Cell-free synthesis of glyoxysomal malate dehydrogenase

Polyadenylated mRNA was isolated from germinating watermelon cotyledons and translated in a wheat germ protein synthesizing system. The synthesis of glyoxysomal malate dehydrogenase was detected by direct immunoprecipitation and electrophoretic analysis of the precipitate. In addition to a small amount of the authentic isoenzyme (subunit molecular weight = 33 000), the major part of the incorporated [35S] methionine was observed in a polypeptide with a molecular weight of 38 000. The possible role of the larger molecule as a precursor of glyoxysomal malate dehydrogenase is discussed.     

Sequence homologies between glyoxysomal and mitochondrial malate dehydrogenase

The comparison of mitochondrial and glyoxysomal malate dehydrogenase (EC 1.1.1.37) from cotyledons of germinating watermelon (Citrullus vulgaris Schrad., cv. Kleckey's Sweet No. 6) by means of serological methods and peptide patterns revealed a high degree of homology. The N-terminal sequence analysis yielded a distinct presequence of eight or nine amino-acid residues, respectively, which is followed by an almost identical stretch of at least 20 amino-acid residues. A very similar domain has been recognized for mitochondrial malate dehydrogenase from porcine heart and yeast, and for Escherichia coli malate dehydrogenase.Abbreviations gMDH glyoxysomal malate dehydrogenase - mMDH mitochondrial malate dehydrogenase - SDS sodium dodecyl sulfate     

A Pisum sativum glyoxysomal malate dehydrogenase induced by cadmium exposure.

The glyoxysomal malate dehydrogenase (gMDH) catalyses the formation of oxaloacetate from malate during beta-oxidation of fatty acids in the glyoxysome. A partial Pisum sativum L. (cv. Greenfeast) cDNA was first isolated from a suppression subtractive hybridisation cDNA library obtained from heavy metal stressed plants. The full length cDNA was then isolated by rapid amplification of cDNA ends. The translated sequence showed strong similarity to Cucumis sativus and Citrullus lanatus gMDH including a typical glyoxysome-targeting presequence comprising the PTS2 motif and a cleavage site for a cystein-directed protease. Exposure of pea plants to Cd2+ induced expression of the gMDH gene in mature pea leaves indicating that the enzyme is under environmental control in addition to the normal developmental regulation pattern.     

Import of in-vitro-synthesized glyoxysomal malate dehydrogenase into isolated watermelon glyoxysomes

Glyoxysomal malate dehydrogenase (gMDH; EC 1.1.1.37) is synthesized by a reticulocyte system in the presence of watermelon mRNA (Citrullus vulgaris Schrad., var. Kleckey's Sweet No 6) as a cytosolic, higher-molecular-weight precursor (41 kdalton). We now show that this precursor is posttranslationally sequestered by a crude glyoxysomal fraction or by glyoxysomes purified on a Percoll^R gradient to a proteolytically protected form (60 min proteinase-K treatment at 4° C) with the size of the gMDH subunit (33 kdalton). In the presence of buffer instead of organelles a complete degradation of the precursor is obtained. The in-vitro organelle import, however, depends upon the presence of proteases such as proteinase K or trypsin. After short proteolytic treatments (e.g. 10 min proteinase K at 4° C), the correct processing of the MDH precursor is obtained even in the absence of organelles. This product, however, is not sequestered in vitro to a protease-resistant form by glyoxysomes. The possibility is discussed that under in-vivo conditions pre-gMDH is processed on the outside of the glyoxysomal membrane and transferred immediately after processing into the organelle presumably as a gMDH monomer followed by refolding and dimerization.Abbreviations gMDH glyoxysomal malate dehydrogenase - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecyl sulfate - TPCK-trypsin trypsin treated with l-1-tosylamide-2-phenylethyl chloromethyl ketone Dedicated to Professor Dr. Hubert Ziegler on the occasion of his 60th birthday     

Targeting and processing of a chimeric protein with the N-terminal presequence of the precursor to glyoxysomal citrate synthase.

Glyoxysomal citrate synthase in pumpkin is synthesized as a precursor that has a cleavable presequence at its N-terminal end. To investigate the role of the presequence in the transport of the protein to the microbodies, we generated transgenic Arabidopsis plants that expressed beta-glucuronidase with the N-terminal presequence of the precursor to the glyoxysomal citrate synthase of pumpkin. Immunogold labeling and cell fractionation studies showed that the chimeric protein was transported into microbodies and subsequently was processed. The chimeric protein was transported to functionally different microbodies, such as glyoxysomes, leaf peroxisomes, and unspecialized microbodies. These observations indicated that the transport of glyoxysomal citrate synthase is mediated by its N-terminal presequence and that the transport system is functional in all plant microbodies. Site-directed mutagenesis of the conserved amino acids in the presequence caused abnormal targeting and inhibition of processing of the chimeric protein, suggesting that the conserved amino acids in the presequence are required for recognition of the target or processing.     

