SummaryThe cellular glutathione redox buffer is assumed to be part of signal transduction pathways transmitting environmental signals during biotic and abiotic stress, and thus is essential for regulation of metabolism and development. Ratiometric redox-sensitive GFP (roGFP) expressed in Arabidopsis thaliana reversibly responds to redox changes induced by incubation with H 2 O 2 or DTT. Kinetic analysis of these redox changes, combined with detailed characterization of roGFP2 in vitro, shows that roGFP2 expressed in the cytosol senses the redox potential of the cellular glutathione buffer via glutaredoxin (GRX) as a mediator of reversible electron flow between glutathione and roGFP2. The sensitivity of roGFP2 toward the glutathione redox potential was tested in vivo through manipulating the glutathione (GSH) content of wild-type plants, through expression of roGFP2 in the cytosol of low-GSH mutants and the endoplasmic reticulum (ER) of wild-type plants, as well as through wounding as an example for stress-induced redox changes. Provided the GSH concentration is known, roGFP2 facilitates the determination of the degree of oxidation of the GSH solution. Assuming sufficient glutathione reductase activity and non-limiting NADPH supply, the observed almost full reduction of roGFP2 in vivo suggests that a 2.5 mM cytosolic glutathione buffer would contain only 25 nM oxidized glutathione disulfide (GSSG). The high sensitivity of roGFP2 toward GSSG via GRX enables the use of roGFP2 for monitoring stressinduced redox changes in vivo in real time. The results with roGFP2 as an artificial GRX target further suggest that redox-triggered changes of biologic processes might be linked directly to the glutathione redox potential via GRX as the mediator.

SNARE (SNAP receptor) proteins drive intracellular membrane fusion and contribute specificity to membrane trafficking. The formation of SNAREpins between membranes is spatially and temporally controlled by a network of sequentially acting accessory components. These regulators add an additional layer of specificity, arrest SNAREpin intermediates, lower the energy required for fusion, and couple membrane fusion to triggering signals. The functional activity of some of these regulators determines the plasticity of regulated exocytosis.

Sec1p/Munc18 proteins and SNAP receptors (SNAREs) are key components of the intracellular membrane fusion machinery. Compartment-specific v-SNAREs on a transport vesicle pair with their cognate t-SNAREs on the target membrane and drive lipid bilayer fusion. In a reconstituted assay that dissects the sequential assembly of t-SNARE (syntaxin 1⅐SNAP-25) and v-/t-SNARE (VAMP2⅐syntaxin 1⅐SNAP-25) complexes, and finally measures lipid bilayer merger, we resolved the inhibitory and stimulatory functions of the Sec1p/Munc18 protein Munc18-1 at the molecular level. Inhibition of membrane fusion by Munc18-1 requires a closed conformation of syntaxin 1. Remarkably, the concurrent preincubation of Munc18-1-inhibited syntaxin 1 liposomes with both VAMP2 liposomes and SNAP-25 at low temperature releases the inhibition and effectively stimulates membrane fusion. VAMP8 liposomes can neither release the inhibition nor exert the stimulatory effect, demonstrating the need for a specific Munc18-1/VAMP2 interaction. In addition, Munc18-1 binds to the N-terminal peptide of syntaxin 1, which is obligatory for a robust stimulation of membrane fusion. In contrast, this interaction is neither required for the inhibitory function of Munc18-1 nor for the release of this block. These results indicate that Munc18-1 and the neuronal SNAREs already have the inherent capability to function as a basic stage-specific off/on switch to control membrane fusion.Membrane fusion in eukaryotic cells is mediated by a conserved machinery consisting of compartment-specific v-SNAREs 3 on transport vesicles and t-SNAREs on the target membrane (1-4). SNAREs are characterized by SNARE motifs, stretches of 60 -70 amino acids, which contain heptad repeats with a central "0" layer and assemble into specific four-helix bundles (5). The formation of SNAREpins, trans v-/t-SNARE complexes bridging two membranes, occurs in a zipper-like manner that starts at the membrane distal (N-terminal) end of the SNAREpins and proceeds toward the (C-terminal) membrane-spanning anchors of the SNAREs (6, 7). Zippering brings the two lipid bilayers in close apposition, finally resulting in membrane merger (2,8). Thus, the energy required for membrane fusion is provided by the exergonic folding of the largely unstructured v-and t-SNARE proteins into stable four-helix bundles (2, 5, 9). Although SNAREs can be considered to be the minimal membrane fusion machinery, in the physiological cellular environment, an array of accessory proteins and lipids controls the spatial and temporal activity of SNARE proteins (10).One class of accessory proteins, the SM (Sec1p/Munc18) proteins, directly bind to SNAREs, control their activity, and are required for membrane fusion in vivo (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). SM proteins contain about 600 amino acids, which are folded into an archshaped structure. At least two SNARE-binding modes have been described. In the first mode, the SM protein binds the t-SNARE component syntaxin in a "closed" conformation, in which the N-terminal three...

The interaction of Munc18-1 helix 11 and 12 with the central region of the VAMP2 SNARE motif is essential for SNARE templating and synaptic transmission

Membrane fusion is fundamental for a broad variety of physiological processes, such as synaptic transmission, fertilization, and viral entry. Intracellular fusion along the secretory and endocytic pathway is mediated by SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. When recombinant v- and t-SNAREs are reconstituted into distinct liposome populations, membrane fusion can be monitored by either lipid or content mixing. The in vitro assays use fluorescence dequenching to measure vesicle fusion. The lipid-mixing assay is based on fluorescence resonance energy transfer between the fluorophores 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD) and rhodamine, which are covalently coupled to lipids. Fusion of labeled v-SNARE liposomes with unlabeled t-SNARE liposomes increases the distance between NBD and rhodamine, increasing the NBD fluorescence. In the content-mixing assay, the water-soluble fluorophore 8-Hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) (pyranine) and its quencher p-Xylene-bis-pyridinium bromide (DPX) are incorporated into v-SNARE vesicles. The fusion of labeled v-SNARE vesicles with unlabeled t-SNARE vesicles dilutes the quencher and thus increases HPTS fluorescence. By controlling the lipid and protein composition, these assays provide important tools to detect fusion intermediates (e.g., hemifusion), and to elucidate the molecular mechanisms that regulate membrane fusion.