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     

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.     

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     

In vivo uptake of mitochondrial malate dehydrogenase from watermelon by mitochondria of Xenopus laevis oocytes

The heterologous in vivo translation system of Xenopus laevis oocytes was used to translate messenger RNA isolated from water-melon cotyledons. Immunocytochemistry was used to localize the translation products in situ within the oocyte. In addition, the translation products were immunoprecipitated from homogenized oocytes, separated on SDS-polyacrylamide electrophoresis and visualized by fluorography. A variety of watermelon proteins encoded in the injected mRNA were translated within the oocytes. Among them was the mitochondrial isoenzyme of malate dehydrogenase (mtMDH). The mtMDH was correctly imported into the mitochondria of the oocytes, as detected by immunocytochemistry.     

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.     

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.     

Organelle and translocatable forms of glyoxysomal malate dehydrogenase. The effect of the N-terminal presequence

Many organelle enzymes coded for by nuclear genes have N-terminal sequences, which directs them into the organelle (precursor) and are removed upon import (mature). The experiments described below characterize the differences between the precursor and mature forms of watermelon glyoxysomal malate dehydrogenase. Using recombinant protein methods, the precursor (p-gMDH) and mature (gMDH) forms were purified to homogeneity using Ni2+-NTA affinity chromatography. Gel filtration and dynamic light scattering have shown both gMDH and p-gMDH to be dimers in solution with p-gMDH having a correspondingly higher molecular weight. p-gMDH also exhibited a smaller translational diffusion coefficient (D(t)) at temperatures between 4 and 32 degrees C resulting from the extra amino acids on the N-terminal. Differential scanning calorimetry described marked differences in the unfolding properties of the two proteins with p-gMDH showing additional temperature dependent transitions. In addition, some differences were found in the steady state kinetic constants and the pH dependence of the K(m) for oxaloacetate. Both the organelle-precursor and the mature form of this glyoxysomal enzyme were crystallized under identical conditions. The crystal structure of p-gMDH, the first structure of a cleavable and translocatable protein, was solved to a resolution of 2.55 A. GMDH is the first glyoxysomal MDH structure and was solved to a resolution of 2.50 A. A comparison of the two structures shows that there are few visible tertiary or quaternary structural differences between corresponding elements of p-gMDH, gMDH and other MDHs. Maps from both the mature and translocatable proteins lack significant electron density prior to G44. While no portion of the translocation sequences from either monomer in the biological dimer was visible, all of the other solution properties indicated measurable effects of the additional residues at the N-terminal.     

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.     

Glyoxysomal and mitochondrial malate dehydrogenase of watermelon (Citrullus vulgaris) cotyledons

Molecular properties of the glyoxysomal and mitochondrial isoenzyme of malate dehydrogenase (EC 1.1.1.37; L-malate: NAD⁺ oxidoreductase) from watermelon cotyledons (Citrullus vulgaris Schrad.) were investigated, using completely purified enzyme preparations. The apparent molecular weights of the glyoxysomal and mitochondrial isoenzymes were found to be 67,000 and 74,000 respectively. Aggregation at high enzyme concentrations was observed with the glyoxysomal but not with the mitochondrial isoenzyme. Using sodium dodecyl sulfate electrophoresis each isoenzyme was found to be composed of two polypeptide chains of identical size (33,500 and 37,000, respectively). The isoenzymes differed in their isoelectric points (gMDH: 8,92, mMDH: 5.39), rate of heat inactivation (gMDH: _1/2 at 40°C=3.0 min; mMDH: stable at 40°C; _1/2 at 60°C=4.5 min), adsorption to dextran gels at low ionic strenght, stability against alkaline conditions and their pH optima for oxaloacetate reduction (gMDH: pH 6.6, mMDH: pH 7.5). Very similar pH optima, however, were observed for L-malate oxidation (pH 9.3–9.5). The results indicate that the glyoxysomal and mitochondrial MDH of watermelon cotyledons are distinct proteins of different structural composition.Abbreviations EDTA ethylene diamine tetraacetic acid - gMDH and mMDH glyoxysomal and mitochondrial malate dehydrogenase, respectively     

