糖尿病大鼠肾脏髓质水通道蛋白2的表达和意义

目的 检测链脲佐菌素诱导的糖尿病（DM）大鼠。肾脏髓质集合管水通道蛋白2（AQP-2）的变化。方法 实验大鼠分为正常对照组（Con组）和糖尿病组（DM组）。在成模12周后，分别用光学显微镜、电子显微镜等方法观察。肾脏形态变化，用免疫组织化学、核酸原位杂交和逆转录PCR方法检测DM大鼠肾髓质集合管AQP-2的表达。结果 （1）实验初DM组与Con组之间血糖、体重差异无统计学意义（P〉0．05），但12周时DM组血糖明显升高（P〈0．01），尿量高于Con组（P〈0．01），体重明显低于Con组（P〈0．01），DM组相对。肾重高于Con组（P〈0．01），二组间血肌酐无明显变化（P〉0．05）。（2）DM组尿渗透压低于Con组（P〈0．05），血渗透压高于Con组（P〈0．05）。（3）DM组大鼠血浆加压素水平比Con组明显升高（P〈0．05）。（4）光镜下。肾脏结构未见明显改变，电镜下可见。肾脏髓质集合管亮细胞和暗细胞结构改变。（5）DM大鼠肾脏髓质集合管AQP-2mRNA及蛋白质的表达增加。结论 DM大鼠肾脏髓质AQP-2mRNA及蛋白质的表达增加，并伴有早期。肾脏病理改变。尿AQP-2有望作为糖尿病。肾病早期诊断指标。     

Factors regulating the abundance and localization of synaptobrevin in the plasma membrane

After synaptic vesicle fusion, vesicle proteins must be segregated from plasma membrane proteins and recycled to maintain a functional vesicle pool. We monitored the distribution of synaptobrevin, a vesicle protein required for exocytosis, in Caenorhabditis elegans motor neurons by using a pH-sensitive synaptobrevin GFP fusion protein, synaptopHluorin. We estimated that 30% of synaptobrevin was present in the plasma membrane. By using a panel of endocytosis and exocytosis mutants, we found that the majority of surface synaptobrevin derives from fusion of synaptic vesicles and that, in steady state, synaptobrevin equilibrates throughout the axon. The surface synaptobrevin was enriched near active zones, and its spatial extent was regulated by the clathrin adaptin AP180. These results suggest that there is a plasma membrane reservoir of synaptobrevin that is supplied by the synaptic vesicle cycle and available for retrieval throughout the axon. The size of the reservoir is set by the relative rates of exo- and endocytosis.     

Loss of AP-3 function affects spontaneous and evoked release at hippocampal mossy fiber synapses

Synaptic vesicle (SV) exocytosis mediating neurotransmitter release occurs spontaneously at low intraterminal calcium concentrations and is stimulated by a rise in intracellular calcium. Exocytosis is compensated for by the reformation of vesicles at plasma membrane and endosomes. Although the adaptor complex AP-3 was proposed to be involved in the formation of SVs from endosomes, whether its function has an indirect effect on exocytosis remains unknown. Using mocha mice, which are deficient in functional AP-3, we identify an AP-3-dependent tetanus neurotoxin-resistant asynchronous release that can be evoked at hippocampal mossy fiber (MF) synapses. Presynaptic targeting of the tetanus neurotoxin-resistant vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) is lost in mocha hippocampal MF terminals, whereas the localization of synaptobrevin 2 is unaffected. In addition, quantal release in mocha cultures is more frequent and more sensitive to sucrose. We conclude that lack of AP-3 results in more constitutive secretion and loss of an asynchronous evoked release component, suggesting an important function of AP-3 in regulating SV exocytosis at MF terminals.     

Cellular and subcellular localization of the vasopressin- regulated urea transporter in rat kidney.

