RESUMEN

Human copper transporters ATP7B and ATP7A deliver copper to biosynthetic pathways and maintain copper homeostasis in the cell. These enzymes combine several challenges for structural biology because they are large low abundance membrane proteins with many highly mobile domains and long disordered loops. No method has yet succeeded in solving the structure of the complete fully functional protein. Still, X-ray crystallography, Cryo-EM and NMR helped to piece together a structure based model of the enzyme activity and regulation by copper. We review the structures of ATP7B and ATP7A with an emphasis on the mechanistic insights into the unique aspects of the transport function and regulation of the human copper ATPases that have emerged from more than twenty years of research.

Asunto(s)

Proteínas de Transporte de Catión , Cobre , Humanos , ATPasas Transportadoras de Cobre/genética , ATPasas Transportadoras de Cobre/metabolismo , Cobre/química , Proteínas de Transporte de Catión/metabolismo , Homeostasis

RESUMEN

As the main organic acid in fruits, malate is produced in the cytoplasm and is then transported into the vacuole. It accumulates by vacuolar proton pumps, transporters, and channels, affecting the taste and flavor of fruits. Among the three types of proton pumps (V-ATPases, V-PPases, and P-ATPases), the P-ATPases play an important role in the transport of malate into vacuoles. In this study, the transcriptome data, collected at different stages after blooming and during storage, were analyzed and the results demonstrated that the expression of MdPH5, a vacuolar proton-pumping P-ATPase, was associated with both pre- and post-harvest malate contents. Moreover, MdPH5 is localized at the tonoplast and regulates malate accumulation and vacuolar pH. In addition, MdMYB73, an upstream MYB transcription factor of MdPH5, directly binds to its promoter, thereby transcriptionally activating its expression and enhancing its activity. In this way, MdMYB73 can also affect malate accumulation and vacuolar pH. Overall, this study clarifies how MdMYB73 and MdPH5 act to regulate vacuolar malate transport systems, thereby affecting malate accumulation and vacuolar pH. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-023-00115-7.

RESUMEN

P-ATPase are a large protein family of integral membrane, playing an important role in plant growth, development and stress. P-ATPase genes family have been identified and characterized in several model plants such as cotton, grapes, tobacco, rice, rubber plant and Arabidopsis. However, still lack of comprehensive study of P-ATPase genes in Chinese pear (Pyrus bretschneideri). A systematic analysis was performed and identified 30 P-ATPase genes from the pear genome to evaluate the qualities and diversity of P-ATPase proteins. Phylogenetic analysis was performed using A. thaliana P-ATPase genes as a model, allowing us to categorize into 4 subfamilies (PbHMA, PbECA, PbACA, and PbAHA) and two subfamilies (ALA and P5) is absent in pear. Even Within the same subclade, P-ATPase genes also shows the similar exon-intron structure and conserved motif structure. Continuing chromosomal localization analysis showed that 23 P-ATPase genes were distributed among 13 chromosome and 7 gene on the scaffold of pear. Promoter regions of P-ATPase genes revealed that several cis-acting elements were involved in plant growth/development, stress responses as well as hormone responses. Additionally, P-ATPase genes were also differentially expressed under hormones treatments of ABA (abscisic acid) and SA (salicylic acid) treatments. Remarkably, the transcriptome data exposed that P-ATPase gene might play an important role in lignin biosynthesis during fruit development. The real time qRT-PCR was performed, and the expression analysis indicated that various P-ATPase genes extremely expressed during different developmental stages of fruit. Our study provides valuable information about the P-ATPase gene family in pear fruit development and lignin polymerization.

Asunto(s)

Adenosina Trifosfatasas/genética , Genes de Plantas/genética , Proteínas de Plantas/genética , Pyrus/enzimología , Adenosina Trifosfatasas/metabolismo , Proteínas de Plantas/metabolismo

RESUMEN

The determination of flower color mainly depends on the anthocyanin biosynthesis pathway and vacuolar pH; however, unlike the former, the mechanism of vacuolar acidification in soybean remains uncharacterized at the molecular level. To investigate this mechanism, we isolated four recessive purple-blue EMS-induced flower mutants from the purple flower soybean cultivar, Pungsannamul. The petals of all the mutants had increased pH compared with those of wild Pungsannamul. One of the mutants had a single nucleotide substitution in GmPH4, a regulator gene encoding an MYB transcription factor, and the substitution resulted in a premature stop codon in its first exon. The other three mutants had nucleotide substitutions in GmPH5, a single new gene that we identified by physical mapping. It corresponds to Glyma.03G262600 in chromosome 3 and encodes a proton pump that belongs to the P3A-ATPase family. The substitutions resulted in a premature stop codon, which may be a defect in the ATP-binding capacity of GmPH5 and possibly a catalytic inefficiency of GmPH5. The result is consistent with their genetic recessiveness as well as the high pH of mutant petals, suggesting that GmPH5 is directly involved in vacuolar acidification. We also found that the expression of GmPH5 and several putative "acidifying" genes in the gmph4 mutant was remarkably reduced, indicating that GmPH4 may regulate the genes involved in determining the vacuolar pH of soybean petals.

