The proteome of higher plant mitochondria.

Plant mitochondria perform a wide range of functions in the plant cell ranging from providing energy and metabolic intermediates, via coenzyme biosynthesis and their own biogenesis to retrograde signaling and programmed cell death. To perform these functions, they contain a proteome of >2000 different proteins expressed in some cells under some conditions. The vast majority of these proteins are imported, in many cases by a dedicated protein import machinery. Recent proteomic studies have identified about 1000 different proteins in both Arabidopsis and potato mitochondria, but even for energy-related proteins, the most well-studied functional protein group in mitochondria, <75% of the proteins are recognized as mitochondrial by even one of six of the most widely used prediction algorithms. The mitochondrial proteomes contain proteins representing a wide range of different functions. Some protein groups, like energy-related proteins, membrane transporters, and de novo fatty acid synthesis, appear to be well covered by the proteome, while others like RNA metabolism appear to be poorly covered possibly because of low abundance. The proteomic studies have improved our understanding of basic mitochondrial functions, have led to the discovery of new mitochondrial metabolic pathways and are helping us towards appreciating the dynamic role of the mitochondria in the responses of the plant cell to biotic and abiotic stress.

The identification of a sequence related to apicomplexan enolase from Sarcocystis neurona.

Equine protozoal myeloencephalitis (EPM) is a neurological disease caused by Sarcocystis neurona, an apicomplexan parasite. S. neurona is also associated with EPM-like diseases in marine and small mammals. The mechanisms of transmission and ability to infect a wide host range remain obscure; therefore, characterization of essential proteins may provide evolutionary information allowing the development of novel chemotherapeutics that target non-mammalian biochemical pathways. In the current study, two-dimensional electrophoresis and matrix-assisted laser desorption ionization-time of flight (MALDI-ToF) mass spectrometry were combined to characterize and identify an enolase protein from S. neurona based on peptide homology to the Toxoplasma gondii protein. Enolase is thought to be a vestigial, non-photosynthetic protein resulting from an evolutionary endosymbiosis event of an apicomplexan ancestor with green algae. Enolase has also been suggested to play a role in parasite stage conversion for T. gondii. Characterization of this protein in S. neurona and comparison to other protozoans indicate a biochemical similarity of S. neurona enolase to other tissue-cyst forming coccidians that cause encephalitis.

ZMPP2, a novel type-2C protein phosphatase from maize.

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.

Brassicaceae express multiple isoforms of biotin carboxyl carrier protein in a tissue-specific manner.

Plastidial acetyl-coenzyme A carboxylase from most plants is a multi-enzyme complex comprised of four different subunits. One of these subunits, the biotin carboxyl carrier protein (BCCP), was previously proposed to be encoded by a single gene in Arabidopsis. We report and characterize here a second Arabidopsis BCCP (AtBCCP2) cDNA with 42% amino acid identity to AtBCCP1 and 75% identity to a class of oilseed rape (Brassica napus) BCCPs. Both Arabidopsis BCCP isoforms were expressed in Escherichia coli and found to be biotinylated and supported carboxylation activity when reconstituted with purified, recombinant Arabidopsis biotin carboxylase. In vitro translated AtBCCP2 was competent for import into pea (Pisum sativum) chloroplasts and processed to a 25-kD polypeptide. Extracts of Arabidopsis seeds contained biotinylated polypeptides of 35 and 25 kD, in agreement with the masses of recombinant AtBCCP1 and 2, respectively. AtBCCP1 protein was present in developing tissues from roots, leaves, flowers, siliques, and seeds, whereas AtBCCP2 protein was primarily expressed in 7 to 10 d-after-flowering seeds at levels approximately 2-fold less abundant than AtBCCP1. AtBCCP1 transcript reflected these protein expression profiles present in all developing organs and highest in 14-d leaves and siliques, whereas AtBCCP2 transcript was present in flowers and siliques. In protein blots, four different BCCP isoforms were detected in developing seeds from oilseed rape. Of these, a 35-kD BCCP was detected in immature leaves and developing seeds, whereas developing seeds also contained 22-, 25-, and 37-kD isoforms highly expressed 21 d after flowering. These data indicate that oilseed plants in the family Brassicaceae contain at least one to three seed-up-regulated BCCP isoforms, depending upon genome complexity.

Pyruvate dehydrogenase kinase from Arabidopsis thaliana: a protein histidine kinase that phosphorylates serine residues.

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.

Histidine modifying agents abolish pyruvate dehydrogenase kinase activity.

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.

Biotin carboxyl carrier protein isoforms in Brassicaceae oilseeds.

De novo fatty acid biosynthesis occurs predominantly in plastids. The committed step for this pathway is the production of malonyl-CoA catalysed by acetyl-CoA carboxylase (ACCase). In most plants, plastidial ACCase is a multisubunit complex minimally comprised of four polypeptides, which catalyse two reactions. In the simple oilseed plant, Arabidopsis thaliana, two cDNAs encoding biotin carboxyl carrier protein (BCCP) isoforms have been identified. The remaining three subunits of ACCase appear to be single gene members in A. thaliana [Mekhedov, Martinez de Ilarduya and Ohlrogge (2000) Plant Physiol. 122, 389-401]. Transcript and protein analyses indicate that BCCP isoform 1 is constitutively expressed while isoform 2 is predominantly expressed in developing seeds. The apparent masses of constitutive and seed-enriched BCCP isoforms agree with the apparent masses of recombinantly expressed isoforms 1 and 2, respectively. In a related oilseed, Brassica napus, multiple putative BCCP polypeptides were also observed in developing seeds. The presence of a divergent class of BCCP genes in A. thaliana and B. napus, coincident with appropriately sized biotin-containing proteins expressed specifically in developing seeds, suggests that these BCCPs play an evolutionarily conserved role in oil deposition.

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.

Molecular cloning and expression analysis of the mitochondrial pyruvate dehydrogenase from maize.

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.

Molecular analysis of two pyruvate dehydrogenase kinases from maize.

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.

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.