The renal urea transporter (RUT) is responsible for urea accumulation in the renal medulla, and consequently plays a central role in the urinary concentrating mechanism. To study its cellular and subcellular localization, we prepared affinity-purified, peptide-derived polyclonal antibodies against rat RUT based on the cloned cDNA sequence. Immunoblots using membrane fractions from rat renal inner medulla revealed a solitary 97-kDa band. Immunocytochemistry demonstrated RUT labeling of the apical and subapical regions of inner medullary collecting duct (IMCD) cells, with no labeling of outer medullary or cortical collecting ducts. Immunoelectron microscopy directly demonstrated labeling of the apical plasma membrane and of subapical intracellular vesicles of IMCD cells, but no labeling of the basolateral plasma membrane. Immunoblots demonstrated RUT labeling in both plasma membrane and intracellular vesicle-enriched membrane fractions from inner medulla, a subcellular distribution similar to that of the vasopressin-regulated water channel, aquaporin-2. In the outer medulla, RUT labeling was seen in terminal portions of short-loop descending thin limbs. Aside from IMCD and descending thin limbs, no other structures were labeled in the kidney. These results suggest that: (i) the RUT provides the apical pathway for rapid, vasopressin-regulated urea transport in the IMCD, (ii) collecting duct urea transport may be increased by vasopressin by stimulation of trafficking of RUT-containing vesicles to the apical plasma membrane, and (iii) the rat urea transporter may provide a pathway for urea entry into the descending limbs of short-loop nephrons.     

Identification of specific apical membrane polypeptides associated with the antidiuretic hormone-elicited water permeability increase in the toad urinary bladder.

Antidiuretic hormone (ADH) increases the water permeability of the toad urinary bladder. The increase occurs in the apical plasma membrane of granular cells that line the urinary surface of the bladder and is produced by the insertion of water permeability units that have been identified by freeze-fracture electron microscopy as intramembrane particle aggregates. Under water-impermeable conditions, particle aggregates reside in intracellular vesicles called "aggrephores." In response to ADH, the aggrephores fuse with the apical plasma membrane and render it water permeable. When ADH is removed, intramembrane particle aggregates and aggrephores are retrieved from the apical membrane, and it returns to a water-impermeable state. To identify proteins involved in the water permeability response, we used lactoperoxidase/glucose oxidase to 125I-label external apical membrane proteins to compare control and ADH-treated bladders. Several polypeptides were consistently labeled in ADH-treated bladders and not in paired controls. After demonstrating that lactoperoxidase behaves as a fluid-phase marker and is sequestered in aggrephore-like vesicles when ADH is withdrawn, we used the technique of Mellman et al. [Mellman, I.S., Steinman, R. M., Unkeless, J. C. & Cohn, Z. A. (1980) J. Cell Biol. 86, 712-722] to label proteins endocytosed when water permeability declines after ADH is withdrawn to test whether the membrane proteins labeled in ADH-treated bladders behaved like particle aggregates. The internalized membranes contained polypeptides of the same molecular weights (55,000, 17,000-14,000, and 7,000) as those labeled on the apical surface of ADH-treated but not control bladders. These polypeptides are evidently involved in the ADH-stimulated water permeability response and may be components of particle aggregates.     

Yeast synaptobrevin homologs are modified posttranslationally by the addition of palmitate.

Yeast possess two homologs of the synaptobrevin family of vesicle-associated membrane proteins that function in membrane recognition and vesicle fusion. Yeast proteins Snc1 and Snc2 localize to secretory vesicles and are required for constitutive exocytosis. They also form a physical complex with a plasma membrane protein, Sec9, which is necessary for vesicle docking and fusion to occur in vivo. Formation of this molecular complex, as a prerequisite for vesicle fusion, appears to have been conserved evolutionarily. Here we demonstrate that Snc proteins undergo a single posttranslational modification with the addition of a palmitate moiety to Cys-95 in Snc1. Modification of Cys-95 (which is located proximal to the transmembrane domain) is rapid, occurs in the endoplasmic reticulum, and is long-lasting. Mutation of Cys-95 to Ser-95 blocks palmitoylation and appears to affect Snc protein stability. This provides evidence that synaptobrevin-like proteins are modified posttranslationally, and we predict that fatty acylation may be common to those found in higher eukaryotes.     

Three-dimensional structure of endosomes in BHK-21 cells.

The organization of the endosome compartment in BHK-21 cells was studied by using horseradish peroxidase as a fluid-phase marker and Semliki Forest virus as an adsorptive marker. Stereo pairs of semithin sections (0.2-0.5 micron) and computer-aided reconstruction and tracing of serial thin sections (CARTOS) were used to obtain three-dimensional images of the labeled compartments. Two types of labeled organelles could be observed: small vesicles and tubules (approximately equal to 50 nm in diameter) and large complex structures consisting of central vesicular elements (with diameters up to 0.5 micron) and associated systems of radiating tubules. The large endosomes were located either in the peripheral cytoplasm or in the perinuclear region, and, importantly, they existed as independent organelles and not as an interconnected reticulum. Each endosomal vacuole had two to seven tubules oriented in random directions from the central vesicle. The tubules were 60-100 nm in diameter and up to 4 micron in length. Morphometric estimates indicated that 60-70% of the endosomal membrane was in the tubules, in contrast to 30-40% of the volume. No structural continuity between endosomes and Golgi cisternae was observed, although elements of the two systems were frequently found in close proximity.     

