overcome the energy barrier required for membrane fusion (Fasshauer et al., 1997;Hanson et al., 1997). Furthermore, Membrane proteins located on vesicles (v-SNAREs) using liposomes reconstituted with t-or v-SNAREs, and on the target membrane (t-SNAREs) mediateRothman and co-workers showed that the v-SNAREspecific recognition and, possibly, fusion between a t-SNARE complex per se fulfills the minimal requirement transport vesicle and its target membrane. The activity for fusion between two membranes (Weber et al., 1998; of SNARE molecules is regulated by several soluble Nickel et al., 1999;Parlati et al., 1999). Yet, based on an cytosolic proteins. We have cloned a bovine brain in vitro system that reconstitutes homotypic fusion of yeast cDNA encoding a conserved 117 amino acid polypepvacuoles, Ungermann and co-workers (1998) deduced tide, denoted Golgi-associated ATPase Enhancer of that the formation of the SNARE complex is only an 16 kDa (GATE-16), that functions as a soluble transport intermediate step in the overall fusion reaction. According factor. GATE-16 interacts with N-ethylmaleimideto this view, SNARE molecules are involved in docking sensitive factor (NSF) and significantly stimulates its between donor and acceptor membranes, while another ATPase activity. It also interacts with the Golgi set of proteins participates in subsequent stages of the v-SNARE GOS-28 in an NSF-dependent manner. We fusion process. This notion is supported by Peters and propose that GATE-16 modulates intra-Golgi transport Mayer (1998), who suggested that calmodulin and other through coupling between NSF activity and SNAREs as yet unidentified factors are involved in mediating late activation.stages of vacuolar fusion.

Transport of proteins between intracellular membrane compartments is a highly regulated process that depends on several cytosolic factors. By using the well characterized intra-Golgi cell-free transport assay, we purified from bovine brain cytosol a 56-kDa protein that shows a significant transport activity. Partial sequencing of four tryptic peptides obtained from the 56-kDa protein revealed its identity to a cytosolic protein previously characterized as a selenium-binding protein, SBP56. Recombinant SBP56 expressed in Escherichia coli exhibited transport activity when added to the cellfree intra-Golgi transport. Affinity purified anti-SBP56 polyclonal antibodies specifically inhibited intra-Golgi transport in vitro. Although SBP56 is predominantly localized in the cytosol, a significant amount is associated with membranes. Subcellular fractionation showed that this protein is peripherally associated with the Golgi membrane. The experiments presented in this study indicate that SBP56 participates in late stages of intraGolgi protein transport.

Intracellular transport of newly synthesized and mature proteins via vesicles is controlled by a large group of proteins. Here we describe a ubiquitous rat protein—endoplasmic reticulum (ER) and Golgi 30-kD protein (ERG30)—which shares structural characteristics with VAP-33, a 33-kD protein from Aplysia californica which was shown to interact with the synaptic protein VAMP. The transmembrane topology of the 30-kD ERG30 corresponds to a type II integral membrane protein, whose cytoplasmic NH2 terminus contains a predicted coiled-coil motif. We localized ERG30 to the ER and to pre-Golgi intermediates by biochemical and immunocytochemical methods. Consistent with a role in vesicular transport, anti-ERG30 antibodies specifically inhibit intra-Golgi transport in vitro, leading to significant accumulation of COPI-coated vesicles. It appears that ERG30 functions early in the secretory pathway, probably within the Golgi and between the Golgi and the ER.

Calcium cations play a critical role in regulating vesicular transport between different intracellular membrane-bound compartments. The role of calcium in transport between the Golgi cisternae, however, remains unclear. Using a well characterized cell-free intra-Golgi transport assay, we now show that changes in free Ca 2؉ concentration in the physiological range regulate this transport process. The calcium-chelating agent 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid blocked transport with an IC 50 of approximately 0.8 mM. The effect of 1,2-bis(2-aminophenoxy)-ethane-N,N,N,N-tetraacetic acid was reversible by addition of fresh cytosol and was irreversible when performed in the presence of a Ca 2؉ ionophore that depletes calcium from lumenal stores. We demonstrate here that intra-Golgi transport is stimulated by low Ca 2؉ concentrations (20 -100 nM) but is inhibited by higher concentrations (above 100 nM). Further, we show that calmodulin antagonists specifically block intraGolgi transport, implying a role for calmodulin in mediating the effect of calcium. Our results suggest that Ca 2؉

The membrane-embedded domain of the unusual electron transporter DsbD (DsbDb) uses two redox-active cysteines to catalyze electron transfer between thioredoxin-fold polypeptides on opposite sides of the bacterial cytoplasmic membrane. How the electrons are transferred across the membrane is unknown. Here, we show that DsbDb displays an inherent functional and structural symmetry: first, the two cysteines of DsbDb can be alkylated from both the cytoplasm and the periplasm. Second, when the two cysteines are disulfide-bonded, cysteine scanning shows that the C-terminal halves of the cysteine-containing transmembrane segments 1 and 4 are exposed to the aqueous environment while the N-terminal halves are not. Third, proline residues located pseudo-symmetrically around the two cysteines are required for redox activity and accessibility of the cysteines. Fourth, mixed disulfide complexes, apparent intermediates in the electron transfer process, are detected between DsbDb and thioredoxin molecules on each side of the membrane. We propose a model where the two redox-active cysteines are located at the center of the membrane, accessible on both sides of the membrane to the thioredoxin proteins.