Sylviane Robineau

Brefeldin A Acts to Stabilize an Abortive ARF–GDP–Sec7 Domain Protein Complex: Involvement of Specific Residues of the Sec7 Domain

We demonstrate that the major in vivo targets of brefeldin A (BFA) in the secretory pathway of budding yeast are the three members of the Sec7 domain family of ARF exchange factors: Gea1p and Gea2p (functionally interchangeable) and Sec7p. Specific residues within the Sec7 domain are important for BFA inhibition of ARF exchange activity, since mutations in these residues of Gea1p (sensitive to BFA) and of ARNO (resistant to BFA) reverse the sensitivity of each to BFA in vivo and in vitro. We show that the target of BFA inhibition of ARF exchange activity is an ARF-GDP-Sec7 domain protein complex, and that BFA acts to stabilize this complex to a greater extent for a BFA-sensitive Sec7 domain than for a resistant one.

Lipid packing sensed by ArfGAP1 couples COPI coat disassembly to membrane bilayer curvature.

Protein coats deform flat lipid membranes into buds and capture membrane proteins to form transport vesicles. The assembly/disassembly cycle of the COPI coat on Golgi membranes is coupled to the GTP/GDP cycle of the small G protein Arf1. At the heart of this coupling is the specific interaction of membrane-bound Arf1–GTP with coatomer, a complex of seven proteins that forms the building unit of the COPI coat. Although COPI coat disassembly requires the catalysis of GTP hydrolysis in Arf1 by a specific GTPase-activating protein (ArfGAP1), the precise timing of this reaction during COPI vesicle formation is not known. Using time-resolved assays for COPI dynamics on liposomes of controlled size, we show that the rate of ArfGAP1-catalysed GTP hydrolysis in Arf1 and the rate of COPI disassembly increase over two orders of magnitude as the curvature of the lipid bilayer increases and approaches that of a typical transport vesicle. This leads to a model for COPI dynamics in which GTP hydrolysis in Arf1 is organized temporally and spatially according to the changes in lipid packing induced by the coat.

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 (ref. 4) and GPS1 (ref. 5), 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, Gea1 and Gea2 (refs 7–9). Here we report the crystal structure of the Sec7 domain of human ARNO at 2 Å resolution and the identification of the site of interaction of ARNO with Arf.

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 [Δ17]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 [Δ17]ARF1‐GDP without inducing the release of GDP. Other mutations in ARNO‐Sec7 and in [Δ17]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.

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 [Δ17]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 [Δ17]ARF1, and a stable complex between Arno Sec7 domain and nucleotide-free [Δ17]ARF1 can be isolated. In contrast to [Δ17]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.

Sequence analysis of three deficient mutants of cytochrome oxidase subunit I of Saccharomyces cerevisiae and their revertants.

Three respiratory-deficient mutants of cytochrome oxidase subunit I in the yeast mitochondrion have been sequenced. They are located in, or near, transmembrane segment VI, the catalytic core of the enzyme. Respiratory-competent revertants have been selected and studied. The mutant V244M was found to revert at the same site in valine (wild-type), isoleucine or threonine. The revertants of the mutant G251R were of three types: glycine (wild-type), serine and threonine at position 251. A search for second-site mutations was carried out but none were found. Among 60 revertants tested, the mutant K265M was found to revert only to the wild-type allele.

28 - Expression, Purification, and Measurements of Activity of ARNO1, a Guanine Nucleotide Exchange Factor for ADP-Ribosylation Factor 1 (ARF1)

The expression in Escherichia coli and purification of ARNO1 and its Sec7 domain ARNO1-Sec7 are described in this chapter. Two different kinds of assay can be used to monitor the exchange activity of ARNO1 or ARNO1-Sec7 on ARFI: classical nucleotide binding measurements with radiolabeled nucleotides or a real-time assay based on the large difference between the tryptophan fluorescence of ARF1-GDP and the tryptophan fluorescence of ARF1-GTP. The advantage of the fluorescence assay is its time resolution. However, this assay requires purified proteins for a good signal-to-noise ratio, and cannot detect futile nucleotide exchanges; as such, exchanges are spectroscopically silent. It is suggested that that although both ARFI and ARNO1 are soluble proteins, they interact with membrane lipids. More importantly, these membrane interactions are necessary for the functional interaction between the two proteins. Therefore, membrane lipids (for instance, artificial lipid vesicles) must be included in functional assays.

ARNO3, a human Sec7-domain guanine nucleotide exchange factor specific for ARF1, controls membrane structure of the golgi complex

conserv&d domain with the yeast Sec7 protein, as a GEF acting on ARFI. Yet, the function of ARNO-like GEFs in the regulation of membrankdynamic in the secret0 We describe a novel human Sec7 2 pathway had not bee; analyzed. omain-containin GEF referred to as ARN03. ARNO and ARNOJ, as well as a t i. trd GEF called cytohesin-1, form a family of highly related proteins with identical structural organization that consists of a central Sec7 domain and a carboxy terminal pleckstrin homology domain. We show that all three proteins act as GEF specific for ARFI in vitro, while they are ineffective onto ARF6, an ARF protein implicated in the early endocytic pathway.