Rat and gerbil pinealocytes contain the synaptosomal-associated protein 25 (SNAP-25)

Abstract: It has recently been established that the neuroendocrine pinealocytes of mammals contain several synaptic membrane proteins that are involved in the regulation of vesicle trafficking in the nerve terminal. In the present study, we have conducted immunoblot and immunocytochemical analyses to demonstrate that another key component of the presynaptic plasmalemma, i.e., protein SNAP-25 (synaptosomal-associated protein 25 kDa), can be detected in pinealocytes. Immunostaining of serial semi-thin sections of plastic-embedded rat and gerbil pineals with monoclonal SNAP-25 antibodies showed that SNAP-25 was present in pinealocytes of both species. We proved its coexpression with other synaptic membrane proteins (synaptophysin, synaptotagmin I, synaptobrevin II, and syntaxin I) at the single cell level. Thus, pinealocytes obviously are endowed with the major proteins that are thought to regulate the targeting and exocytosis of secretory vesicles, in particular of synaptic -like microvesicles.     

Minireview: aquaporin 2 trafficking

In the kidney aquaporin-2 (AQP2) provides a target for hormonal regulation of water transport by vasopressin. Short-term control of water permeability occurs via vesicular trafficking of AQP2 and long-term control through changes in the abundance of AQP2 and AQP3 water channels. Defective AQP2 trafficking causes nephrogenic diabetes insipidus, a condition characterized by the kidney inability to produce concentrated urine because of the insensitivity of the distal nephron to vasopressin. AQP2 is redistributed to the apical membrane of collecting duct cells through activation of a cAMP signaling cascade initiated by the binding of vasopressin to its V2-receptor. Protein kinase A-mediated phosphorylation of AQP2 has been proposed to be essential in regulating AQP2-containing vesicle exocytosis. Cessation of the stimulus is followed by endocytosis of the AQP2 proteins exposed on the plasma membrane and their recycling to the original stores, in which they are retained. Soluble N-ethylmaleimide sensitive fusion factor attachment protein receptors (SNARE) and actin cytoskeleton organization regulated by small GTPase of the Rho family were also proved to be essential for AQP2 trafficking. Data for functional involvement of the SNARE vesicle-associated membrane protein 2 in AQP2 targeting has recently been provided. Changes in AQP2 expression/trafficking are of particular importance in pathological conditions characterized by both dilutional and concentrating defects. One of these conditions, hypercalciuria, has shown to be associated with alteration of AQP2 urinary excretion. More precisely, recent data support the hypothesis that, in vivo external calcium, through activation of calcium-sensing receptors, modulates the expression/trafficking of AQP2. Together these findings underscore the importance of AQP2 in kidney pathophysiology.     

Conformation of the synaptobrevin transmembrane domain

The synaptic vesicle protein synaptobrevin (also called VAMP, vesicle-associated membrane protein) forms part of the SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) complex, which is essential for vesicle fusion. Additionally, the synaptobrevin transmembrane domain can promote lipid mixing independently of complex formation. Here, the conformation of the transmembrane domain was studied by using circular dichroism and attenuated total reflection Fourier-transform infrared spectroscopy. The synaptobrevin transmembrane domain has an alpha-helical structure that breaks in the juxtamembrane region, leaving the cytoplasmic domain unstructured. In phospholipid bilayers, infrared dichroism data indicate that the transmembrane domain adopts a 36 degrees angle with respect to the membrane normal, similar to that reported for viral fusion peptides. A conserved aromatic/basic motif in the juxtamembrane region may be causing this relatively high insertion angle.     

