RESUMO
In various cellular membrane systems, vacuolar ATPases (V-ATPases) function as proton pumps, which are involved in many processes such as bone resorption and cancer metastasis, and these membrane proteins represent attractive drug targets for osteoporosis and cancer. The hydrophilic V(1) portion is known as a rotary motor, in which a central axis DF complex rotates inside a hexagonally arranged catalytic A(3)B(3) complex using ATP hydrolysis energy, but the molecular mechanism is not well defined owing to a lack of high-resolution structural information. We previously reported on the in vitro expression, purification and reconstitution of Enterococcus hirae V(1)-ATPase from the A(3)B(3) and DF complexes. Here we report the asymmetric structures of the nucleotide-free (2.8 Å) and nucleotide-bound (3.4 Å) A(3)B(3) complex that demonstrate conformational changes induced by nucleotide binding, suggesting a binding order in the right-handed rotational orientation in a cooperative manner. The crystal structures of the nucleotide-free (2.2 Å) and nucleotide-bound (2.7 Å) V(1)-ATPase are also reported. The more tightly packed nucleotide-binding site seems to be induced by DF binding, and ATP hydrolysis seems to be stimulated by the approach of a conserved arginine residue. To our knowledge, these asymmetric structures represent the first high-resolution view of the rotational mechanism of V(1)-ATPase.
Assuntos
Enterococcus/enzimologia , Modelos Moleculares , ATPases Vacuolares Próton-Translocadoras/química , Sítios de Ligação , Cristalização , Enterococcus/genética , Mutação , Nucleotídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas , Rotação , ATPases Vacuolares Próton-Translocadoras/genéticaRESUMO
In yeast cells growing under nutrient-rich condition approximately 50% of total amino acids are accumulated in the vacuoles; however, the composition of amino acids in the cytosol and in the vacuoles is quite different. The vacuoles, like lysosomes, degrade proteins transported into their lumen and produce amino acids. These amino acids should be quickly excreted to the cytosol under nutrient starvation condition and recycled for de novo protein synthesis. These suggest that specific machineries that transport amino acids into and out of the vacuoles operate at the vacuolar membrane. Several families of transporter involved in the vacuolar compartmentalization of amino acids have been identified and characterized using budding yeast Saccharomyces cerevisiae. In this review, we describe the vacuolar amino acid transporters identified so far and introduce recent findings on their activity and physiological function.
Assuntos
Aminoácidos/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Vacúolos/metabolismo , Transporte Biológico , Meios de Cultura , Citosol/metabolismo , Lisossomos/metabolismo , Nutrientes , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismoRESUMO
The vacuolar membrane proteins Ypq1p, Ypq2p, and Ypq3p of Saccharomyces cerevisiae are known as the members of the PQ-loop protein family. We found that the ATP-dependent uptake activities of arginine and histidine by the vacuolar membrane vesicles were decreased by ypq2Δ and ypq3Δ mutations, respectively. YPQ1 and AVT1, which are involved in the vacuolar uptake of lysine/arginine and histidine, respectively, were deleted in addition to ypq2Δ and ypq3Δ. The vacuolar membrane vesicles isolated from the resulting quadruple deletion mutant ypq1Δypq2Δypq3Δavt1Δ completely lost the uptake activity of basic amino acids, and that of histidine, but not lysine and arginine, was evidently enhanced by overexpressing YPQ3 in the mutant. These results suggest that Ypq3p is specifically involved in the vacuolar uptake of histidine in S. cerevisiae. The cellular level of Ypq3p-HA(3) was enhanced by depletion of histidine from culture medium, suggesting that it is regulated by the substrate.
