RESUMEN
The sequence of the atDjC6 chaperone protein includes three potential nuclear localization signal (NLS) sequences (A-C) and three potential nuclear export signal (NES) sequences (X-Z). The subcellular localization of atDjC6 was studied by scanning laser confocal microscopy of chimera with the green-fluorescent protein (GFP) transiently expressed in tobacco BY-2 cells. The localization of the atDjC6::GFP chimera was coincident with that of the nuclear stain propidium iodide. Site-directed mutagenesis was used to verify the predicted NLS sequences. Each was individually fused to GFP and tested for protein localization. The individual NLS sequences were sufficient to direct partial nuclear localization of GFP, although the targeting information within NLS-B is apparently conformation sensitive. Site-directed mutagenesis of the NES sequences increased the amount of each chimera that was nuclearly localized, indicating a decrease in nuclear export. When any pair of NLS sequences were appended to GFP, the chimera were entirely nuclearly localized. Quantitative two-hybrid analysis was used to verify that the decoding of NLS sequence information involves interaction with the NLS-receptor protein importin-alpha. Each of the NLS sequences is flanked by a site of potential Ser phosphorylation, and recombinant atDjC6 could be phosphorylated in vitro. Mutagenesis of Ser residues to the P-Ser mimic Asp interfered with nuclear targeting, apparently by preventing recognition or binding by importin-alpha. Our results are consistent with a regulated nucleocytoplasmic localization of the atDjC6 chaperone protein.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Chaperonas Moleculares/metabolismo , Señales de Localización Nuclear/metabolismo , Algoritmos , Secuencia de Aminoácidos , Arabidopsis/ultraestructura , Proteínas de Arabidopsis/química , Núcleo Celular/metabolismo , Células Cultivadas , Microscopía Confocal , Chaperonas Moleculares/química , Datos de Secuencia Molecular , Fosforilación , Nicotiana/metabolismo , Técnicas del Sistema de Dos Híbridos , alfa Carioferinas/metabolismoRESUMEN
A total of 89 J-domain proteins were identified in the genome of the model flowering plant Arabidopsis thaliana. The deduced amino acid sequences of the J-domain proteins were analyzed for an assortment of structural features and motifs. Based on the results of sequence comparisons and structure and function predictions, 51 distinct families were identified. The families ranged in size from 1 to 6 members. Subcellular localizations of the A thaliana J-domain proteins were predicted; species were found in both the soluble and membrane compartments of all cellular organelles. Based on digital Northern analysis, the J-domain proteins could be separated into groups of low, medium, and moderate expression levels. This genomics-based analysis of the A thaliana J-domain proteins establishes a framework for detailed studies of biological function and specificity. It additionally provides a comprehensive basis for evolutionary comparisons.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Choque Térmico/genética , Chaperonas Moleculares/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Bases de Datos Factuales , Etiquetas de Secuencia Expresada , Genoma de Planta , Proteínas del Choque Térmico HSP40 , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/clasificación , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/clasificación , Chaperonas Moleculares/metabolismo , Filogenia , Estructura Terciaria de ProteínaRESUMEN
A cDNA clone was selected as a candidate for the catalytic subunit of phospho-pyruvate dehydrogenase phosphatase (PDP) by screening a Zea mays expressed sequence tag database with the bovine PDP deduced amino acid sequence. Both strands of the cDNA were completely sequenced. The maize clone contains an open reading frame of 1098 base pairs that encodes a polypeptide of 40 127 Da, ZMPP2. The deduced amino acid sequence of ZMPP2 contains the five PP2C signature domains, as does PDP. However, the expression pattern of ZMPP2, determined by reverse transcriptase-polymerase chain reaction, was different from those of the maize pyruvate dehydrogenase E1 alpha subunit and pyruvate dehydrogenase kinase. Additionally, the predicted subcellular location of ZMPP2 is cytoplasmic, while the pyruvate dehydrogenase complex, regulated by reversible phosphorylation, is mitochondrial. Thus, ZMPP2 is a PP2C-type protein phosphatase related to but distinct from PDP.
