Silicon is beneficial to plant growth and helps plants to overcome abiotic and biotic stresses by preventing lodging (falling over) and increasing resistance to pests and diseases, as well as other stresses. Silicon is essential for high and sustainable production of rice, but the molecular mechanism responsible for the uptake of silicon is unknown. Here we describe the Low silicon rice 1 (Lsi1) gene, which controls silicon accumulation in rice, a typical silicon-accumulating plant. This gene belongs to the aquaporin family and is constitutively expressed in the roots. Lsi1 is localized on the plasma membrane of the distal side of both exodermis and endodermis cells, where casparian strips are located. Suppression of Lsi1 expression resulted in reduced silicon uptake. Furthermore, expression of Lsi1 in Xenopus oocytes showed transport activity for silicon only. The identification of a silicon transporter provides both an insight into the silicon uptake system in plants, and a new strategy for producing crops with high resistance to multiple stresses by genetic modification of the root's silicon uptake capacity.

No abstract

Summary Iron acquisition of graminaceous plants is characterized by the synthesis and secretion of the iron‐chelating phytosiderophore, mugineic acid (MA), and by a specific uptake system for iron(III)–phytosiderophore complexes. We identified a gene specifically encoding an iron–phytosiderophore transporter (HvYS1) in barley, which is the most tolerant species to iron deficiency among graminaceous plants. HvYS1 was predicted to encode a polypeptide of 678 amino acids and to have 72.7% identity with ZmYS1, a first protein identified as an iron(III)–phytosiderophore transporter in maize. Real‐time RT‐PCR analysis showed that the HvYS1 gene was mainly expressed in the roots, and its expression was enhanced under iron deficiency. In situ hybridization analysis of iron‐deficient barley roots revealed that the mRNA of HvYS1 was localized in epidermal root cells. Furthermore, immunohistological staining with anti‐HvYS1 polyclonal antibody showed the same localization as the mRNA. HvYS1 functionally complemented yeast strains defective in iron uptake on media containing iron(III)–MA, but not iron–nicotianamine (NA). Expression of HvYS1 in Xenopus oocytes showed strict specificity for both metals and ligands: HvYS1 transports only iron(III) chelated with phytosiderophore. The localization and substrate specificity of HvYS1 is different from those of ZmYS1, indicating that HvYS1 is a specific transporter for iron(III)–phytosiderophore involved in primary iron acquisition from soil in barley roots.

Vacuolar-type H+-ATPase (V-ATPase) is a multi-subunit enzyme that has important roles in the acidification of a variety of intracellular compartments and some extracellular milieus. Four isoforms for the membrane-intrinsic subunit (subunit a) of the V-ATPase have been identified in mammals, and they confer distinct cellular localizations and activities on the proton pump. We found that V-ATPase with the a3 isoform is highly expressed in pancreatic islets, and is localized to membranes of insulin-containing secretory granules in β-cells. oc/oc mice, which have a null mutation at the a3 locus, exhibited a reduced level of insulin in the blood, even with high glucose administration. However, islet lysates contained mature insulin, and the ratio of the amount of insulin to proinsulin in oc/oc islets was similar to that of wild-type islets, indicating that processing of insulin was normal even in the absence of the a3 function. The insulin contents of oc/oc islets were reduced slightly, but this was not significant enough to explain the reduced levels of the blood insulin. The secretion of insulin from isolated islets in response to glucose or depolarizing stimulation was impaired. These results suggest that the a3 isoform of V-ATPase has a regulatory function in the exocytosis of insulin secretion.