RESUMEN

Plant P-type H⁺-ATPase (P-ATPase) is a membrane protein existing in the plasma membrane that plays an important role in the transmembrane transport of plant cells. To understand the variety and quantity of P-ATPase proteins in different cotton species, we combined four databases from two diploid cotton species (Gossypium raimondii and G. arboreum) and two tetraploid cotton species (G. hirsutum and G. barbadense) to screen the P-ATPase gene family and resolved the evolutionary relationships between the former cotton species. We identified 53, 51, 99 and 98 P-ATPase genes from G. arboretum, G. raimondii, G. barbadense and G. hirsutum, respectively. The structural and phylogenetic analyses revealed that the gene structure was consistent between P-ATPase genes, with a close evolutionary relationship. The expression analysis of P-ATPase genes showed that many P-ATPase genes were highly expressed in various tissues and at different fiber developmental stages in G. hirsutum, suggesting that they have potential functions during growth and fiber development in cotton.

Asunto(s)

Regulación de la Expresión Génica de las Plantas , Genoma de Planta , Gossypium/genética , ATPasa Intercambiadora de Hidrógeno-Potásio/genética , Proteínas de Plantas/genética , Evolución Biológica , Mapeo Cromosómico , Fibra de Algodón , Regulación del Desarrollo de la Expresión Génica , Gossypium/clasificación , Gossypium/enzimología , Gossypium/crecimiento & desarrollo , ATPasa Intercambiadora de Hidrógeno-Potásio/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Familia de Multigenes , Especificidad de Órganos , Filogenia , Proteínas de Plantas/metabolismo , Ploidias , Especificidad de la Especie

RESUMEN

P-type ATPases form a large and ubiquitous superfamily of ion and lipid transporters that use ATP (adenosine triphosphate) to carry out their function. The IB subclass (PIB-ATPases) allows flux of heavy metals and are key players in metal detoxification, critical for human health, crops, and survival of pathogens. Nevertheless, PIB-ATPases remain poorly understood at a molecular level. In this study, nanobodies (Nbs) are selected against the zinc-transporting PIB-ATPase ZntA from Shigella sonnei (SsZntA), aiming at developing tools to assist the characterization of the structure and function of this class of transporters. We identify six different Nbs that bind detergent stabilized SsZntA. We further assess the effect of the Nbs on the catalytic function of SsZntA, and find that five nanobodies associate without affecting the function, while one nanobody significantly reduces the ATPase activity. This study paves the way for more refined mechanistical and structural studies of zinc-transporting PIB-ATPases.

RESUMEN

The P-type ATPases (P-ATPases) are present in all living cells where they mediate ion transport across membranes on the expense of ATP hydrolysis. Different ions which are transported by these pumps are protons like calcium, sodium, potassium, and heavy metals such as manganese, iron, copper, and zinc. Maintenance of the proper gradients for essential ions across cellular membranes makes P-ATPases crucial for cell survival. In this study, characterization of two families of P-ATPases including P-ATPase 13A1 and P-ATPase 13A3 protein was compared in two different insect species from different orders. According to the conserved motifs found with MEME, nine motifs were shared by insects of 13A1 family but eight in 13A3 family. Seven different insect species from 13A1 and five samples from 13A3 family were selected as the representative samples for functional and structural analyses. The structural and functional analyses were performed with ProtParam, SOPMA, SignalP 4.1, TMHMM 2.0, ProtScale and ProDom tools in the ExPASy database. The tertiary structure of Bombus terrestris as a sample of each family of insects were predicted by the Phyre2 and TM-score servers and their similarities were verified by SuperPose server. The tertiary structures were predicted via the "c3b9bA" model (PDB Accession Code: 3B9B) in P-ATPase 13A1 family and "c2zxeA" model (PDB Accession Code: 2ZXE) in P-ATPase 13A3 family. A phylogenetic tree was constructed with MEGA 6.06 software using the Neighbor-joining method. According to the results, there was a high identity of P-ATPase families so that they should be derived from a common ancestor however they belonged to separate groups. In protein-protein interaction analysis by STRING 10.0, six common enriched pathways of KEGG were identified in B. terrestris in both families. The obtained data provide a background for bioinformatic studies of the function and evolution of other insects and organisms.