Asunto(s)
Fosfoproteínas Fosfatasas/genética , Proteínas de Saccharomyces cerevisiae , Zea mays/enzimología , Animales , Citoplasma/metabolismo , ADN Complementario/genética , ADN de Plantas , Datos de Secuencia Molecular , Péptidos/genética , Proteína Fosfatasa 2 , Proteína Fosfatasa 2C , ARN de PlantaRESUMEN
Sequence comparisons were used to identify cDNAs potentially encoding the alpha- and beta-subunits of chloroplast pyruvate dehydrogenase. Coupled in-vitro transcription plus translation was used to synthesize radiolabeled pyruvate dehydrogenase alpha- and beta-subunit precursor proteins. When the precursors were incubated with intact pea (Pisum sativum L.) seedling chloroplasts in the presence of ATP, they were imported and proteolytically processed. In contrast, there was no import or processing of the precursors by isolated, intact pea seedling mitochondria. Monospecific antibodies to the recombinant pyruvate dehydrogenase alpha-subunit were additionally able to co-precipitate radiolabeled pyruvate dehydrogenase beta-subunit, indicating association between subunits after import and processing. Furthermore, size-exclusion chromatography was used to identify an alphabeta heterodimer that is an intermediate in the assembly of the native alpha2beta2 heterotetrameric enzyme.
Asunto(s)
Cloroplastos/enzimología , Piruvato Deshidrogenasa (Lipoamida) , Complejo Piruvato Deshidrogenasa/metabolismo , Pisum sativum/enzimología , Biosíntesis de Proteínas , Complejo Piruvato Deshidrogenasa/biosíntesis , Transcripción GenéticaRESUMEN
Little is known of the plant branched-chain alpha-ketoacid dehydrogenase complex. We have undertaken a detailed study of the structure of the dihydrolipoyl acyltransferase (BCE2) subunit that forms the core of the complex, to which two other enzymes attach. Mature Arabidopsis thaliana BCE2 was expressed in Escherichia coli. The soluble recombinant protein was purified using a Superose 6 size-exclusion column to >90% homogeneity and was catalytically active. The recombinant protein formed a stable complex with a native molecular mass of 0.95 MDa and an S coefficient of 19.4, consistent with formation of a 24-mer. Negative-staining transmission electron microscopy of the recombinant protein confirmed that BCE2 forms a core with octagonal symmetry. Despite divergence of mammalian and plant BCE2s, there is clearly conservation of structure that is independent of primary sequence.
Asunto(s)
Aciltransferasas/química , Proteínas de Plantas/química , Aciltransferasas/genética , Aciltransferasas/metabolismo , Microscopía Electrónica , Complejos Multienzimáticos/química , Proteínas de Plantas/metabolismo , Conformación Proteica , Proteínas Quinasas/química , Subunidades de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Ultracentrifugación/métodosAsunto(s)
Inhibidores Enzimáticos/farmacología , Complejo Piruvato Deshidrogenasa/antagonistas & inhibidores , Reactivos de Sulfhidrilo/farmacología , Secuencia de Aminoácidos , Animales , Dominio Catalítico/genética , Secuencia Conservada , Cisteína/química , Humanos , Cinética , Mitocondrias/enzimología , Datos de Secuencia Molecular , Pisum sativum/enzimología , Pisum sativum/genética , Plastidios/enzimología , Complejo Piruvato Deshidrogenasa/química , Complejo Piruvato Deshidrogenasa/genética , Homología de Secuencia de AminoácidoRESUMEN
Pyruvate dehydrogenase kinase (PDK) is the primary regulator of flux through the mitochondrial pyruvate dehydrogenase complex (PDC). Although PDKs inactivate mitochondrial PDC by phosphorylating specific Ser residues, the primary amino acid sequence indicates that they are more closely related to prokaryotic His kinases than to eukaryotic Ser/Thr kinases. Unlike Ser/Thr kinases, His kinases use a conserved His residue for phosphotransfer to Asp residues. To understand these unique kinases better, a presumptive PDK from Arabidopsis thaliana was heterologously expressed and purified for this investigation. Purified, recombinant A. thaliana PDK could inactivate kinase-depleted maize mitochondrial PDC by phosphorylating Ser residues. Additionally, A. thaliana PDK was capable of autophosphorylating Ser residues near its N-terminus, although this reaction is not part of the phosphotransfer pathway. To elucidate the mechanism involved, we performed site-directed mutagenesis of the canonical His residue likely to be involved in phosphotransfer. When His-121 was mutated to Ala or Gln, Ser-autophosphorylation was decreased by 50% and transphosphorylation of PDC was decreased concomitantly. We postulate that either (1) His-121 is not the sole phosphotransfer His residue or (2) mutagenesis of His-121 exposes an additional otherwise cryptic phosphotransfer His residue. Thus His-121 is one residue involved in kinase function.