The vacuolar-type H؉ -ATPase (V-ATPase) translocates protons across membranes. Here, we have identified a mouse cDNA coding for a fourth isoform (a4) of the membrane sector subunit a of V-ATPase. This isoform was specifically expressed in kidney, but not in the heart, brain, spleen, lung, liver, muscle, or testis. Immunoprecipitation experiments, together with sequence similarities for other isoforms (a1, a2, and a3), indicate that the a4 isoform is a component of V-ATPase. Moreover, histochemical studies show that a4 is localized in the apical and basolateral plasma membranes of cortical ␣-and ␤-intercalated cells, respectively. These results suggest that the V-ATPase, with the a4 isoform, is important for renal acid/base homeostasis. A ubiquitous vacuolar-type Hϩ -ATPase (V-ATPase) 1 translocates protons across membranes utilizing the energy of ATP hydrolysis (for reviews, see Refs. 1-5). The organellar acidic pH generated by V-ATPase is responsible for various processes including zymogen activation, receptor-mediated endocytosis, macromolecule degradation, and protein sorting. The enzyme is also found in plasma membranes, where it transports protons outside cells such as osteoclasts (6, 7), renal-intercalated cells (8), and epithelial cells of the seminal duct and bladder (9, 10).V-ATPase has a membrane peripheral V 1 sector for ATP hydrolysis and an integral V o for proton translocation (1-5). The V o sector consists of at least five subunits (a, c, cЈ, cЉ, and d) (11). Subunit a is the largest (116 kDa) of the V-ATPase subunits, and its isoforms have been found in yeast (12), chicken (13), mouse (7, 14, 15), cow (16, 17), and human (18). These isoforms exhibit different distributions in organelles and tissues. Yeast isoforms (Vph1p and Stv1p) are localized in vacuoles and Golgi/endosomes, respectively (12). Three isoforms (a1, a2, and a3) have been found previously in mammals (7, 14 -18). These isoforms may be important for the localization of V-ATPase in various organelles or plasma membranes.In this study, we have isolated a mouse cDNA coding for a fourth isoform (a4) of V-ATPase subunit a. Isoform a4 exhibits high similarities with the a1, a2, and a3 isoforms and is expressed exclusively in kidney. Furthermore, a4 was localized immunochemically in the apical and basolateral plasma membranes of cortical ␣-and ␤-intercalated cells, respectively. These results suggest that the V-ATPase with the a4 isoform is a kidney-specific proton pump important for acid/base homeostasis.

Osteoclasts generate a massive acid flux to mobilize bone calcium. Local extracellular acidification is carried out by vacuolar type H ؉ -ATPase (V-ATPase) localized in the plasma membrane. We have shown that a3, one of the four subunit a isoforms (a1, a2, a3, and a4), is a component of the plasma membrane V-ATPase (Toyomura, T., Oka, T., Yamaguchi, C., Wada, Y., and Futai, M. (2000) J. Biol. Chem. 275, 8760 -8765). To establish the unique localization of V-ATPase, we have used a murine macrophage cell line, RAW 264.7, that can differentiate into multinuclear osteoclast-like cells on stimulation with RANKL (receptor activator of nuclear factor B ligand). The V-ATPase with the a3 isoform was localized to late endosomes and lysosomes, whereas those with the a1 and a2 isoforms were localized to organelles other than lysosomes. After stimulation, the V-ATPase with the a3 isoform was immunochemically colocalized with lysosome marker lamp2 and was detected in acidic organelles. These organelles were also colocalized with microtubules, and the signals of lamp2 and a3 were dispersed by nocodazole, a microtubule depolymerizer. In RAW-derived osteoclasts cultured on mouse skull pieces, the a3 isoform was transported to the plasma membrane facing the bone and accumulated inside podosome rings. These findings indicate that V-ATPases with the a3 isoform localized in late endosomes/lysosomes are transported to the cell periphery during differentiation and finally assembled into the plasma membrane of mature osteoclasts.Osteoclasts are multinuclear bone-resorbing cells derived from hematopoietic stem cells (1, 2) that form an extracellular compartment (resorption lacunae) between the plasma membrane (ruffled border) and the bone surface. The acidic pH of the lacunae (3, 4) is essential for mineral solubilization and hydrolysis of bone matrix by enzymes, including collagenase and cathepsin K (5). The degraded matrix is transported in luminal acidic vesicles (organelles) to the secretory domain facing the extracellular space (6). Vacuolar type H ϩ -ATPase (V-ATPase) 1 functions in the ruffled border as a proton pump that acidifies the resorption lacunae. Although the important role of V-ATPase in bone resorption has been established, much less is known about the mechanism of targeting the enzyme during osteoclast differentiation. Meanwhile, V-ATPases function in the membranes of ubiquitous organelles, including secretory vesicles, endosomes, the Golgi apparatus, and lysosomes. Thus, it is of interest to know how V-ATPases are localized to the various membranes and whether or not they have different compositions depending on the cellular location (7).V-ATPase is a multisubunit complex formed from a catalytic V 1 sector and a membrane-spanning V O sector (7-13). The V O sector may have a pertinent role in localizing V-ATPases to various cellular membranes. The yeast V O sector is composed of five subunits (a, c, cЈ, cЉ, and d) and has two subunit a isoforms, Stv1p and Vph1p. The nematode Caenorhabditis elegans has two c (14 -16) ...

