Crystal structure of Sar1-GDP at 1.7 A resolution and the role of the NH2 terminus in ER export.

The Sar1 GTPase is an essential component of COPII vesicle coats involved in export of cargo from the ER. We report the 1.7-A structure of Sar1 and find that consistent with the sequence divergence of Sar1 from Arf family GTPases, Sar1 is structurally distinct. In particular, we show that the Sar1 NH2 terminus contains two regions: an NH2-terminal extension containing an evolutionary conserved hydrophobic motif that facilitates membrane recruitment and activation by the mammalian Sec12 guanine nucleotide exchange factor, and an alpha1' amphipathic helix that contributes to interaction with the Sec23/24 complex that is responsible for cargo selection during ER export. We propose that the hydrophobic Sar1 NH2-terminal activation/recruitment motif, in conjunction with the alpha1' helix, mediates the initial steps in COPII coat assembly for export from the ER.

Dual interaction of ADP ribosylation factor 1 with Sec7 domain and with lipid membranes during catalysis of guanine nucleotide exchange.

Sec7 domains catalyze the replacement of GDP by GTP on the G protein ADP-ribosylation factor 1 (myrARF1) by interacting with its switch I and II regions and by destabilizing, through a glutamic finger, the beta-phosphate of the bound GDP. The myristoylated N-terminal helix that allows myrARF1 to interact with membrane lipids in a GTP-dependent manner is located some distance from the Sec7 domain-binding region. However, these two regions are connected. Measuring the binding to liposomes of functional or abortive complexes between myrARF1 and the Sec7 domain of ARNO demonstrates that myrARF1, in complex with the Sec7 domain, adopts a high affinity state for membrane lipids, similar to that of the free GTP-bound form. This tight membrane attachment does not depend on the release of GDP induced by the Sec7 domain but is partially inhibited by the uncompetitive inhibitor brefeldin A. These results suggest that the conformational switch of the N-terminal helix of myrARF1 to the membrane-bound form is an early event in the nucleotide exchange pathway and is a prerequisite for a structural rearrangement at the myrARF1-GDP/Sec7 domain interface that allows the glutamic finger to expel GDP from myrARF1.

A glutamic finger in the guanine nucleotide exchange factor ARNO displaces Mg2+ and the beta-phosphate to destabilize GDP on ARF1.

The Sec7 domain of the guanine nucleotide exchange factor ARNO (ARNO-Sec7) is responsible for the exchange activity on the small GTP-binding protein ARF1. ARNO-Sec7 forms a stable complex with the nucleotide-free form of [Delta17]ARF1, a soluble truncated form of ARF1. The crystal structure of ARNO-Sec7 has been solved recently, and a site-directed mutagenesis approach identified a hydrophobic groove and an adjacent hydrophilic loop as the ARF1-binding site. We show that Glu156 in the hydrophilic loop of ARNO-Sec7 is involved in the destabilization of Mg2+ and GDP from ARF1. The conservative mutation E156D and the charge reversal mutation E156K reduce the exchange activity of ARNO-Sec7 by several orders of magnitude. Moreover, [E156K]ARNO-Sec7 forms a complex with the Mg2+-free form of [Delta17]ARF1-GDP without inducing the release of GDP. Other mutations in ARNO-Sec7 and in [Delta17]ARF1 suggest that prominent hydrophobic residues of the switch I region of ARF1 insert into the groove of the Sec7 domain, and that Lys73 of the switch II region of ARF1 forms an ion pair with Asp183 of ARNO-Sec7.

Structure of the Sec7 domain of the Arf exchange factor ARNO.

Small G proteins switch from a resting, GDP-bound state to an active, GTP-bound state. As spontaneous GDP release is slow, guanine-nucleotide-exchange factors (GEFs) are required to promote fast activation of small G proteins through replacement of GDP with GTP in vivo. Families of GEFs with no sequence similarity to other GEF families have now been assigned to most families of small G proteins. In the case of the small G protein Arf1, the exchange of bound GDP for GTP promotes the coating of secretory vesicles in Golgi traffic. An exchange factor for human Arf1, ARNO, and two closely related proteins, named cytohesin 1 and GPS1, have been identified. These three proteins are modular proteins with an amino-terminal coiled-coil, a central Sec7-like domain and a carboxy-terminal pleckstrin homology domain. The Sec7 domain contains the exchange-factor activity. It was first found in Sec7, a yeast protein involved in secretion, and is present in several other proteins, including the yeast exchange factors for Arf, Geal and Gea2. Here we report the crystal structure of the Sec7 domain of human ARNO at 2 A resolution and the identification of the site of interaction of ARNO with Arf.

Role of protein-phospholipid interactions in the activation of ARF1 by the guanine nucleotide exchange factor Arno.