Asunto(s)
Arabidopsis/enzimología , Proteínas Quinasas/metabolismo , Serina/metabolismo , Alanina/química , Secuencia de Aminoácidos , Ácido Aspártico/química , ADN Complementario/metabolismo , Glutamina/química , Histidina/química , Histidina Quinasa , Cinética , Mitocondrias/enzimología , Modelos Biológicos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Ácidos Fosfoaminos/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas , Estructura Terciaria de Proteína , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Serina/química , Tripsina/farmacología , Zea mays/enzimologíaRESUMEN
A vector was constructed for intracellular expression of the Arabidopsis thaliana DnaJ homologue AtJ2 in the methylotrophic yeast Pichia pastoris. The vector includes DNA encoding an amino-terminal histidine-tag, to simplify protein purification. Shake-flask cultures could be induced to produce approximately 250 mg/ L of AtJ2. Purified recombinant AtJ2 was able to stimulate the ATPase activities of both the Escherichia coli and Zea mays cytoplasmic Stress70 chaperone proteins five- to ninefold. The carboxy terminus of AtJ2 is -CAQQ, a protein farnesylation motif. When transformed P. pastoris was induced to synthesize AtJ2 in the presence of [(3)H]mevalonolactone, radioactivity was incorporated into the protein, suggesting farnesylation.
Asunto(s)
Arabidopsis/química , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Pichia/metabolismo , Adenosina Trifosfatasas/química , Proteínas de Arabidopsis , Proteínas de Escherichia coli , Proteínas del Choque Térmico HSP40 , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/aislamiento & purificación , Histidina/química , Pichia/química , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Pea (Pisum sativum) mitochondrial pyruvate dehydrogenase (E1) was produced by coexpression of the mature alpha and beta subunits in the cytoplasm of the yeast Pichia pastoris. Size-exclusion chromatography of recombinant E1, using a Superose 12 column, yielded a peak at M(r) 160,000 that contained both alpha and beta subunits as well as E1 activity. This corresponds to the size of native alpha(2)beta(2) E1. Recombinant E1 alpha (His(6))-E1 beta was purified by affinity chromatography using immobilized Ni(+), with a yield of 2.8 mg L(-1). The pyruvate-decarboxylating activity of recombinant E1 was dependent upon added Mg(2+) and thiamin-pyrophosphate and was enhanced by the oxidant potassium ferricyanide. Native pea mitochondrial E1-kinase catalyzed phosphorylation of Ser residues in the alpha-subunit of recombinant E1, with concomitant loss of enzymatic activity. Thus, mitochondrial pyruvate dehydrogenase can be assembled in the cytoplasm of P. pastoris into an alpha(2)beta(2) heterotetramer that is both catalytically active and competent for regulatory phosphorylation.