Plasma antidiuretic hormone levels and kidney responsiveness to vasopressin in the jerboa, Jaculus orientalis

The plasma antidiuretic hormone (ADH) concentration and the kidney medulla responsiveness to vasopressin were measured in adult jerboas ( Jaculus orientalis) in different states of hydration. In 15 jerboas adapted to 30 degrees and fed a dry diet, the average ADH concentration in blood plasma was 479 +/- 59 pg/ml, as measured by a radioimmunoassay. About 6 hr after receiving a 5% body wt water load by gavage, the plasma ADH concentration fell to 130 +/- 30 pg/ml in the 5 jerboas still producing hypertonic urine (1022 +/- 267 mosmol/liter) and to 41.5 +/- 8.4 pg/ml in the 6 jerboas producing hypoosmotic urine (157 +/- 6 mosmol/liter). In vitro biochemical experiments were performed on the kidney medullas from two groups of 5 jerboas fed a dry diet (group I) or a water-enriched diet (group II), respectively, for 4 to 7 weeks. Compared to group II, group I animals exhibited (a) higher plasma ADH values, 372 +/- 86 versus 76 +/- 25 pg/ml; (b) higher urine osmolarities (3817 +/- 638 versus 647 +/- 90 mosmol/liter); (c) some decrease in [3H]lysine-vasopressin (LVP) binding capacity to kidney membrane fractions (maximal binding: 0.4 versus 0.6 pmol [3H]LVP bound/mg protein); d) decreased adenylate cyclase responses to arginine-vasopressin, lysine-vasopressin, and oxytocin in kidney membrane fractions; and (e) weaker adenylate cyclase responses to arginine-vasopressin in microdissected pieces of the medullary thick ascending limb of Henle's loop. The values found for (a) the dissociation constant of [3H]lysine-vasopressin binding to membranes (KD); (b) adenylate cyclase sensitivity to the three neurohypophyseal hormones (KA); and (c) adenylate cyclase sensitivity to arginine-vasopressin (KA) in medullary collecting tubules and medullary thick ascending limbs are similar in the two groups of jerboas and roughly comparable to those previously reported for the rat kidney medulla. The reduced maximal adenylate cyclase responses to vasopressin in the jerboas fed a dry diet might indicate some physiological "down regulation" of the number of vasopressin-specific receptors in the kidney as a result of the huge ADH concentration present in blood plasma under these conditions. However, this desensitization is not sufficient to account for the production of hypoosmotic urine in spite of the relatively high ADH plasma levels which persisted after acute overhydration.     

Mouse VAP33 is associated with the endoplasmic reticulum and microtubules

VAMP/synaptobrevin is a synaptic vesicle protein that is essential for neurotransmitter release. Intracellular injection of antisera against the Aplysia californica VAMP/synaptobrevin-binding protein ApVAP33 inhibited evoked excitatory postsynaptic potentials (EPSPs) in cultured cells, suggesting that this association may regulate the function of VAMP/synaptobrevin. We have identified and characterized a mouse homologue of ApVAP33, mVAP33. The overall domain structure of the proteins is conserved, and they have similar biochemical properties. mVAP33 mRNA is detectable in all mouse tissues examined, in contrast to the more restricted expression seen in A. californica. We analyzed the cellular distribution of mVAP33 protein in brain slices and cultured cortical cells by light and electron microscopy. Although present at higher levels in neurons, immunoreactivity was detected throughout both neurons and glia in a reticular pattern similar to that of endoplasmic reticulum-resident proteins. mVAP33 does not colocalize with VAMP/synaptobrevin at synaptic structures, but expression overlaps with lower levels of VAMP/synaptobrevin in the soma. Ultrastructural analysis revealed mVAP33 associated with microtubules and intracellular vesicles of heterogeneous size. In primary neuronal cultures, large aggregates of mVAP33 are also detected in short filamentous structures, which are occasionally associated with intracellular membranes. There is no evidence for accumulation of mVAP33 on synaptic vesicles or at the plasma membrane. These data suggest that mVAP33 is an endoplasmic-reticulum-resident protein that associates with components of the cytoskeleton. Any functional interaction between mVAP33 and VAMP/synaptobrevin, therefore, most likely involves the delivery of components to synaptic terminals rather than a direct participation in synaptic vesicle exocytosis.     

