The dihydrolipoamide S-acetyltransferase subunit of the mitochondrial pyruvate dehydrogenase complex from maize contains a single lipoyl domain.

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.

Cloning and molecular analyses of the Arabidopsis thaliana plastid pyruvate dehydrogenase subunits.

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.

The mitochondrial pyruvate dehydrogenase complex: nucleotide and deduced amino-acid sequences of a cDNA encoding the Arabidopsis thaliana E1 alpha-subunit.

A cDNA encoding the E1 alpha subunit of the Arabidopsis thaliana (At) mitochondrial (mt) pyruvate dehydrogenase complex (PDC) was sequenced. The 1435-bp cDNA consists of a 1167-bp open reading frame encoding a 43.0-kDa polypeptide of 389 amino acids (aa) (pI 7.1). The plant E1 alpha subunit has 47-51% aa sequence identity with other eukaryotic sequences. Among the regions that are highly conserved are the aa surrounding phosphorylation sites 1 and 2 of the mammalian sequence, including the conserved Ser292 residue of At at site 1. An essential active site residue, Cys62 of the bovine subunit, is also conserved. A 32-aa presumptive mt targeting sequence is present at the N terminus.

Purification, Characterization, and Submitochondrial Localization of a 58-Kilodalton NAD(P)H Dehydrogenase.

An NADH dehydrogenase activity from red beet (Beta vulgaris L.) root mitochondria was purified to a 58-kD protein doublet. An immunologically related dehydrogenase was partially purified from maize (Zea mays L. B73) mitochondria to a 58-kD protein doublet, a 45-kD protein, and a few other less prevalent proteins. Polyclonal antibodies prepared against the 58-kD protein of red beet roots were found to immunoprecipitate the NAD(P)H dehydrogenase activity. The antibodies cross-reacted to similar proteins in mitochondria from a number of plant species but not to rat liver mitochondrial proteins. The polyclonal antibodies were used in conjunction with maize mitochondrial fractionation to show that the 58-kD protein was likely part of a protein complex loosely associated with the membrane fraction. A membrane-impermeable protein cross-linking agent was used to further show that the majority of the 58-kD protein was located on the outer surface of the inner mitochondrial membrane or in the intermembrane space. Analysis of the cross-linked 58-kD NAD(P)H dehydrogenase indicated that specific proteins of 64, 48, and 45 kD were cross-linked to the 58-kD protein doublet. The NAD(P)H dehydrogenase activity was not affected by ethyleneglycol-bis([beta]-aminoethyl ether)-N,N[prime] -tetraacetic acid or CaCl2, was stimulated somewhat (21%) by flavin mononucleotide, was inhibited by p-chloromercuribenzoic acid (49%) and mersalyl (40%), and was inhibited by a bud scale extract of Platanus occidentalis L. containing platanetin (61%).

Purification, Characterization, and Submitochondrial Localization of the 32-Kilodalton NADH Dehydrogenase from Maize.

Plant mitochondria have the unique ability to directly oxidize exogenous NAD(P)H. We recently separated two NAD(P)H dehydrogenase activities from maize (Zea mays L.) mitochondria using anion-exchange (Mono Q) chromatography. The first peak of activity oxidized only NADH, whereas the second oxidized both NADH and NADPH. In this paper we describe the purification of the first peak of activity to a 32-kD protein. Polyclonal antibodies to the 32-kD protein were used to show that it was present in mitochondria from several plant species. Two-dimensional gel analysis of the 32-kD NADH dehydrogenase indicated that it consisted of two major and one minor isoelectric forms. Immunoblot analysis of submitochondrial fractions indicated that the 32-kD protein was enriched in the soluble protein fraction after mitochondrial disruption and fractionation; however, some association with the membrane fraction was observed. The membrane-impermeable protein cross-linking agent 3,3[prime] -dithiobis-(sulfosuccinimidylpropionate) was used to further investigate the submitochondrial location of the 32-kD NADH dehydrogenase. The 32-kD protein was localized to the outer surface of the inner mitochondrial membrane or to the intermembrane space. The pH optimum for the enzyme was 7.0. The activity was found to be severely inhibited by p-chloromercuribenzoic acid, mersalyl, and dicumarol, and stimulated somewhat by flavin mononucleotide.

The nucleotide and deduced amino acid sequences of a cDNA encoding the E1 beta-subunit of the Arabidopsis thaliana mitochondrial pyruvate dehydrogenase complex.

A cDNA encoding the E1 beta subunit of the Arabidopsis thaliana mitochondrial pyruvate dehydrogenase complex was sequenced. The 1230 bp cDNA contains a 1089-base open reading frame encoding a polypeptide of 363 amino acids with a predicted molecular mass of 39,190 Da and an isoelectric point of 4.9. A 29-residue presumptive mitochondrial targeting sequence is present at the amino terminus.

