; hydroxylapatite (Bio-Gel HTP gel) was from Bio-Rad; phosphatidylserine was from Sigma; BFA was from Epicentre Technologies (Madison, WI); and 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) was from Boehringer Mannheim. Sources of other materials have been published (5, 6).Purification of BFA-Sensitive GEP. Soluble proteins from bovine brain cortex (830 g) in 300 ml of buffer A (20 mM Tris, pH 8.0͞1 mM EDTA͞1 mM NaN 3 ͞1 mM DTT͞0.25 M sucrose) containing leupeptin, aprotinin, and soybean and lima bean inhibitors (each 1 g͞ml) with 0.5 mM AEBSF were precipitated with 45% saturated (NH 4 ) 2 SO 4 . Precipitated proteins (3.75 g) were dissolved in buffer B (buffer A plus 2 mM MgCl 2 and 0.5 mM AEBSF), dialyzed against the same buffer, and applied to a column (5 ϫ 44 cm, 850 ml) of DEAESephacel equilibrated with buffer B. After washing with 850 ml of buffer B containing 50 mM NaCl, proteins were eluted with a linear gradient of 50-250 mM NaCl in buffer B (total 3.4 liters). Fractions containing BFA-sensitive GEP activity (eluted with 160-190 mM NaCl) were pooled and adjusted to pH 7.5 and 200 mM NaCl (based on conductivity) before application to a column of hydroxylapatite (5 ϫ 9 cm, 180 ml) equilibrated with buffer B containing 200 mM NaCl followed ...