The clinical manifestations of classical Menkes disease, mild Menkes disease and occipital horn syndrome are reviewed. Menkes disease is a neurodegenerative disease with X-linked recessive inheritance. Orally administered copper accumulates in the intestine, resulting in the failure of copper absorption. The primary metabolic defect that causes copper accumulation in the intestine is present in almost all extrahepatic tissues. The blood, liver and brain are in a state of copper deficiency, which is due to defective copper absorption. The characteristic features, including neurological disturbances, arterial degeneration and hair abnormalities, can be explained by the decrease in cuproenzyme activities. DNA-based diagnosis is now possible. Mild Menkes disease and occipital horn syndrome, which show milder forms than Menkes disease, have been identified as genetic disorders resulting from mutations in the Menkes disease gene. Because the clinical spectrum of Menkes disease is wide, males with mental retardation and connective tissue abnormalities should be evaluated for biochemical evidence of defective copper transport. The treatment accepted currently is parenteral administration of copper. When treatment is started in patients with classical Menkes disease above the age of 2 months, it does not improve the neurological degeneration. When the treatment is initiated in newborn babies affected with this disease, the neurological degeneration can be prevented in some, but not all, cases. Moreover, early treatment cannot improve non-neurological problems, such as connective tissue laxity. Therefore, alternative therapies for Menkes disease and occipital horn syndrome should be studied.

The vacuolar-type H؉ -ATPases (V-ATPases) are multimeric proton pumps involved in a wide variety of physiological processes. We have identified two alternative splicing variants of C2 subunit isoforms: C2-a, a lungspecific isoform containing a 46-amino acid insertion, and C2-b, a kidney-specific isoform without the insert. Immunohistochemistry with isoform-specific antibodies revealed that V-ATPase with C2-a is localized specifically in lamellar bodies of type II alveolar cells, whereas the C2-b isoform is found in the plasma membranes of renal ␣ and ␤ intercalated cells. Immunoprecipitation combined with immunohistological analysis revealed that C2-b together with other kidney-specific isoforms was selectively assembled to form a unique proton pump in intercalated cells. Furthermore, a chimeric yeast VATPase with mouse the C2-a or C2-b isoform showed a lower K m(ATP) and lower proton transport activity than that with C1 or Vma5p (yeast C subunit). These results suggest that V-ATPases with the C2-a and C2-b isoform are involved in luminal acidification of lamellar bodies and regulation of the renal acid-base balance, respectively.Highly differentiated endomembrane organelles, including the Golgi apparatus, lysosomes, endosomes, and secretory vesicles, have a luminal acidic pH, which is required for various cellular functions. The acidic pH is established by a ubiquitously expressed multisubunit proton pump, vacuolar type H ϩ -ATPase (V-ATPase) 1 (for reviews, see Refs. 1-5). In addition to the intracellular organelles, the V-ATPase is localized in the plasma membranes of highly differentiated cells, including osteoclasts (6), kidney intercalated cells (7), and male tract epithelial cells (8), where it is required for bone metabolism, urine acidification, and spermatogenesis, respectively. Furthermore, the same enzyme is required for the acidification of specialized organelles, including synaptic vesicles (9, 10) and acrosomes (11). Thus, assembly of V-ATPase, its targeting to final destinations, and its proper regulation are essential for the diverse physiological functions.V-ATPase has a membrane peripheral V 1 sector for ATP hydrolysis and an integral V O sector for proton translocation and exhibits similarity to F-ATPase (ATP synthase) in both structure and catalytic mechanism. The ATP-dependent conformational changes are transmitted between the peripheral complex (V 1 or F 1 ) and the proton pore (V O or F O ) through a number of subunits forming a stalk (1, 3, 5). We have demonstrated that the catalytic mechanism involving subunit rotation is conserved in V-and F-ATPases (12-16). Deletion of mammalian V O subunit c, which is encoded by a single gene (17, 18), has been shown to cause an embryonic lethal phenotype (19), indicating that the enzyme is essential in early development. The subunits in the stalk region are required for activity and assembly in yeast (20) and mammals (11,21).Recent studies suggested that the diverse physiological functions of V-ATPase are established through the utilization of speci...