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

Malate dehydrogenases of leaf tissue from Spinacia oleracea: Properties of three isoenzymes

Autophagy is a conserved degradative process that is crucial for cellular homeostasis and cellular quality control via the selective removal of subcellular structures such as mitochondria. We demonstrate that a regulatory link exists between mitochondrial function and autophagy in Saccharomyces cerevisiae. During amino-acid starvation, the autophagic response consists of two independent regulatory arms-autophagy gene induction and autophagic flux-and our analysis indicates that mitochondrial respiratory deficiency severely compromises both. We show that the evolutionarily conserved protein kinases Atg1, target of rapamycin kinase complex I, and protein kinase A (PKA) regulate autophagic flux, whereas autophagy gene induction depends solely on PKA. Within this regulatory network, mitochondrial respiratory deficiency suppresses autophagic flux, autophagy gene induction, and recruitment of the Atg1-Atg13 kinase complex to the pre-autophagosomal structure by stimulating PKA activity. Our findings indicate an interrelation of two common risk factors-mitochondrial dysfunction and autophagy inhibition-for ageing, cancerogenesis, and neurodegeneration.

Nuclei, mitochondria, microsomes and cytosol were obtained from rat liver homogenates in sucrose solution by centrifugal fractionation. Dise electrophoresis exhibits the existence of two separately migrating malate dehydrogenases in these particulate fractions. One of them is preferably found in the mitochondria, the other one in the cytosol. The two malate dehydrogenase separated by disc electrophoresis are probably identical with the mitochondrial and cytoplasmic malate dehydrogenases differentiated earlier by other authors observing their different reaction kinetics with oxaloacetate.In starvation the malate dehydrogenase activity in the cytoplasm decreased, whereas that in the mitochondria increased. The increase in mitochondrial activity is prevented by actinomycin. The decrease of cytoplasmic malate dehydrogenase caused by starvation is aggravated by actinomycin. All these effects are similar if the malate dehydrogenase activities are expressed as units per g liver fresh weight, per g body weight or per mg protein content in the extract.The regulation of malate dehydrogenase in the mitochondria is discussed as repression by carbohydrates (or a metabolite originating from carbohydrates). The increase of the enzyme in starvation can therefore be understood as “derepression.” As was found by other authors [2, 16, 17a], mitochondrial malate dehydrogenase participates in gluconeogenesis in the liver; thus a repression of this enzyme by carbohydrates appears “useful for the cell economy”. Probably only the fraction of malate dehydrogenase activity additionally arising in starvation is necessary for gluconeogenesis, whereas the mitochondrial malate dehydrogenase present in normal nutrition is functioning for carbohydrate catabolism in the citric acid cycle.

1. The mitochondrial malate dehydrogenase from rat liver has been purified to a state of homogeneity as judged by starch-gel electrophoresis and the cytoplasmic isoenzyme has been obtained in a partically purified state. 2. Inhibition of the isoenzymes by sulphite has been studied. 3. In mitochondria loaded with sulphite, the catalytic activity of the (partially inhibited) internal malate dehydrogenase has been measured by addition of oxaloacetate to the suspension medium and observation of the consequent decrease in fluorescence of NADH. 4. Addition of mitochondrial malate dehydrogenase to suspensions of mitochondria loaded with sulphite resulted in an increase in the level of intramitochondrial enzymic activity as measured by the above technique. Addition of the cytoplasmic isoenzyme did not result in such an increase. 5. These results show that mitochondria in suspension are permeable to the mitochondrial malate dehydrogenase but not to the cytoplasmic isoenzyme. 6. This conclusion has been confirmed by direct measurement of a decrease of enzyme activity in solution and an increase inside the mitochondria after incubation of organelles in solutions containing mitochondrial malate dehydrogenase. No such effect was observed with the cytoplasmic isoenzyme. 7. Some features of the permeation process have been studied.

The metabolon theory describes the protein interactions of enzymes regulating a metabolic pathway. Through these interactions proteins can share substrates through direct channeling. One set of enzymes that are involved in metabolon complexes are citrate synthase and malate dehydrogenase. This study attempts to understand the interactions regulating the metabolon of malate dehydrogenase (MDH) and citrate synthase (CS), which are integral intermediate metabolic enzymes. Using size exclusion chromatography and sedimentation, previous studies identified the interaction between CS and MDH to be isoform specific, but no further work has been conducted to obtain affinity values or the biophysical nature of the interaction. Codon optimized His tagged recombinant proteins were expressed and purified using affinity chromatography. These enzymes include: glyoxysomal malate dehydrogenase (gMDH), human cytosolic malate dehydrogenase (MDH1), human mitochondrial malate dehydrogenase (MDH2), and human mitochondrial citrate synthase (CS). We analyzed the protein interactions using fast thermal melts which further explain the thermal stability of the the dimerization of the metabolons. We first show that protein concentration did not induce oligomerization of CS or any MDH isoform, indicating that any changes are not from MDH or CS homodimerization. Next, we investigated specific CS‐MDH interactions using fast thermal melts. The results show that from the initial melting temperature of CS, the addition of mitochondrial MDH had a greater shift in its melting temperature than the addition of cytosolic MDH, by 3–4°C. The melting point of CS was not altered in the presence of the plant MDH isoform. This indicates a specific and dramatic set of contacts for mitochondrial MDH and CS. However unlike the information found in the literature there was still a measurable shift in melting point when cytosolic MDH was incubated with CS. Overall this shows for the first time a quantifiable interaction between proteins in a well‐established pathway that will allow the further dissection of these interactions.Support or Funding InformationSupport for this project was from NSF REU 146045This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Physical properties and chemical compositions of cytoplasmic and mitochondrial malate dehydrogenase from Physarum polycephalum