Assuntos
Sistemas de Transporte de Aminoácidos/genética , Antiporters/genética , Vesículas Citoplasmáticas/metabolismo , Regulação Fúngica da Expressão Gênica , Histidina/metabolismo , Proteínas de Membrana/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Trifosfato de Adenosina/metabolismo , Sistemas de Transporte de Aminoácidos/deficiência , Antiporters/deficiência , Arginina/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Deleção de Genes , Lisina/metabolismo , Proteínas de Membrana/deficiência , Isoformas de Proteínas/deficiência , Isoformas de Proteínas/genética , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismoRESUMO
In the vacuolar basic amino acid (VBA) transporter family of Saccharomyces cerevisiae, VBA4 encodes a vacuolar membrane protein with 14 putative transmembrane helices. Transport experiments with isolated vacuolar membrane vesicles and estimation of the amino acid contents in vacuoles showed that Vba4p is not likely involved in the transport of amino acids. We found that the vba4Δ cells, as well as vba1Δ and vba2Δ cells, showed increased susceptibility to several drugs, particularly to azoles. Although disruption of the VBA4 gene did not affect the salt tolerance of the cells, vacuolar fragmentation observed under high salt conditions was less prominent in vba4Δ cells than in wild type, vba1Δ, and vba2Δ cells. Vba4p differs from Vba1p and Vba2p as a vacuolar transporter but is important for the drug resistance and vacuolar morphology of S. cerevisiae.
Assuntos
Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Antifúngicos/farmacologia , Farmacorresistência Fúngica/genética , Membranas Intracelulares/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Sistemas de Transporte de Aminoácidos Básicos/genética , Aminoácidos/metabolismo , Transporte Biológico , Fluconazol/farmacologia , Expressão Gênica , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/ultraestrutura , Cetoconazol/farmacologia , Miconazol/farmacologia , Forma das Organelas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Tolerância ao Sal , Cloreto de Sódio/farmacologia , Vacúolos/efeitos dos fármacos , Vacúolos/ultraestruturaRESUMO
Avt3p, a vacuolar amino acid exporter (656 amino acid residues) that is important for vacuolar amino acid compartmentalization as well as spore formation in Schizosaccharomyces pombe, has an extremely long hydrophilic region (approximately 290 amino acid residues) at its N-terminus. Because known functional domains have not been found in this region, its functional role was examined with a deletion mutant avt3(∆1-270) expressed in S. pombe avt3∆ cells. The deletion of this region did not affect its intracellular localization or vacuolar contents of basic amino acids as well as neutral ones. The defect of avt3Δ cells in spore formation was rescued by the expression of avt3+ but was not completely rescued by the expression of avt3(∆1-270). The N-terminal region is thus dispensable for the function of Avt3p as an amino acid exporter, but it is likely to be involved in the role of Avt3p under nutritional starvation conditions.
Assuntos
Aminoácidos/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/metabolismo , Vacúolos/metabolismo , Transporte Proteico , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Deleção de Sequência , Esporos Fúngicos/metabolismoRESUMO
Fusarium oxysporum causes wilt disease in many plant families, and many genes are involved in its development or growth in host plants. A recent study revealed that vacuolar amino acid transporters play an important role in spore formation in Schizosaccharomyces pombe and Saccharomyces cerevisiae. To investigate the role of vacuolar amino acid transporters of this phytopathogenic fungus, the FOXG_11334 (FoAVT3) gene from F. oxysporum was isolated and its function was characterized. Transcription of FoAVT3 was upregulated after rapamycin treatment. A green fluorescent protein fusion of FoAvt3p was localized to vacuolar membranes in both S. cerevisiae and F. oxysporum. Analysis of the amino acid content of the vacuolar fraction and amino acid transport activities using vacuolar membrane vesicles from S. cerevisiae cells heterologously expressing FoAVT3 revealed that FoAvt3p functions as a vacuolar amino acid transporter, exporting neutral amino acids. We conclude that the FoAVT3 gene encodes a vacuolar neutral amino acid transporter.
Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Proteínas Fúngicas/metabolismo , Fusarium/citologia , Fusarium/genética , Saccharomyces cerevisiae/genética , Vacúolos/metabolismo , Sequência de Aminoácidos , Sistemas de Transporte de Aminoácidos/química , Sistemas de Transporte de Aminoácidos/genética , Aminoácidos/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Genoma Fúngico/genética , Dados de Sequência Molecular , Transporte Proteico , Saccharomyces cerevisiae/metabolismo , Alinhamento de SequênciaRESUMO
Several genes for vacuolar amino acid transport were reported in Saccharomyces cerevisiae, but have not well been investigated. We characterized AVT1, a member of the AVT vacuolar transporter family, which is reported to be involved in lifespan of yeast. ATP-dependent uptake of isoleucine and histidine by the vacuolar vesicles of an AVT exporter mutant was lost by introducing avt1∆ mutation. Uptake activity was inhibited by the V-ATPase inhibitor: concanamycin A and a protonophore. Isoleucine uptake was inhibited by various neutral amino acids and histidine, but not by γ-aminobutyric acid, glutamate, and aspartate. V-ATPase-dependent acidification of the vesicles was declined by the addition of isoleucine or histidine, depending upon Avt1p. Taken together with the data of the amino acid contents of vacuolar fractions in cells, the results suggested that Avt1p is a proton/amino acid antiporter important for vacuolar compartmentalization of various amino acids.
Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos Neutros/metabolismo , Antiporters/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Sistemas de Transporte de Aminoácidos/genética , Antiporters/genética , Ácido Aspártico/metabolismo , Transporte Biológico , Ácido Glutâmico/metabolismo , Histidina/metabolismo , Membranas Intracelulares/metabolismo , Isoleucina/metabolismo , Prótons , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Ácido gama-Aminobutírico/metabolismoRESUMO
Active transport systems for various amino acids operate in the vacuolar membrane of Saccharomyces cerevisiae. The gene families for vacuolar amino acid transporters were identified by reverse genetics experiments. In the AVT transporter family, Avt1p works for active uptake of amino acid into vacuole, and Avt3p, Avt4p, and Avt6p for active extrusion of amino acid from vacuole to cytosol. Here, we found green fluorescent protein-tagged Avt7p, an unidentified member of the AVT family, localized to the vacuolar membrane of S. cerevisiae. Disruption of the AVT7 gene enhanced both vacuolar contents of several amino acids and uptake activities of glutamine and proline by vacuolar membrane vesicles. Efficiency of spore formation was impaired by the disruption of the AVT7 gene, suggesting the physiological importance of Avt7p-dependent efflux of amino acid from vacuoles under nutrient-poor condition.
Assuntos
Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Aminoácidos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Esporos Fúngicos/fisiologia , Membranas Intracelulares/metabolismo , Nitrogênio/deficiência , Transporte Proteico , Saccharomyces cerevisiae/fisiologia , Vacúolos/metabolismoRESUMO
Saccharomyces cerevisiae Ypq1p is a vacuolar membrane protein of the PQ-loop protein family. We found that ATP-dependent uptake activities of amino acids by vacuolar membrane vesicles were impaired by ypq1∆ mutation. Loss of lysine uptake was most remarkable, and the uptake was recovered by overproduction of Ypq1p. Ypq1p is thus involved in transport of amino acids into vacuoles.
Assuntos
Trifosfato de Adenosina/metabolismo , Membranas Intracelulares/metabolismo , Lisina/metabolismo , Proteínas de Membrana/metabolismo , Mutação , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Vacúolos/metabolismo , Transporte Biológico/genética , Proteínas de Membrana/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Basic amino acids (lysine, histidine and arginine) accumulated in Saccharomyces cerevisiae vacuoles should be mobilized to cytosolic nitrogen metabolism under starvation. We found that the decrease of vacuolar basic amino acids in response to nitrogen starvation was impaired by the deletion of AVT4 gene encoding a vacuolar transporter. In addition, overexpression of AVT4 reduced the accumulation of basic amino acids in vacuoles under nutrient-rich condition. In contrast to AVT4, the deletion and overexpression of AVT3, which encodes the closest homologue of Avt4p, did not affect the contents of vacuolar basic amino acids. Consistent with these, arginine uptake into vacuolar membrane vesicles was decreased by Avt4p-, but not by Avt3p-overproduction, whereas various neutral amino acids were excreted from vacuolar membrane vesicles in a manner dependent on either Avt4p or Avt3p. These results suggest that Avt4p is a vacuolar amino acid exporter involving in the recycling of basic amino acids.