Glyoxysomal malate dehydrogenase of watermelon cotyledons: De novo synthesis on cytoplasmic ribosomes

The development of glyoxysomal malate dehydrogenase (gMDH, EC 1.1.1.37) during early germination of watermelon seedlings (Citrullus vulgaris Schrad.) was determined in the cotyledons by means of radial immunodiffusion. The active isoenzyme was found to be absent in dry seeds. By density labelling with deuterium oxide and incorporation of [¹⁴C] amino acids it was shown that the marked increase of gMDH activity in the cotyledons during the first 4 days of germination was due to de novo synthesis of the isoenzyme. The effects of protein synthesis inhibitors (cycloheximide and chloramphenicol) on the synthesis of gMDH indicated that the glyoxysomal isoenzyme was synthesized on cytoplasmic ribosomes. Possible mechanisms by which the glyoxysomal malate dehydrogenase isoenzyme reaches its final location in the cell are discussed.Abbreviations mMDH mitochondrial malate dehydrogenase - gMDH glyoxysomal malate dehydrogenase - D₂O deuterium oxide - EDTA ethylenediaminetetraacetic acid, disodium salt     

Immunolocalization of glyoxysomal malate synthase from soybean cotyledons (Glycine max L)

Summary— Malate synthase (MS; EC 4.1.3.2), an enzyme specific to the glyoxylate cycle, was studied in cotyledons of dark-grown soybean (Glycine max L) seedlings with light and electron microscopy techniques. Immunogold localization confirmed biochemical evidence that MS from soybean is a glyoxysomal matrix enzyme.     

Mitochondrial malate dehydrogenase from watermelon: sequence of cDNA clones and primary structure of the higher-plant precursor protein

The isolation and sequence of a cDNA clone encoding the complete mitochondrial malate dehydrogenase (mMDH) of watermelon cotyledons is presented. Taking advantage of the polymerase chain reaction technology partial cDNA clones from the central part, the 3 part and the 5 part of the mRNA were obtained with oligonucleotides based on directly determined amino acid sequences. Subsequently, two complete cDNA clones for mMDH were synthesized with a sense primer corresponding to the nucleotide sequence of the amino terminal end of pre-mMDH and two antisense primers corresponding to the major alternative adenylation sites found in the mRNA.The amino acid residues for substrate and cofactor binding identified by X-ray crystallography for pig heart cytoplasmic MDH are conserved in the 320 amino acid long mature higher-plant mMDH. A presequence of 27 amino acids is present at the amino terminal end of the precursor protein.     

Purification and comparative properties of microsomal and glyoxysomal malate synthase from castor bean endosperm

Sucrose density gradient centrifugation was employed to separate microsomes, mitochondria, and glyoxysomes from homogenates prepared from castor bean (Ricinus communis) endosperm. In the case of tissue removed from young seedlings, a significant proportion of the characteristic glyoxysomal enzyme malate synthase was recovered in the microsomal fraction. Malate synthase was purified from both isolated microsomes and glyoxysomes by a procedure involving osmotic shock, KCI solubilization, and sucrose density gradient centrifugation. All physical and catalytic properties examined were identical for the enzyme isolated from both organelle fractions. These properties include a molecular weight of 575,000, with a single subunit type of molecular weight 64,000, a pH optimum of 8, apparent K_m for acetyl-CoA of 10 μm and glyoxylate of 2 mm. Microsomal and glyoxysomal malate synthases showed identical responses to various inhibitors. Adenine nucleotides were competitive inhibitors with respect to acetyl-CoA, and oxalate (K_i 110 μm) and glycolate (K_i 150 μm) were competitive inhibitors with respect to glyoxylate. Antiserum raised in rabbits against purified glyoxysomal malate synthase was used to confirm serological identity between the microsomal and glyoxysomal enzymes, and was capable of specifically precipitating ³⁵S-labeled malate synthase from KCI extracts of both microsomes and glyoxysomes isolated from [³⁵S]methionine-labeled endosperm tissue.     