Asunto(s)
Citoplasma/enzimología , Mitocondrias/química , Pichia/enzimología , Pisum sativum/química , Complejo Piruvato Deshidrogenasa/metabolismo , Autorradiografía , Cromatografía en Gel , Pisum sativum/ultraestructura , Fosforilación , Pichia/ultraestructura , Pliegue de Proteína , Piruvato Deshidrogenasa (Lipoamida) , Complejo Piruvato Deshidrogenasa/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Staphylococcal protein A (SPA)-based vectors were constructed to direct secretion of the E1alpha and E1beta subunits of Pisum sativum mitochondrial pyruvate dehydrogenase from Bacillus subtilis. These proteins were not exported when the signal peptide from levansucrase (SacBSP) was fused to their N-termini. Both SacBSP-E1alpha and SacBSP-E1beta fusion proteins were insoluble in the cytoplasm. However, when the SPA open-reading frame was inserted between SacBSP and E1alpha or E1beta, corresponding fusion proteins were secreted from the cells. The first (E) IgG-binding domain of SPA was sufficient to direct low level secretion of both fusion proteins (SacBSP-E-E1alpha and SacBSP-E-E1beta). Adding the second (D) IgG-binding domain improved extracellular protein yields 3- to 4-fold over E alone, but was not as efficient as secretion of the full-length (EDABC) SPA-fusion proteins. All constructs were based on the pUB110-derived multicopy plasmid pWB705. Separate B. subtilis strains transformed with SacBSP-E-E1alpha-His(6) or SacBSP-E1beta were cocultivated in the presence of Ni-NTA agarose. The native pyruvate dehydrogenase alpha2beta2 structure was bound to the affinity matrix, demonstrating assembly after secretion. The use of SPA as a fusion partner during expression of heterologous proteins by B. subtilis provides the basis of a versatile system that can be used to study both secretion and protein:protein interactions.
Asunto(s)
Bacillus subtilis/metabolismo , Mitocondrias/enzimología , Pisum sativum/metabolismo , Proteínas de Plantas/metabolismo , Piruvato Deshidrogenasa (Lipoamida) , Complejo Piruvato Deshidrogenasa/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteína Estafilocócica A/metabolismo , Animales , ADN Recombinante/genética , Vectores Genéticos , Unión Proteica , Estructura Terciaria de Proteína , ConejosRESUMEN
Pyruvate dehydrogenase kinase (PDK) specifically phosphorylates the E1alpha subunit of the pyruvate dehydrogenase complex (PDC). Sequence analysis of cloned PDKs led to the proposal that they are mechanistically related to prokaryotic 2-component His-kinases. The reaction mechanism of protein His-kinases involves autophosphorylation of a specific His residue followed by phosphotransfer to an Asp residue. Treatment of recombinant Arabidopsis thaliana PDK with the His-directed reagents diethyl pyrocarbonate (DEPC) and dichloro-(2,2':6', 2"-terpyridine)-platinum(II) dihydrate led to a marked inhibition of autophosphorylation. In addition, DEPC treatment abolished the ability of PDK to trans-phosphorylate and inactivate PDC. These results validate the prediction that PDKs require His residues for activity.
Asunto(s)
Histidina/metabolismo , Inhibidores de Proteínas Quinasas , Piruvato Deshidrogenasa (Lipoamida) , Arabidopsis/enzimología , Arabidopsis/genética , Dietil Pirocarbonato/farmacología , Histidina Quinasa , Compuestos Organoplatinos/farmacología , Fosforilación , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , Complejo Piruvato Deshidrogenasa/metabolismo , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
The peroxisomal isoform of ascorbate peroxidase (APX) is a novel membrane isoform that functions in the regeneration of NAD(+) and protection against toxic reactive oxygen species. The intracellular localization and sorting of peroxisomal APX were examined both in vivo and in vitro. Epitope-tagged peroxisomal APX, which was expressed transiently in tobacco BY-2 cells, localized to a reticular/circular network that resembled endoplasmic reticulum (ER; 3,3'-dihexyloxacarbocyanine iodide-stained membranes) and to peroxisomes. The reticular network did not colocalize with other organelle marker proteins, including three ER reticuloplasmins. However, in vitro, peroxisomal APX inserted post-translationally into the ER but not into other purified organelle membranes (including peroxisomal membranes). Insertion into the ER depended on the presence of molecular chaperones and ATP. These results suggest that regions of the ER serve as a possible intermediate in the sorting pathway of peroxisomal APX. Insight into this hypothesis was obtained from in vivo experiments with brefeldin A (BFA), a toxin that blocks vesicle-mediated protein export from ER. A transiently expressed chloramphenicol acetyltransferase-peroxisomal APX (CAT-pAPX) fusion protein accumulated only in the reticular/circular network in BFA-treated cells; after subsequent removal of BFA from these cells, the CAT-pAPX was distributed to preexisting peroxisomes. Thus, plant peroxisomal APX, a representative enzymatic peroxisomal membrane protein, is sorted to peroxisomes through an indirect pathway involving a preperoxisomal compartment with characteristics of a distinct subdomain of the ER, possibly a peroxisomal ER subdomain.