Arno is a 47-kDa human protein recently identified as a guanine nucleotide exchange factor for ADP ribosylation factor 1 (ARF1) with a central Sec7 domain responsible for the exchange activity and a carboxyl-terminal pleckstrin homology (PH) domain (Chardin, P., Paris, S., Antonny, B., Robineau, S., Béraud-Dufour, S., Jackson, C. L., and Chabre, M. (1996) Nature 384, 481-484). Binding of the PH domain to phosphatidylinositol 4,5-bisphosphate (PIP2) greatly enhances Arno-mediated activation of myristoylated ARF1. We show here that in the absence of phospholipids, Arno promotes nucleotide exchange on [Delta17]ARF1, a soluble mutant of ARF1 lacking the first 17 amino acids. This reaction is unaffected by PIP2, which suggests that the PIP2-PH domain interaction does not directly regulate the catalytic activity of Arno but rather serves to recruit Arno to membranes. Arno catalyzes the release of GDP more efficiently than that of GTP from [Delta17]ARF1, and a stable complex between Arno Sec7 domain and nucleotide-free [Delta17]ARF1 can be isolated. In contrast to [Delta17]ARF1, full-length unmyristoylated ARF1 is not readily activated by Arno in solution. Its activation requires the presence of phospholipids and a reduction of ionic strength and Mg2+ concentration. PIP2 is strongly stimulatory, indicating that binding of Arno to phospholipids is involved, but in addition, electrostatic interactions between phospholipids and the amino-terminal portion of unmyristoylated ARF1GDP seem to be important. We conclude that efficient activation of full-length ARF1 by Arno requires a membrane surface and two distinct protein-phospholipid interactions: one between the PH domain of Arno and PIP2, and the other between amino-terminal cationic residues of ARF1 and anionic phospholipids. The latter interaction is normally induced by insertion of the amino-terminal myristate into the bilayer but can also be artificially facilitated by decreasing Mg2+ and salt concentrations.

N-terminal hydrophobic residues of the G-protein ADP-ribosylation factor-1 insert into membrane phospholipids upon GDP to GTP exchange.

GDP/GTP exchange modulates the interaction of the small G-protein ADP-ribosylation factor-1 with membrane lipids: if ARF(GDP) is mostly soluble, ARF(GTP) binds tightly to lipid vesicles. Previous studies have shown that this GTP-dependent binding persists upon removal of the N-terminal myristate but is abolished following further deletion of the 17 N-terminal residues. This suggests a role for this amphipathic peptide in lipid membrane binding. In the ARF(GDP) crystal structure, the 2-13 peptide is helical, with its hydrophobic residues buried in the protein core. When ARF switches to the GTP state, these residues may insert into membrane lipids. We have studied the binding of ARF to model unilamellar vesicles of defined composition. ARF(GDP) binds weakly to vesicles through hydrophobic interaction of the myristate and electrostatic interaction of cationic residues with anionic lipids. Phosphatidylinositol 4,5-bis(phosphate) shows no specific effects other than strictly electrostatic. By using fluorescence energy transfer, the strength of the ARF(GTP)-lipid interaction is assessed via the dissociation rate of ARF(GTPgammaS) from labeled lipid vesicles. ARF(GTPgammaS) dissociates slowly (tau(off) approximately 75 s) from neutral PC vesicles. Including 30% anionic phospholipids increases tau(off) by only 3-fold. Reducing the N-terminal peptide hydrophobicity by point mutations had larger effects: F9A and L8A-F9A substitutions accelerate the dissociation of ARF(GTPgammaS) from vesicles by factors of 7 and 100, respectively. This strongly suggests that, upon GDP/GTP exchange, the N-terminal helix is released from the protein core so its hydrophobic residues can interact with membrane phospholipids.

A human exchange factor for ARF contains Sec7- and pleckstrin-homology domains.

The small G protein ARF1 is involved in the coating of vesicles that bud from the Golgi compartments. Its activation is controlled by as-yet unidentified guanine-nucleotide exchange factors. Gea1, the first ARF exchange factor to be discovered in yeast, is a large protein containing a domain of homology with Sec7, another yeast protein that is also involved in secretion. Here we characterized a smaller human protein (relative molecular mass 47K) named ARNO, which contains a central Sec7 domain that promotes guanine-nucleotide exchange on ARF1. ARNO also contains an amino-terminal coiled-coil motif and a carboxy-terminal pleckstrin-homology (PH) domain. The PH domain mediates an enhancement of ARNO exchange activity by negatively charged phospholipid vesicles supplemented with phosphatidylinositol bisphosphate. The exchange activity of ARNO is not inhibited by brefeldin A, an agent known to block vesicular transport and inhibit the exchange activity on ARF1 in cell extracts. This suggests that a regulatory component which is sensitive to brefeldin A associates with ARNO in vivo, possibly through the amino-terminal coiled-coil. We propose that other proteins with a Sec7 domain regulate different members of the ARF family.