Biosynthesis and intracellular transport of glyoxysomal malate dehydrogenase in germinating pumpkin cotyledons

Glyoxysomal malate dehydrogenase was synthesized as a larger molecular mass precursor in germinating pumpkin cotyledons. In pulse-chase experiments, the radioactive larger molecular mass precursor (38 kDa) disappeared and was converted to the mature form (33 kDa) of the enzyme. When the radiolabeled cotyledon was fractionated into cytosolic and organellar fractions, the larger molecular mass precursor was first recovered in the cytosolic fraction and then only after a 20 min chase the mature form was found in the organellar fraction. This indicates that the higher molecular mass precursor is synthesized in the cytosol and the processing of the transient precursor is coupled to the transport into glyoxysomes.     

Import of glyoxysomal malate dehydrogenase precursor into glyoxysomes: A heterologous in-vitro system

A heterologous in-vitro system is described for the import of the precursor to glyoxysomal malate dehydrogenase from watermelon (Citrullus vulgaris Schrad., cv. Kleckey's Sweet No. 6) cotyledons into glyoxysomes from castor-bean (Ricinus communis L.) endosperm. The 41-kDa precursor is posttranslationally sequestered and correctly processed to the mature 33-kDa subunit by a crude glyoxysomal fraction or by glyoxysomes purified on a sucrose gradient. The import and the cleavage of the extrasequence is not inhibited by metal chelators such as 1,10-phenanthroline and ethylenediaminetetraacetic acid. Uncouplers (carbonylcyanide m-chlorophenylhydrazone), ionophores (valinomycin), or inhibitors of oxidative phosphorylation (oligomycin) and ATP-ADP translocation (carboxyatractyloside) do not interfere, thus indicating the independence of the process of import by the organelle from the energization of the glyoxysomal membrane.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - EDTA ethylenediaminetetraacctic acid - gMDH glyoxysomal malate dehydrogenase - PMSF phenylmethylsulfonyl fluoride     

A cysteine endopeptidase isolated from castor bean endosperm microbodies processes the glyoxysomal malate dehydrogenase precursor protein.

A plant cysteine endopeptidase with a molecular mass of 35 kD was purified from microbodies of germinating castor bean (Ricinus communis) endosperm by virtue of its capacity to specifically process the glyoxysomal malate dehydrogenase precursor protein to the mature subunit in vitro. Processing of the glyoxysomal malate dehydrogenase precursor occurs sequentially in three steps, the first intermediate resulting from cleavage after arginine-13 within the presequence and the second from cleavage after arginine-33. The endopeptidase is unable to remove the presequences of prethiolases from rape (Brassica napus) glyoxysomes and rat peroxisomes at the expected cleavage site. Protein sequence analysis of N-terminal and internal peptides revealed high identity to the mature papain-type cysteine endopeptidases from cotyledons of germinating mung bean (Vigna mungo) and French bean (Phaseolus vulgaris) seeds. These endopeptidases are synthesized with an extended pre-/prosequence at the N terminus and have been considered to be processed in the endoplasmic reticulum and targeted to protein-storing vacuoles.     

Dispensable presequence for cellular localization and function of mitochondrial malate dehydrogenase from Saccharomyces cerevisiae

The nucleotide sequence corresponding to codons for the 17-amino acid residues in the presumed targeting presequence for yeast mitochondrial malate dehydrogenase was removed by oligonucleotide-directed mutagenesis of the isolated gene (MDH1). Integrative transformation was used to insert the "leaderless" gene (mdhl-) into the MDH1 chromosomal locus of a strain containing a disrupted MDH1 gene. Expression of the mature form of malate dehydrogenase as a primary translation product was verified by demonstrating that the mature form is synthesized in mdhl- cells at the same rate as the precursor form in MDH1 cells in the presence of carbonyl cyanide m-chlorophenylhydrazone and by comparison of in vitro translation products of RNAs from mdhl- and MDH1 cells. Expression of mdhl- restores total cellular malate dehydrogenase activity to levels comparable to those in wild type cells and reverses the phenotype associated with strains containing MDH1 disruptions by restoring wild type rates of growth in media containing acetate as a carbon source. Immunochemical analyses and enzyme assays show comparable levels of malate dehydrogenase in the matrix fractions from mitochondria isolated from mdhl- and MDH1 cells and give no evidence for accumulation of the mature enzyme in the cytosol of mdhl- cells. These results indicate that the presequence for malate dehydrogenase is not essential for efficient mitochondrial localization or function in yeast.     