Asunto(s)
Nicotiana/enzimología , Peroxidasas/metabolismo , Peroxisomas/enzimología , Plantas Tóxicas , Secuencia de Aminoácidos , Ascorbato Peroxidasas , Brefeldino A/farmacología , Fraccionamiento Celular , Células Cultivadas , Retículo Endoplásmico/enzimología , Membranas Intracelulares/enzimología , Datos de Secuencia Molecular , Peroxidasas/química , Peroxidasas/genética , Biosíntesis de Proteínas , Transcripción GenéticaAsunto(s)
Chaperonas Moleculares/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Pliegue de Proteína , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Modelos Moleculares , Células Vegetales , Conformación ProteicaRESUMEN
The dihydrolipoamide S-acetyltransferase (E2) subunit of the maize mitochondrial pyruvate dehydrogenase complex (PDC) was postulated to contain a single lipoyl domain based upon molecular mass and N-terminal protein sequence (Thelen, J. J., Miernyk, J. A., and Randall, D. D. (1998) Plant Physiol. 116, 1443-1450). This sequence was used to identify a cDNA from a maize expressed sequence tag data base. The deduced amino acid sequence of the full-length cDNA was greater than 30% identical to other E2s and contained a single lipoyl domain. Mature maize E2 was expressed in Escherichia coli and purified to a specific activity of 191 units mg(-1). The purified recombinant protein had a native mass of approximately 2.7 MDa and assembled into a 29-nm pentagonal dodecahedron as visualized by electron microscopy. Immunoanalysis of mitochondrial proteins from various plants, using a monoclonal antibody against the maize E2, revealed 50-54-kDa cross-reacting polypeptides in all samples. A larger protein (76 kDa) was also recognized in an enriched pea mitochondrial PDC preparation, indicating two distinct E2s. The presence of a single lipoyl-domain E2 in Arabidopsis thaliana was confirmed by identifying a gene encoding a hypothetical protein with 62% amino acid identity to the maize homologue. These data suggest that all plant mitochondrial PDCs contain an E2 with a single lipoyl domain. Additionally, A. thaliana and other dicots possess a second E2, which contains two lipoyl domains and is only 33% identical at the amino acid level to the smaller isoform. The reason two distinct E2s exist in dicotyledon plants is uncertain, although the variability between these isoforms, particularly within the subunit-binding domain, suggests different roles in assembly and/or function of the plant mitochondrial PDC.