Malate Dehydrogenase (MDH) stands as a pivotal enzyme crucial for cellular energy metabolism, orchestrating the conversion of malate to oxaloacetate in both prokaryotic and eukaryotic organisms. This study delves into the evolutionary trajectories of MDH1 (cytoplasmic) and MDH2 (mitochondrial), offering substantial evidence supporting the archaebacterial origin of eukaryotes. The dataset spans nine groups, encompassing human MDH1 and MDH2, mammalian MDH1 and MDH2, amphibian MDH2, arthropod MDH2, amoeba MDH1, archaea, and bacteria. Protein BLAST analysis revealed significant sequence homology in mammalian MDH1, particularly among primates, underlining a close evolutionary connection. Conversely, lower eukaryotes, including amoeba, arthropods, and amphibians, exhibited marked divergence from human MDH1 and MDH2. Phylogenetic analysis unveils distinct clusters for MDH1 and MDH2, accentuating significant genetic diversity between mitochondrial and cytoplasmic MDH enzymes. Prokaryotic MDH sequences cluster with human mitochondrial MDH2, while MDH1 forms a separate cluster. Staphylococcus MDH aligns with archaeal MDH, emphasizing the diversity within bacterial MDH evolution. Protein variability analysis indicates noteworthy divergence of human MDH1 from prokaryotic MDH, while MDH2 displays comparatively lower divergence. Pairwise evolutionary divergence analysis sheds light on complex relationships among MDH protein sequences. Human MDH1 shows close evolutionary ties to mammalian MDH1, whereas MDH2 exhibits a unique pattern, aligning closely with mammalian MDH2, arthropods, and amphibians. Furthermore, MDH2 demonstrates closer proximity to bacterial MDH, supporting a bacterial origin of mitochondrial MDH. In contrast, MDH1 displays less divergence to archaeal MDH than its bacterial counterpart, endorsing an archaeal origin for cytoplasmic MDH. In conclusion, this study provides compelling support for the archaebacterial origin of eukaryotes, suggesting a bacterial endosymbiont within an archaeal host that evolved into mitochondria. It contributes valuable insights into MDH evolution, unraveling the intricate relationships and unique adaptations shaping the evolutionary history of eukaryotic cells.
 Bangladesh J Microbiol, Volume 40, Number 1, June 2023, pp 15-24

Malate dehydrogenase fractions of the chick embryo were demonstrated after starch gel electrophoresis of homogenates of liver, brain and spleen. A total of seven malate dehydrogenase fractions were observed to occur in the chick embryo in an organ specific pattern. Treatment of the homogenates with urea, sodium chloride-sodium phosphate, and p-chloromercuribenzoate prior to electrophoresis revealed that only three distinct malate dehydrogenase-active proteins were presence. Two of these proteins exhibited properties similar to those previously reported for the supernatant malate dehydrogenase and mitochondrial malate dehydrogenase of other species. Becuase of the differing properties of chick malate and lactate dehydrogenase it is concluded that the molecular basis for malate dehydrogenase isozymes is different from that reported for lactate dehydrogenase isozymes.

Electrophoretic patterns of malate and lactate dehydrogenases in a freshwater catfish

Kinetic measurements of the uptake of native mitochondrial aspartate aminotransferase and malate dehydrogenase into mitochondria in vitro were carried out. The uptake of both the enzymes is essentially complete in 1 min and shows saturation characteristics. The rate of uptake of aspartate aminotransferase into mitochondria is decreased by malate dehydrogenase, and vice versa. The inhibition is exerted by isoenzyme remaining outside the mitochondria rather than by isoenzyme that has been imported. The thiol compound beta-mercaptoethanol decreases the rate of uptake of the tested enzymes; inhibition is a result of interaction of beta-mercaptoethanol with the mitochondria and not with the enzymes themselves. The rate of uptake of aspartate aminotransferase is inhibited non-competitively by malate dehydrogenase, but competitively by beta-mercaptoethanol. The rate of uptake of malate dehydrogenase is inhibited non-competitively by aspartate aminotransferase and by beta-mercaptoethanol. beta-Mercaptoethanol prevents the inhibition of the rate of uptake of malate dehydrogenase by aspartate aminotransferase. These results are interpreted in terms of a model system in which the two isoenzymes have separate but interacting binding sites within a receptor in the mitochondrial membrane system.