Monoclonal Antibodies to the [alpha]- and [beta]-Subunits of the Plant Mitochondrial F1-ATPase.

We have generated nine monoclonal antibodies against subunits of the maize (Zea mays L.) mitochondrial F1-ATPase. These monoclonal antibodies were generated by immunizing mice against maize mitochondrial fractions and randomly collecting useful hybridomas. To prove that these monoclonal antibodies were directed against ATPase subunits, we tested their cross-reactivity with purified F1-ATPase from pea cotyledon mitochondria. One of the antibodies ([alpha]-ATPaseD) cross-reacted with the pea F1-ATPase [alpha]-subunit and two ([beta]-ATPaseD and [beta]-ATPaseE) cross-reacted with the pea F1-ATPase [beta]-subunit. This established that, of the nine antibodies, four react with the maize [alpha]-ATPase subunit and the other five react with the maize [beta]-ATPase subunit. Most of the monoclonal antibodies cross-react with the F1-ATPase from a wide range of plant species. Each of the four monoclonal antibodies raised against the [alpha]-subunit recognizes a different epitope. Of the five [beta]-subunit antibodies, at least three different epitopes are recognized. Direct incubation of the monoclonal antibodies with the F1-ATPase failed to inhibit the ATPase activity. The monoclonal antibodies [alpha]-ATPaseD and [beta]-ATPaseD were bound to epoxide-glass QuantAffinity beads and incubated with a purified preparation of pea F1-ATPase. The ATPase activity was not inhibited when the antibodies bound the ATPase. The antibodies were used to help map the pea F1-ATPase subunits on a two-dimensional map of whole pea cotyledon mitochondrial protein. In addition, the antibodies have revealed antigenic similarities between various isoforms observed for the [alpha]- and [beta]-subunits of the purified F1-ATPase. The specificity of these monoclonal antibodies, along with their cross-species recognition and their ability to bind the F1-ATPase without inhibiting enzymic function, makes these antibodies useful and invaluable tools for the further purification and characterization of plant mitochondrial F1-ATPases.

Partial Purification and Characterization of Three NAD(P)H Dehydrogenases from Beta vulgaris Mitochondria.

Mitochondria isolated from the taproot of beet (Beta vulgaris) were used in an effort to identify and partially purify the proteins constituting the exogenous NADH dehydrogenase. Three NAD(P)H dehydrogenases are released from these mitochondria by sonication, and these enzymes were partially purified using fast protein liquid chromatography. One of the enzymes, designated peak I, is capable of oxidizing NADPH and the beta form of NADH. The other two activities, peaks II and III, oxidize only beta-NADH. All three peaks are insensitive to divalent cation chelators and a complex I inhibitor, rotenone. The major component to peak I is a polypeptide with an apparent molecular mass of approximately 42 kilodaltons. Peak I activity was insensitive to platanetin, a specific inhibitor of the exogenous dehydrogenase, and insensitive to added Ca(2+) or Mg(2+). Peak I displayed a broad pH activity profile with an optimum between 7.5 and 8.0 for both NADPH and NADH. Purified peak II gave a single polypeptide of about 32 kilodaltons, had a pH optimum between 7.0 and 7.5, and was slightly stimulated by Ca(2+) and Mg(2+). As with peak I, platanetin had no effect on peak II activity. Peak III was not purified completely, but contained two major polypeptides with apparent molecular masses of 55 and 40 kilodaltons. This enzyme was not affected by Ca(2+) and Mg(2+), but was inhibited by platanetin. The peak III enzyme had a rather sharp pH optimum of approximately 6.5 to 6.6. The above data indicate that peak III activity is likely the exogenous NADH dehydrogenase.

Mitochondrial malate dehydrogenase from corn : purification of multiple forms.

A method to fractionate corn (Zea mays L. B73) mitochondria into soluble proteins, high molecular weight soluble proteins, and membrane proteins was developed. These fractions were analyzed by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and assays of mitochondrial enzyme activities. The Krebs cycle enzymes were enriched in the soluble fraction. Malate dehydrogenase has been purified from the soluble fraction by a two-step fast protein liquid chromatography method. Six different malate dehydrogenase peaks were obtained from the Mono Q column. These peaks were individually purified using a Phenyl Superose column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified peaks showed that three of the isoenzymes consisted of different homodimers (I, III, VI) and three were different heterodimers (II, IV, V). Apparent molecular masses of the three different monomer subunits were 37, 38, and 39 kilodaltons. Nondenaturing gel analysis of the malate dehydrogenase peaks showed that each Mono Q peak contained a band of malate dehydrogenase activity with different mobility. These observations are consistent with three nuclear genes encoding corn mitochondrial malate dehydrogenase. Polyclonal antibodies raised against purified malate dehydrogenase were used to identify the gene products using Western blots of two-dimensional gels.