Asunto(s)
Acetiltransferasas/química , Acetiltransferasas/genética , Mitocondrias/enzimología , Complejo Piruvato Deshidrogenasa/química , Complejo Piruvato Deshidrogenasa/genética , Zea mays/enzimología , Secuencia de Aminoácidos , Arabidopsis/enzimología , Sitios de Unión , Dominio Catalítico , Clonación Molecular , ADN Complementario , Acetiltransferasa de Residuos Dihidrolipoil-Lisina , Etiquetas de Secuencia Expresada , Variación Genética , Humanos , Sustancias Macromoleculares , Datos de Secuencia Molecular , Peso Molecular , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Saccharomyces cerevisiae/enzimología , Alineación de Secuencia , Eliminación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
An Arabidopsis cDNA encoding the dihydrolipoamide S-acetyltransferase subunit of the plastid pyruvate dehydrogenase complex (E2) was isolated from a lambdaPRL2 library. The cDNA is 1709 bp in length, with a continuous open reading frame of 1440 bp encoding a protein of 480 amino acids with a calculated molecular mass of 50,079 D. Southern analysis suggests that a single gene encodes plastid E2. The amino acid sequence has characteristic features of an acetyltransferase, namely, distinct lipoyl, subunit-binding, and catalytic domains, although it is unusual in having only a single lipoyl domain. The in vitro synthesized plastid E2 precursor protein has a relative molecular weight of 67,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Upon incubation of the precursor with pea (Pisum sativum) chloroplasts, it was imported and processed to a mature-sized relative molecular weight of 60,000. The imported protein was located in the chloroplast stroma, associated with the endogenous pyruvate dehydrogenase. Catalytically active recombinant plastid E2 was purified as a glutathione S-transferase fusion protein. Analysis of plastid E2 mRNA by reverse transcriptase-polymerase chain reaction showed highest expression in flowers, followed by leaves, siliques, and roots. The results of immunoblot analysis indicate that protein expression was similar in roots and flowers, less similar in leaves, and even less similar in siliques. This is the first report, to our knowledge, describing a plastid E2.
Asunto(s)
Acetiltransferasas/genética , Arabidopsis/enzimología , Arabidopsis/genética , Complejo Piruvato Deshidrogenasa/genética , Secuencia de Aminoácidos , Animales , Cloroplastos/enzimología , Clonación Molecular , ADN Complementario/genética , ADN de Plantas/genética , Acetiltransferasa de Residuos Dihidrolipoil-Lisina , Genes de Plantas , Humanos , Datos de Secuencia Molecular , Filogenia , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Planta/genética , ARN de Planta/metabolismo , Homología de Secuencia de Aminoácido , Fracciones Subcelulares/enzimología , Distribución TisularRESUMEN
Four cDNAs, one encoding an alpha-subunit and three encoding beta-subunits of the mitochondrial pyruvate dehydrogenase, were isolated from maize (Zea mays L.) libraries. The deduced amino acid sequences of both alpha- and beta-subunits are approximately 80% identical with Arabidopsis and pea (Pisum sativum L.) homologs. The mature N terminus was determined for the beta-subunit by microsequencing the protein purified from etiolated maize shoot mitochondria and was resolved by two-dimensional gel electrophoresis. This single isoelectric species comprised multiple isoforms. Both alpha- and beta-subunits are encoded by multigene families in maize, as determined by Southern-blot analyses. RNA transcripts for both alpha- and beta-subunits were more abundant in roots than in young leaves or etiolated shoots. Pyruvate dehydrogenase activity was also higher in roots (5-fold) compared with etiolated shoots and leaves. Both subunits were present at similar levels in all tissues examined, indicating coordinated gene regulation. The protein levels were highest in heterotrophic organs and in pollen, which contained about 2-fold more protein than any other organ examined. The relative abundance of these proteins in nonphotosynthetic tissues may reflect a high cellular content of mitochondria, a high level of respiratory activity, or an extra plastidial requirement for acetate.