Although pyruvate carboxylase associated with both mitochondrial aspartate aminotransferase and malate dehydrogenase, it had a higher affinity for the amino-transferase. Furthermore, the aminotransferase enhanced dissociation of malate dehydrogenase from pyruvate carboxylase. Glutamate dehydrogenase did not associate with pyruvate carboxylase alone, but it apparently associated with the pyruvate carboxylase-aminotransferase complex, and malate dehydrogenase associated with the resulting ternary complex. Citrate synthase and other proteins tested did not associate with pyruvate carboxylase. However, citrate synthase associated with the pyruvate carboxylase-malate dehydrogenase complex. Apparently as a consequence of these heteroenzyme interactions, the rate of the pyruvate carboxylase reaction was slightly greater when coupled with malate dehydrogenase or both malate dehydrogenase and citrate synthase than when coupled with citrate synthase alone. In addition, in the presence of both coupling enzymes, the rate of conversion of pyruvate to citrate was higher than predicted on the basis of the Michaelis-Menten relationship of the two coupling enzymes. Therefore, binding of malate dehydrogenase to pyruvate carboxylase enhances pyruvate carboxylase activity. Association of citrate synthase with the malate dehydrogenase-pyruvate carboxylase binary complex does not alter activation of pyruvate carboxylase but results in citrate synthase being more reactive than free citrate synthase with oxalacetate.

Using CUREs to integrate research into the teaching environment has significant impact on motivation and persistence of STEM students as seen from traditional undergraduate research experiences. Specifically, involvement in undergraduate research provides opportunities to think and act as scientists, bolsters feelings of belonging, and improves confidence in STEM. THE MDH CURES Community is an NSF funded community of faculty using malate dehydrogenase (MDH) to engage students in CUREs. One of several themes of MDH CUREs is protein‐protein interactions and the impact of post‐translational modification. Here we present how several primarily undergraduate and comprehensive universities and a community college, several of which are Hispanic Serving Institutions (HSIs), have included MDH‐CS interactions in their classroom. Interactions between cytosolic MDH and four other proteins thought to share substrate/product were explored in silico at a community college in a first‐year chemistry course. At Malone university, students used MS phosphorylation information to predict potential interactions which they explored by mutagenesis. Students at Mercyhust University have used in‐silico and experimental methods to design and then test CS and MDH interactions using pull down assays. Students at Suffolk University created a novel bacterial two‐hybrid model to test interactions between cytosolic and mitochondrial MDH with CS in a genetics course. In a horizontally integrated sequence, students at University of San Diego modeled potential interactions between MDH and CS and the effects of various mutations. In a subsequent semester, students used that information to analyze the mutation’s functional impact and effect on interactions with MDH‐CS using a pull‐down assay. Each project is an ongoing effort working as part of the MCC network. Approaches and lessons learned will be shared.

Diurnal variations in enzymic activities in subcellular fractions of cactus phylloclades

The resistance of mitochondrial respiration and enzymatic activity to elevated temperatures differs among species in parallel with differences in their maximal body temperatures. To examine the causes of thermal disruption of mitochondrial respiration, we have measured rates of succinate-supported respiration and activities of the mitochondrial enzymes cytochrome c oxidase, succinic dehydrogenase, and malate dehydrogenase in mitochondria from the hydrothermal vent tube worm Riftia pachyptila and the coastal bivalve Solemya reidi. Intact mitochondria and mitochondrial membrane preparations were incubated for 10 min at temperatures between 0° and 70° C, and function was examined at 20° C (R. pachyptila) or 15° C (S. reidi). Incubation at similar elevated temperatures caused inactivation of respiration and caused the activities of several mitochondrial enzymes to either increase or decrease sharply. Fluorescence polarization measurements using the hydrophobic probe 1,6-diphenyl-1,3,5-hexatriene showed that no sharp changes in membrane fluidity occurred at the temperatures at which respiration and membrane-associated enzymes altered activity. Reducing bulk membrane fluidity by enrichment with saturated phospholipids had no effect on the thermal inactivation of membrane-associated enzymes. However, disruption of membrane hydrophobic interactions with detergents, 1-5 mM deoxycholate or 5-25 mM octyl glucoside, significantly reduced the temperatures at which loss of enzyme activity occurred. We conclude that disruption of hydrophobic interactions between membrane proteins and their closely associated lipids, or between membrane proteins themselves, is at least partially responsible for high temperature-induced loss of mitochondrial function.

Salt stress was shown to cause changes in the activity and isozyme composition of mitochondrial malate dehydrogenase (MDH) in leaves of amaranth (C4-type); these changes were manifested in the induction of an additional MDH isoform. Exposure of spinach plants (C3-type) to a NaCl solution did not cause significant changes in the isozyme composition and activity of the enzymes studied. Succinate dehydrogenase (SDH) activity in amaranth leaves under salt stress conditions increased several times, while the isoenzyme composition of SDH remained unchanged. The levels of gene transcripts in leaves of amaranth and spinach clearly correlated with changes in the MDH and SDH activity. Salt stress enhanced expression of the mdh-mtx (mitochondrial) and mdh-cyt (cytoplasmic) genes in amaranth leaves during the whole experiment. In the case of the sdha gene, a decrease in the content of transcripts was observed that clearly correlated with the reduction in SDH activity.