Fate of glyoxysomal malate dehydrogenase from watermelon after heterologous in-vivo translation in xenopus oocytes

Total poly A⁺-mRNA from watermelon cotyledons was translated in Xenopus laevis oocytes. Watermelon glyoxysomal malate dehydrogenase was found as its higher molecular weight precursor (pre-gMDH) and accumulates over at least 48 hours of translation. Organelle separation and immunocytochemistry located the watermelon pre-gMDH in the cytosol of the oocyte. The heterologous translation product from oocytes can be imported into isolated glyoxysomes from endosperm of castor bean. This import was correct in terms of protection against proteolysis and cleavage of the presequence within the limits of accuracy. We conclude that watermelon pre-gMDH accumulates in mRNA-injected oocytes as an import competent cytosolic precursor.     

Expression and function of heterologous forms of malate dehydrogenase in yeast.

The structure of the tricarboxylic acid cycle enzyme malate dehydrogenase is highly conserved in various organisms. To test the extent of functional conservation, the rat mitochondrial enzyme and the enzyme from Escherichia coli were expressed in a strain of Saccharomyces cerevisiae containing a disruption of the chromosomal MDH1 gene encoding yeast mitochondrial malate dehydrogenase. The authentic precursor form of the rat enzyme, expressed using a yeast promoter and a multicopy plasmid, was found to be efficiently targeted to yeast mitochondria and processed to a mature active form in vivo. Mitochondrial levels of the polypeptide and malate dehydrogenase activity were found to be similar to those for MDH1 in wild-type yeast cells. Efficient expression of the E. coli mdh gene was obtained with multicopy plasmids carrying gene fusions encoding either a mature form of the procaryotic enzyme or a precursor form with the amino terminal mitochondrial targeting sequence from yeast MDH1. Very low levels of mitochondrial import and processing of the precursor form were obtained in vivo and activity could be demonstrated for only the expressed precursor fusion protein. Results of in vitro import experiments suggest that the percursor form of the E. coli protein associates with yeast mitochondria but is not efficiently internalized. Respiratory rates measured for isolated yeast mitochondria containing the mammalian or procaryotic enzyme were, respectively, 83 and 62% of normal, suggesting efficient delivery of NADH to the respiratory chain. However, expression of the heterologous enzymes did not result in full complementation of growth phenotypes associated with disruption of the yeast MDH1 gene.     

Purification procedure and N-terminal amino acid sequence of yeast malate dehydrogenase isoenzymes

A method has been devised for the rapid isolation of malate dehydrogenase isoenzymes. First, anionic proteins were precipitated with polyethyleneimine, whilst hydrophobic malate dehydrogenase remained in the supernatant fluid. Secondly, the supernatant was 30% saturated with ammonium sulfate and the two isoenzymes were separated by hydrophobic phenyl-Sepharose CL-4B chromatography. For further purification the enzymes were chromatofocused, and polybuffer was removed by hydrophobic chromatography. Affinity chromatography with blue Sepharose CL-6B [1] was used as final purification step. The purified isoenzymes were homogeneous as shown by isoelectric focusing and could be used for N-terminal sequencing. 34 amino acid residues could be identified for the cytoplasmic isoenzyme and 56 amino acid residues for the mitochondrial isoenzyme. Although there are regions of strong homology between both isoenzymes, the sequence differences clearly showed support that both isoenzymes are coded by different genes. Sequence comparison clearly indicated that the N-terminus of the cytoplasmic enzyme extended that of the mitochondrial enzyme by 12 amino acid residues. The amino acid sequence of the extending sequence resembled that of leading sequences known for enzymes which are transported into the mitochondria. The assumed leading sequence is discussed with respect to its possible role in glucose inactivation.     

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.     

Comparison of the precursor and mature forms of rat heart mitochondrial malate dehydrogenase

The complete amino acid sequence of mitochondrial malate dehydrogenase from rat heart has been determined by chemical methods. Peptides used in this study were purified after digestions with cyanogen bromide, trypsin, endoproteinase Lys C, and staphylococcal protease V-8. The amino acid sequence of this mature enzyme is compared with that of the precursor form, which includes the primary structure of the transit peptide. The transit peptide is required for incorporation into mitochondria and appears to be homologous to the NH2-terminal arm of a related cytoplasmic enzyme, pig heart lactate dehydrogenase. The amino acid differences between the rat heart and pig heart mitochondrial malate dehydrogenases are analyzed in terms of the three-dimensional structure of the latter. Only 12/314 differences are found; most are conservative changes, and all are on or near the surface of the enzyme. We propose that the transit peptide is located on the surface of the mitochondrial malate dehydrogenase precursor.     

Kinetic mechanism of the molecular forms of chicken liver mitochondrial malate dehydrogenase

• 1.1. The reaction kinetic mechanism (pH 7.4) of the molecular forms of chicken liver m-MDH is of the ordered bi-bi ternary complex type with the existence of the E-oxaloacetate, E-L-malate, E-NAD⁺ oxaloacetate, E-NADH-l-malate, E-NAD⁺-NADH, E-NAD⁺-NAD⁺, E-NADH-NAD⁺ and E-NADH-NADH abortive complexes.
• 2.2. The saturating concentration values of the substrates are notably modified, in certain cases, in the presence of the reaction products.