Expression Of aquaporin-1 (AQP-1) in rat heart

Background. AQP-1, a channel-forming integral membrane protein of 28 kDa prevalent in red blood cells and renal proximal tubules, was recently shown to be expressed in rat heart.Aims: Our purpose was to charaterise the experssion of the AQO-1 gene in rat heart with respect to cell type, developmental stage and pathophysiological condition. Methods: To determine in which heart cell type the water channel was expressed, we measured AQP-1 mRNA and protein levels and immunolocalised protein in freshly isolated myocytes from adult rats and cultured myocytes and fibroblasts from neonatal rats. Results: Northern blot analysis showed that AQP-1 mRNA is expressed in adult cardiac myocytes, neonatal myocytes, and neonatal cardiac fibroblasts of rats at levels nearly as high as those observed in rat kidney. Western blot analysis, however, detected AQP-1 protein only in purified adult and neonatal cardiac myocytes at levels markedly less than kidney. Immunohistochemistry and confocal imaging localised AQP-1 protein to the sarcolemmal membrane of myocytes. Since the expression of other membrane-spanning proteins is altered during hypertrophy, we determined the level of AQP-1 mRNA in hearts of aortic-constricted (AC) rats. The level of AQP-1 mRNA decreased progressively after AC, and was 42% less than the level in left ventricles (LVs) of sham-operated control rats after 3 days of AC. Conclusions: These data indicate that AQP-1 is expressed in cardiac myocytes of adult and neonatal rats, and its expression is modulated in the rat heart during pressure-overload hypertrophy.     

水通道蛋白与糖尿病肾病

水通道蛋白即存在于细胞膜上的特异性水转运孔道,目前发现的水通道蛋白已有11种。有研究证明,在糖尿病中水通道蛋白-1,2,3的表达是上调的,这有助于防止糖尿病时机体溶质和水的进一步丢失。有学者认为糖尿病肾病中尿排泄水通道蛋白-2减少,这可能与集合管细胞对血管加压素应答的细胞信号转导途径受损有关,而且在糖尿病酮症酸中毒患者治疗期间,可以通过尿排泄水通道蛋白-2来估计循环血容量的恢复情况。     

SNARE motif-mediated sorting of synaptobrevin by the endocytic adaptors clathrin assembly lymphoid myeloid leukemia (CALM) and AP180 at synapses

Neurotransmission depends on the exo-endocytosis of synaptic vesicles at active zones. Synaptobrevin 2 [also known as vesicle-associated membrane protein 2 (VAMP2)], the most abundant synaptic vesicle protein and a major soluble NSF attachment protein receptor (SNARE) component, is required for fast calcium-triggered synaptic vesicle fusion. In contrast to the extensive knowledge about the mechanism of SNARE-mediated exocytosis, little is known about the endocytic sorting of synaptobrevin 2. Here we show that synaptobrevin 2 sorting involves determinants within its SNARE motif that are recognized by the ANTH domains of the endocytic adaptors AP180 and clathrin assembly lymphoid myeloid leukemia (CALM). Depletion of CALM or AP180 causes selective surface accumulation of synaptobrevin 2 but not vGLUT1 at the neuronal surface. Endocytic sorting of synaptobrevin 2 is mediated by direct interaction of the ANTH domain of the related endocytic adaptors CALM and AP180 with the N-terminal half of the SNARE motif centered around M46, as evidenced by NMR spectroscopy analysis and site-directed mutagenesis. Our data unravel a unique mechanism of SNARE motif-dependent endocytic sorting and identify the ANTH domain proteins AP180 and CALM as cargo-specific adaptors for synaptobrevin endocytosis. Defective SNARE endocytosis may also underlie the association of CALM and AP180 with neurodevelopmental and cognitive defects or neurodegenerative disorders.     

Angiotensin II increases H+-ATPase B1 subunit expression in medullary collecting ducts

Metabolic alkalosis is a common feature of hypokalemic hypertensive syndromes associated with angiotensin II excess. The alkalosis-generating effect of angiotensin II is usually ascribed to its stimulatory effect on aldosterone secretion, a hormone that upregulates collecting duct hydrogen ion secretion. We studied the effect of angiotensin II infusions on the expression of B1 and a4 protein, subunits of the renal H+-ATPase in adrenalectomized rats. Adrenalectomized rats were given either angiotensin II or vehicle for 7 days via osmotic mini-pumps. H+-ATPase B1 protein expression was evaluated by Western blot analysis in isolated medulla and cortex plasma membrane preparations from one kidney, whereas the contralateral kidney was used for immunostaining. By Western blotting, the relative abundance of B1 protein was 2-fold higher in renal medulla membranes from rats with intact adrenal glands (sham surgery) than from adrenalectomized rats (219+/-47%, n=12; P<0.05). In contrast to renal medulla, adrenalectomy did not significantly alter the relative abundance of B1 protein in renal cortex. Angiotensin II also did not significantly alter the relative levels of B1 protein in the cortex, but it increased it significantly in renal medullary membranes (231+/-56%, n=8; P<0.005). Moreover, enhanced H+-ATPase B1 subunit protein immunoreactivity was found in medullary collecting duct segments of rats infused with angiotensin II. In contrast to B1, expression of a4, another subunit of the H+-ATPase was not altered by adrenalectomy or angiotensin II. We conclude that adrenalectomy decreases whereas angiotensin II increases H+-ATPase B1 subunit expression in medullary, but not in cortical collecting ducts. By increasing the relative abundance of the B1 subunit of H+-ATPase in the collecting duct, angiotensin II excess may lead to increased hydrogen ion secretion and thus metabolic alkalosis-a common feature of hypertensive syndromes associated with angiotensin II overactivity.     