Assuntos
Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Aminoácidos Básicos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sistemas de Transporte de Aminoácidos Básicos/química , Transporte Biológico , Membranas Intracelulares/metabolismo , Dados de Sequência Molecular , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
The prokaryotic V-ATPase of Enterococcus hirae, closely related to the eukaryotic enzymes, provides a unique opportunity to study the ion-translocation mechanism because it transports Na(+), which can be detected by radioisotope (22Na(+)) experiments and X-ray crystallography. In this study, we demonstrated that the binding affinity of the rotor ring (K ring) for 22Na(+) decreased approximately 30-fold by reaction with N,N(')-dicyclohexylcarbodiimide (DCCD), and determined the crystal structures of Na(+)-bound and Na(+)-unbound K rings modified with DCCD at 2.4- and 3.1-Å resolutions, respectively. Overall these structures were similar, indicating that there is no global conformational change associated with release of Na(+) from the DCCD-K ring. A conserved glutamate residue (E139) within all 10 ion-binding pockets of the K ring was neutralized by modification with DCCD, and formed an "open" conformation by losing hydrogen bonds with the Y68 and T64 side chains, resulting in low affinity for Na(+). This open conformation is likely to be comparable to that of neutralized E139 forming a salt bridge with the conserved arginine of the stator during the ion-translocation process. Based on these findings, we proposed the ion-translocation model that the binding affinity for Na(+) decreases due to the neutralization of E139, thus releasing bound Na(+), and that the structures of Na(+)-bound and Na(+)-unbound DCCD-K rings are corresponding to intermediate states before and after release of Na(+) during rotational catalysis of V-ATPase, respectively.
Assuntos
Biocatálise , Dicicloexilcarbodi-Imida/química , Sódio/metabolismo , ATPases Vacuolares Próton-Translocadoras/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Transporte Biológico , Enterococcus/enzimologia , Ligação Proteica , Conformação Proteica , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
V-ATPases function as ATP-dependent ion pumps in various membrane systems of living organisms. ATP hydrolysis causes rotation of the central rotor complex, which is composed of the central axis D subunit and a membrane c ring that are connected by F and d subunits. Here we determined the crystal structure of the DF complex of the prokaryotic V-ATPase of Enterococcus hirae at 2.0-Å resolution. The structure of the D subunit comprised a long left-handed coiled coil with a unique short ß-hairpin region that is effective in stimulating the ATPase activity of V(1)-ATPase by twofold. The F subunit is bound to the middle portion of the D subunit. The C-terminal helix of the F subunit, which was believed to function as a regulatory region by extending into the catalytic A(3)B(3) complex, contributes to tight binding to the D subunit by forming a three-helix bundle. Both D and F subunits are necessary to bind the d subunit that links to the c ring. From these findings, we modeled the entire rotor complex (DFdc ring) of V-ATPase.
Assuntos
Enterococcus/enzimologia , Células Procarióticas/enzimologia , Subunidades Proteicas/química , ATPases Vacuolares Próton-Translocadoras/química , Sequência de Aminoácidos , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Subunidades Proteicas/metabolismo , Alinhamento de Sequência , Eletricidade Estática , Homologia Estrutural de Proteína , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
A vacuolar membrane protein, Vba2p of Schizosaccharomyces pombe, is involved in basic amino acid uptake by intact cells. Here we found evidence that Vba2p mediated ATP-dependent lysine uptake by vacuolar membrane vesicles of Saccharomyces cerevisiae. Vba2p was also responsible for quinidine sensitivity, and the addition of lysine improved cell growth on quinidine-containing media. These findings should be useful for further characterization of Vba2p.