Asunto(s)

Insectos/enzimología , ATPasas Tipo P/química , ATPasas Tipo P/metabolismo , Animales , Insectos/clasificación , ATPasas Tipo P/clasificación , ATPasas Tipo P/genética , Filogenia

RESUMEN

Petunia mutants (Petunia hybrida) with blue flowers defined a novel vacuolar proton pump consisting of two interacting P-ATPases, PH1 and PH5, that hyper-acidify the vacuoles of petal cells. PH5 is similar to plasma membrane H(+) P3A -ATPase, whereas PH1 is the only known eukaryoticP3B -ATPase. As there were no indications that this tonoplast pump is widespread in plants, we investigated the distribution and evolution of PH1 and PH5. We combined database mining and phylogenetic and synteny analyses of PH1- and PH5-like proteins from all kingdoms with functional analyses (mutant complementation and intracellular localization) of homologs from diverse angiosperms. We identified functional PH1 and PH5 homologs in divergent angiosperms. PH5 homologs evolved from plasma membrane P3A -ATPases, acquiring an N-terminal tonoplast-sorting sequence and new cellular function before angiosperms appeared. PH1 is widespread among seed plants and related proteins are found in some groups of bacteria and fungi and in one moss, but is absent in most algae, suggesting that its evolution involved several cases of gene loss and possibly horizontal transfer events. The distribution of PH1 and PH5 in the plant kingdom suggests that vacuolar acidification by P-ATPases appeared in gymnosperms before flowers. This implies that, next to flower color determination, vacuolar hyper-acidification is required for yet unknown processes.

Asunto(s)

Ácidos/metabolismo , Evolución Molecular , Proteínas de Transporte de Membrana/metabolismo , Petunia/enzimología , ATPasas de Translocación de Protón/metabolismo , Vacuolas/enzimología , Secuencia de Aminoácidos , Sitios de Unión , Cationes , Membrana Celular/metabolismo , Regulación de la Expresión Génica de las Plantas , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , ATPasas de Translocación de Protón/química , Rosa/genética , Homología de Secuencia de Aminoácido , Vacuolas/metabolismo , Vitis/genética

RESUMEN

Membrane transport P-type ATPases display two characteristic enzymatic activities: a principal ATPase activity provides the driving force for ion transport across biological membranes, whereas a promiscuous secondary activity catalyzes the hydrolysis of phosphate monoesters. This last activity is usually denoted as the phosphatase activity of P-ATPases. In the present study, we characterize the phosphatase activity of the Cu(+)-transport ATPase from Archaeglobus fulgidus (Af-CopA) and compare it with the principal ATPase activity. Our results show that the phosphatase turnover number was 20 times higher than that corresponding to the ATPase activity, but it is compensated by a high value of Km, producing a less efficient catalysis for pNPP. This secondary activity is enhanced by Mg(2+) (essential activator) and phospholipids (non-essential activator), and inhibited by salts and Cu(+). Transition state analysis of the catalyzed and noncatalyzed hydrolysis of pNPP indicates that Af-CopA enhances the reaction rates by a factor of 10(5) (ΔΔG(‡)=38 kJ/mol) mainly by reducing the enthalpy of activation (ΔΔH(‡)=30 kJ/mol), whereas the entropy of activation is less negative on the enzyme than in solution. For the ATPase activity, the decrease in the enthalpic component of the barrier is higher (ΔΔH(‡)=39 kJ/mol) and the entropic component is small on both the enzyme and in solution. These results suggest that different mechanisms are involved in the transference of the phosphoryl group of p-nitrophenyl phosphate and ATP.