Asunto(s)
Complejo Piruvato Deshidrogenasa/genética , Zea mays/enzimología , Zea mays/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , ADN de Plantas/genética , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Humanos , Mitocondrias/enzimología , Datos de Secuencia Molecular , Familia de Multigenes , Filogenia , Conformación Proteica , Complejo Piruvato Deshidrogenasa/química , Homología de Secuencia de AminoácidoRESUMEN
Two maize cDNAs were isolated and sequenced that had open reading frames with approximately 37% amino acid identity to mammalian pyruvate dehydrogenase kinases. Both maize kinase sequences contain the five domains with conserved signature residues typical of procaryotic two-component histidine kinases. Sequence comparisons identified six other highly conserved motifs that are proposed to be specific to pyruvate dehydrogenase kinases. In addition, specific Trp and Cys residues are also invariant in these sequences. The maize cDNAs are 1332 (PDK1) and 1602 (PDK2) nucleotides in length, encoding polypeptides with calculated molecular masses of 38,867 and 41,327 Da that share 77% amino acid identity. Reverse transcriptase-polymerase chain reaction analysis with oligonucleotide-specific primers revealed a differential expression pattern for the two isoforms. PDK1 and PDK2 were expressed in Escherichia coli with N-terminal His6 tags to facilitate purification. The recombinant proteins migrated at 44 and 48 kDa, respectively, during SDS-polyacrylamide gel electrophoresis. Anti-PDK1 antibodies immunoprecipitated 75% of pyruvate dehydrogenase kinase activity from a maize mitochondrial matrix fraction, and recognized a matrix protein of 43 kDa. Recombinant PDK2, expressed as a fusion with the maltose-binding protein, inactivated kinase-depleted maize pyruvate dehydrogenase complex when incubated with MgATP, coincident with incorporation of 32P from [gamma-32P]ATP into the alpha subunit of pyruvate dehydrogenase.
Asunto(s)
Isoenzimas/genética , Proteínas Quinasas/genética , Zea mays/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , ADN Complementario , Isoenzimas/química , Datos de Secuencia Molecular , Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Homología de Secuencia de AminoácidoRESUMEN
Herein we report the first molecular description of the pyruvate dehydrogenase component of the higher plant plastid pyruvate dehydrogenase complex. The full-length cDNAs for the E1 alpha (1530 bp) and E1 beta (1441 bp) subunits of the Arabidopsis thaliana plastid pyruvate dehydrogenase contain open reading frames that encode polypeptides of 428 and 406 amino acids, respectively, with calculated molecular weight values of 47,120 and 44,208. The deduced amino acid sequences for Arabidopsis plastid E1 alpha and E1 beta have 61% and 68% identity to the odpA and odpB genes of the red alga Porphyra purpurea, respectively, but only 31% and 32% identity to the plant mitochondrial counterparts. Results of Southern analyses suggest that each subunit is encoded by a single gene. Northern blot analyses indicate expression of mRNAs of the appropriate size in Arabidopsis leaves.
Asunto(s)
Arabidopsis/enzimología , Complejo Piruvato Deshidrogenasa/genética , Secuencia de Aminoácidos , Clonación Molecular , Genes de Plantas , Datos de Secuencia Molecular , Filogenia , Proteínas de Plantas/genética , ARN de Planta/genética , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
The nucleotide sequence of a cDNA clone from Arabidopsis thaliana ecotype Columbia was determined, and the corresponding amino sequence deduced. The open reading frame encodes a protein, AtJ1, of 368 residues with a molecular mass of 41 471 Da and an isoelectric point of 9.2. The predicted sequence contains regions homologous to the J- and cysteine-rich domains of Escherichia coli DnaJ, but the glycine/phenylalanine-rich region is not present. Based upon Southern analysis, Arabidopsis appears to have a single atJ1 structural gene. A single species of mRNA, of 1.5 kb, was detected when Arabidopsis poly(A)+ RNA was hybridized with the atJ1 cDNA. The function of atJ1 was tested by complementation of a dnaJ deletion mutant of E. coli, allowing growth in minimal medium at 44 degrees C. The AtJ1 protein was expressed in E. coli as a fusion with the maltose binding protein. This fusion protein was purified by amylose affinity chromatography, then cleaved by digestion with the activated factor X protease. The recombinant AtJ1 protein was purified to electrophoretic homogeneity. In vitro, recombinant AtJ1 stimulated the ATPase activity of both E. coli DnaK and maize endosperm cytoplasmic Stress70. The deduced amino acid sequence of AtJ1 contains a potential mitochondrial targeting sequence at the N-terminus. Radioactive recombinant AtJ1 was synthesized in E. coli and purified. When the labeled protein was incubated with intact pea cotyledon mitochondria, it was imported and proteolytically processed in a reaction that depended upon an energized mitochondrial membrane.