Exiting the endoplasmic reticulum

The movement of nascent proteins from sites of synthesis to final cellular or extracellular destinations involves their transport through a distinct series of vesicular compartments. Vesicle biogenesis is regulated by specific proteins and co-factors that control distinct steps including budding, transport, docking, and fusion with target membranes. Budding requires assembly of a coat protein complex on the membrane, membrane deformation and the subsequent cleavage of the nascent vesicle from donor membrane. Coat proteins may also mediate vesicle interactions with the cytoskeleton or insulate the vesicles from fusion with unwanted compartments. Three classes of cytoplasmic coats have been identified. (1) Clathrin, interacting with different adaptor proteins, participates in endocytosis, lysosome biogenesis and as yet unidentified vesicular transport processes that arise in the trans-Golgi region of cells [reviewed in (Kreis, T.E., Lowe, M., Pepperkok, R., 1995. COPs regulating membrane traffic. Ann. Rev. Cell. Dev. Biol. 11, 677--706.)]. (2) The COPI coatomer is involved in retrograde traffic within the Golgi and from the cis-Golgi region to the endoplasmic reticulum (ER). It may also participate in anterograde transport from the ER [reviewed in (Aridor, M., Balch, W.E., 1999. Integration of endoplasmic reticulum signaling in health and disease. Nature 5, 745--751.)]. (3) COPII coats mediate anterograde transport of cargo out of the ER [Barlowe, C., Orci, L., Yeung, T., Hosobuchi, M., Hamamoto, S., Salama, N., Rexach, M.F., Ravazazola, M., Amherdt, M., Schekman, R., 1994. COPII: a membrane coat formed by sec proteins that drive vesicle budding from the endoplasmic reticulum. Cell 77, 895--907; Scales, S.J., Gomez, M., Kreis, T.E., 2000. Coat proteins regulating membrane traffic. Int. Rev. Cytol. 195, 67--144.]. The COPII coat is required for budding from the ER and ER to Golgi trafficking. Further, COPII proteins also participate in cargo selection and concentrate some nascent proteins in the budding vesicle. Recent studies have shown that human disease may result from mutations that affect proteins in COPII vesicles.     

Suppression of Arabidopsis vesicle-SNARE expression inhibited fusion of H2O2-containing vesicles with tonoplast and increased salt tolerance

Intracellular vesicle trafficking performs essential functions in eukaryotic cells, such as membrane trafficking and delivery of molecules to their destinations. A major endocytotic route in plants is vesicle trafficking to the vacuole that plays an important role in plant salt tolerance. The final step in this pathway is mediated by the AtVAMP7C family of vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptors (v-SNAREs) that carry out the vesicle fusion with the tonoplast. Exposure to high-salt conditions causes immediate ionic and osmotic stresses, followed by production of reactive oxygen species. Here, we show that the reactive oxygen species are produced intracellularly, in endosomes that were targeted to the central vacuole. Suppression of the AtVAMP7C genes expression by antisense AtVAMP711 gene or in mutants of this family inhibited fusion of H2O2-containing vesicles with the tonoplast, which resulted in formation of H2O2-containing megavesicles that remained in the cytoplasm. The antisense and mutant plants exhibited improved vacuolar functions, such as maintenance of DeltapH, reduced release of calcium from the vacuole, and greatly improved plant salt tolerance. The antisense plants exhibited increased calcium-dependent protein kinase activity upon salt stress. Improved vacuolar ATPase activity during oxidative stress also was observed in a yeast system, in a DeltaVamp7 knockout strain. Interestingly, a microarray-based analysis of the AtVAMP7C genes showed a strong down-regulation of most genes in wild-type roots during salt stress, suggesting an evolutionary molecular adaptation of the vacuolar trafficking.