Asunto(s)

Adenosina Trifosfatasas/química , Adenosina Trifosfato/química , Proteínas Arqueales/química , Archaeoglobus fulgidus/química , Cobre/química , Monoéster Fosfórico Hidrolasas/química , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Archaeoglobus fulgidus/enzimología , Biocatálisis , Dominio Catalítico , Cationes Bivalentes , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Calor , Cinética , Magnesio/química , Modelos Moleculares , Nitrofenoles/química , Compuestos Organofosforados/química , Fosfolípidos/química , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Termodinámica

RESUMEN

P5-ATPases are important for processes associated with the endosomal-lysosomal system of eukaryotic cells. In humans, the loss of function of P5-ATPases causes neurodegeneration. In the yeastSaccharomyces cerevisiae, deletion of P5-ATPase Spf1p gives rise to endoplasmic reticulum stress. The reaction cycle of P5-ATPases is poorly characterized. Here, we showed that the formation of the Spf1p catalytic phosphoenzyme was fast in a reaction medium containing ATP, Mg(2+), and EGTA. Low concentrations of Ca(2+)in the phosphorylation medium decreased the rate of phosphorylation and the maximal level of phosphoenzyme. Neither Mn(2+)nor Mg(2+)had an inhibitory effect on the formation of the phosphoenzyme similar to that of Ca(2+) TheKmfor ATP in the phosphorylation reaction was ∼1 µmand did not significantly change in the presence of Ca(2+) Half-maximal phosphorylation was attained at 8 µmMg(2+), but higher concentrations partially protected from Ca(2+)inhibition. In conditions similar to those used for phosphorylation, Ca(2+)had a small effect accelerating dephosphorylation and minimally affected ATPase activity, suggesting that the formation of the phosphoenzyme was not the limiting step of the ATP hydrolytic cycle.

Asunto(s)

Transportadoras de Casetes de Unión a ATP/metabolismo , Calcio/metabolismo , Estrés del Retículo Endoplásmico/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Transportadoras de Casetes de Unión a ATP/genética , Fosforilación/fisiología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética

RESUMEN

Intracellular pH homeostasis is essential for all living cells. In plants, pH is usually maintained by three structurally distinct and differentially localized types of proton pump: P-type H(+) -ATPases in the plasma membrane, and multimeric vacuolar-type H(+) -ATPases (V-ATPases) and vacuolar H(+) -pyrophosphatases (H(+) -PPases) in endomembranes. Here, we show that reduced accumulation of proanthocyanidins (PAs) and hence the diminished brown seed coloration found in the Arabidopsis thaliana mutant transparent testa 13 (tt13) is caused by disruption of the gene encoding the P3A -ATPase AHA10. Identification of the gene encoded by the tt13 locus completes the molecular characterization of the classical set of transparent testa mutants. Cells of the tt13 seed coat endothelium do not contain PA-filled central vacuoles as observed in the wild-type. tt13 phenocopies tt12, a mutant that is defective in vacuolar import of the PA precursor epicatechin. Our data show that vacuolar loading with PA precursors depends on TT13. Consistent with the tt13 phenotype, but in contrast to other isoforms of P-type H(+) -ATPases, TT13 localizes to the tonoplast. PA accumulation in tt13 is partially restored by expression of the tonoplast localized H(+) -PPase VHP1. Our findings indicate that the P3A -ATPase TT13 functions as a proton pump in the tonoplast of seed coat endothelium cells, and generates the driving force for TT12-mediated transport of PA precursors to the vacuole.

Asunto(s)

Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proantocianidinas/metabolismo , ATPasas de Translocación de Protón/metabolismo , Semillas/metabolismo , Vacuolas/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Transporte Biológico , Regulación de la Expresión Génica de las Plantas , Prueba de Complementación Genética , Mutación , Petunia/genética , Plantas Modificadas Genéticamente , ATPasas de Translocación de Protón/genética , Semillas/genética , Vacuolas/genética

RESUMEN

Brassinosteroids (BRs) are plant hormones regulating growth and development. In interaction with other hormones, they are involved in environmental cue responses. The present model of the BR response pathway in Arabidopsis includes the perception of the hormone by the plasma membrane (PM) receptor brassinosteroid insensitive 1 (BRI1) and its hetero-oligomerization with the co-receptor BRI1-associated receptor kinase 1 (BAK1), followed by the activation of a signaling-cascade finally resulting in the expression of BR-responsive genes. New findings have shed light on the receptor density in the PM and on the molecular mechanism of BR perception, which includes the hormone-induced formation of a platform in the BRI1 extracellular domain for interaction with BAK1. Furthermore, new knowledge on early, BRI1-initiated signaling events at the PM-cytoplasm interface has recently been gained. In addition, a fast BR response pathway that modifies the membrane potential and the expansion of the cell wall - both crucial processes preceding cell elongation growth - have been identified. In this review, these latest findings are summarized and discussed against the background of the present model of BRI1 signaling.