Crystallization of halophilic malate dehydrogenase from Halobacterium marismortui

Malate dehydrogenase from the extreme halophile Halobacterium marismortui crystallizes in highly concentrated phosphate solution in space group 12 with cell dimensions a = 113.8 A, b = 122.8 A, c = 126.7 A, beta = 98.1 degrees. The halophilic enzyme was found to be unstable at lower concentrations of phosphate. It associates with unusually large amounts of water and salt, and the combined particle volume shows a tight fit in the unit cell.     

Reversible dissociation of malate dehydrogenase from plants]

It was demonstrated that 0.2 M citric acid (pH 2.5) inactivates highly-purified malate dehydrogenase from tea leaves; the degree of inactivation depends on temperature and time of incubation. The enzyme activity is restored by certain inorganic salts, the degree of reactivation being dependent on pH, ionic strengths of salts and duration of enzyme incubation with both inactivating and reactivating agents. Urea and guanidine hydrochloride also have a reversibly inactivating effect on the enzyme. The degree of inactivation depends on their concentration and incubation time. In the latter case reactivation of enzyme is achieved by dialysis or 20-40-fold dilution of the enzyme preparation. A kinetic study demonstrated that inactivation of enzyme by the above-mentioned agents is due to the enzyme dissociation into 4 catalytically inactive subunits with molecular weights of 17 500 +/- 1000, which under certain conditions are capable of reassociating into an active molecule of enzyme with completely restored native conformation.     

Microheterogeneity of the malate dehydrogenase from several sources

Different homogeneously purified cytosolic malate dehydrogenases gave, on isoelectric focusing, several active bands. The phenomenon could not be assigned to differences in their molecular weights or to alterations in the enzyme preparations during the purification procedure. Resolution of the multiple malate dehydrogenase active bands was achieved by chromatofocusing. The aged isolated subforms always yielded the original electrofocusing pattern. This fact suggests that conformational isomerism is a likely explanation for the charge heterogeneity of the enzymes studied.     

Fluorescence immunohistochemical localization of malate dehydrogenase isoenzymes in watermelon cotyledons : a developmental study of glyoxysomes and mitochondria

Monospecific antibodies to glyoxysomal, mitochondrial, and cytosolic I malate dehydrogenase were used for the fluorescence immunohistochemical localization of these isoenzymes in dark-grown watermelon (Citrullus vulgaris Schrad.) cotyledons. It was demonstrated that, with cell organelles isolated by sucrose density gradient centrifugation, antibodies to glyoxysomal malate dehydrogenase were specific markers for glyoxysomes, and similarly, antibodies to mitochondrial malate dehydrogenase were markers for mitochondria. The time course of the glyoxysomal malate dehydrogenase appearance and decline was not synchronous for the individual tissues and differed completely from that of the mitochondria. The cytosolic malate dehydrogenase I was confined to restricted regions of the lower epidermis. The activity which was definitively localized outside the cell organelles decreased during the first days of germination.     

Mitochondrial malate dehydrogenase of watermelon cotyledons: Time course and mode of enzyme activity changes during germination

Summary Specific antibodies were prepared against the purified mitochondrial malate dehydrogenase (EC 1.1.1.37) from cotyledons of watermelon seedlings (Citrullus vulgaris Schrad.). The isoenzyme was assayed by means of quantitative radial immunodiffusion. Cotyledons of ungerminated seeds were found to contain mitochondrial MDH. During the first 4 days of germination the enzyme activity increased threefold finally contributing 16% to the total MDH activity extracted from cotyledon tissue. Isopycnic CsCl density centrifugation was used to investigate the mode of activity increase. After a four-day period of labelling with deuterium oxide and purification of the mitochondrial isoenzyme, a density shift of 0.021kgx1^-1, accompanied by considerable band broadening of the enzyme profile was observed. These findings are evidence for the de novo synthesis of mitochondrial MDH and its relatively slow turnover in germinating seeds.Abbreviations mMDH mitochondrial malate dehydrogenase - D₂O deuterium oxide