Assuntos
Sistemas de Transporte de Aminoácidos Básicos/genética , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Membranas Intracelulares/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Vacúolos/genética , Trifosfato de Adenosina/metabolismo , Expressão Gênica , Lisina/metabolismoRESUMO
The crystal structures of the Na(+)- and Li(+)-bound NtpK rings of Enterococcus hirae V-ATPase have been obtained. The coupling ion (Na(+) or Li(+)) was surrounded by five oxygen atoms contributed by residues T64, Q65, Q110, E139, and L61, and the hydrogen bonds of the side chains of Q110, Y68, and T64 stabilized the position of the E139 γ carboxylate essential for ion occlusion (PDB accession numbers 2BL2 and 2CYD). We previously indicated that an NtpK mutant strain (E139D) lost tolerance to sodium but not to lithium at alkaline pHs and suggested that the E139 residue is indispensable for the enzymatic activity of E. hirae V-ATPase linked with the sodium tolerance of this bacterium. In this study, we examined the activities of V-ATPase in which these four residues, except for E139, were substituted. The V-ATPase activities of the Q65A and Y68A mutants were slightly retained, but those of the T64A and Q110A mutants were negligible. Among the residues, T64 and Q110 are indispensable for the ion coupling of E. hirae V-ATPase, in addition to the essential residue E139.
Assuntos
Enterococcus/enzimologia , ATPases Vacuolares Próton-Translocadoras/química , Substituição de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Enterococcus/genética , Ligação de Hidrogênio , Lítio/química , Mutagênese Sítio-Dirigida , Mutação , Oxigênio , Subunidades Proteicas/química , Subunidades Proteicas/genética , Sódio/química , ATPases Vacuolares Próton-Translocadoras/genéticaRESUMO
In Schizosaccharomyces pombe, neither intracellular sorting nor ubiquitination of amino acid permeases is well understood. In the present study, we show that intracellular sorting of the amino acid permease Aat1p in S. pombe depends on the presence of a nitrogen source in the growth medium. Under nitrogen-sufficient conditions, Aat1p appeared to be stably localized at the Golgi apparatus. In contrast, under nitrogen-insufficient conditions, Aat1p was sorted to the plasma membrane. Over time, plasma membrane-localized Aat1p was internalized and sorted to the lumen of the vacuole, where it was degraded. Sorting of Aat1p to the vacuolar lumen was dependent on the ESCRT (endosomal sorting complex required for transport) complex, which is required for formation of the multivesicular body. S. pombe has three genes (pub1(+), pub2(+) and pub3(+)) that are homologous to the ubiquitin ligase RSP5. Under nitrogen-sufficient conditions, Aat1-GFP was missorted to the plasma membrane in pub1Δ cells and ubiquitinated Aat1p was not detected. These results suggest that Pub1p-mediated ubiquitination is required for retention of Aat1 at the Golgi under nitrogen-sufficient conditions. The Aat1p lysine mutant Aat1(K18, 26, 27) was completely missorted to the plasma membrane under nitrogen-rich conditions. Furthermore, Aat1(K4, 18R), Aat1(K4, 26, 27R) and Aat1(K18, 26, 27K) mutants were severely blocked in endocytosis. These results indicate that ubiquitination is an important determinant for localization and regulation of the Aat1p permease in S. pombe.
Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Schizosaccharomyces/enzimologia , Schizosaccharomyces/metabolismo , Ubiquitinação , Membrana Celular/química , Meios de Cultura/química , Complexo de Golgi/química , Nitrogênio/metabolismo , Transporte Proteico , Schizosaccharomyces/química , Vacúolos/químicaRESUMO
Amino acid analysis of Saccharomyces cerevisiae cells indicated that neutral amino acids such as glycine and alanine were probably excluded from the vacuoles, and that vacuolar H(+)-ATPase (V-ATPase) was involved in the vacuolar compartmentalization of these amino acids. We found that vacuolar membrane vesicles export neutral amino acids in an ATP-dependent manner. This is important in identifying vacuolar transporters for neutral amino acids.
Assuntos
Trifosfato de Adenosina/metabolismo , Aminoácidos Neutros/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Vesículas Transportadoras/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Transporte Biológico , Membranas Intracelulares/química , Cinética , Vesículas Transportadoras/química , Vacúolos/químicaRESUMO
Vba5p is closest to Vba3p in the vacuolar transporter for basic amino acids (VBA) family of Saccharomyces cerevisiae. We found that green fluorescence protein (GFP)-tagged Vba5p localized exclusively to the plasma membrane. The uptake of lysine and arginine by whole cells was little affected by deletion of the VBA5 gene, but was stimulated by overexpression of the VBA5 gene. The inhibitory effect of 4-nitroquinoline N-oxide on cell growth was accelerated by expression of the VBA5 gene, and was lessened by the addition of arginine. These results suggest that Vba5p is a plasma membrane protein involved in amino acid uptake and drug sensitivity.
Assuntos
Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Aminoácidos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , 4-Nitroquinolina-1-Óxido/farmacologia , Sequência de Aminoácidos , Sistemas de Transporte de Aminoácidos Básicos/química , Transporte Biológico/efeitos dos fármacos , Proteínas de Membrana/química , Dados de Sequência Molecular , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
A Glu139Asp mutant of the NtpK subunit (kE139D) of Enterococcus hirae vacuolar-type ATPase (V-ATPase) lost tolerance to sodium but not to lithium at pH 10. Purified kE139D V-ATPase retained relatively high specific activity and affinity for the lithium ion compared to the sodium ion. The kE139 residue of V-ATPase is indispensable for its enzymatic activity that is linked with the salt tolerance of enterococci.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Enterococcus/enzimologia , Tolerância ao Sal , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/metabolismo , Proteínas de Bactérias/genética , Catálise , Enterococcus/química , Enterococcus/genética , Enterococcus/fisiologia , Glutamatos/química , Glutamatos/genética , Glutamatos/metabolismo , Lítio/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Sódio/metabolismo , ATPases Vacuolares Próton-Translocadoras/genéticaRESUMO
We identified a gene product of At5g19500 (At5g19500p) from Arabidopsis thaliana that is homologous to EcTyrP, a tyrosine-specific transporter from Escherichia coli. Computational analyses of the amino acid sequence of At5g19500p predicted 11 transmembrane domains (TMDs) and a potential plastid targeting signal at its amino terminus. As a first step toward understanding the possible role of At5g19500p in plant cells, we attempted to determine the localization of At5g19500p by an in vitro chloroplastic import assay using At5g19500p translated in a cell-free wheat germ system (Madin et al., Proc. Natl. Acad. Sci. USA, 97, 559-564 (2000)), followed by subfractionation of the chloroplasts. At5g19500p was successfully imported into chloroplasts, and the newly transported mature form of At5g19500p was recovered from the inner envelope membrane.
Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cloroplastos/metabolismo , Sequência de Aminoácidos , Sistemas de Transporte de Aminoácidos/química , Sistemas de Transporte de Aminoácidos/genética , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sistema Livre de Células , Cloroplastos/genética , Clonagem Molecular , Escherichia coli/genética , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Homologia de SequênciaRESUMO
We have found that (-)-virgatusin and related compounds have antimicrobial and antifungal activity. To identify further biological activities of these compounds, we tested the activity of acridine orange efflux, which shows ionophore-like disruption of cellular ion homeostasis activity. After testing 31 compounds, we found that verrucosin and a related compound had disruption activity.