Enzymes of Glycolysis Are Functionally Associated with the Mitochondrion in Arabidopsis Cells
Philippe GiegéJoshua L. HeazlewoodUte RoessnerA. Harvey MillarAlisdair R. FernieChristopher J. LeaverLee Sweetlove
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Abstract:
Mitochondria fulfill a wide range of metabolic functions in addition to the synthesis of ATP and contain a diverse array of proteins to perform these functions. Here, we present the unexpected discovery of the presence of the enzymes of glycolysis in a mitochondrial fraction of Arabidopsis cells. Proteomic analyses of this mitochondrial fraction revealed the presence of 7 of the 10 enzymes that constitute the glycolytic pathway. Four of these enzymes (glyceraldehyde-3-P dehydrogenase, aldolase, phosphoglycerate mutase, and enolase) were also identified in an intermembrane space/outer mitochondrial membrane fraction. Enzyme activity assays confirmed that the entire glycolytic pathway was present in preparations of isolated Arabidopsis mitochondria, and the sensitivity of these activities to protease treatments indicated that the glycolytic enzymes are present on the outside of the mitochondrion. The association of glycolytic enzymes with mitochondria was confirmed in vivo by the expression of enolase– and aldolase–yellow fluorescent protein fusions in Arabidopsis protoplasts. The yellow fluorescent protein fluorescence signal showed that these two fusion proteins are present throughout the cytosol but are also concentrated in punctate regions that colocalized with the mitochondrion-specific probe Mitotracker Red. Furthermore, when supplied with appropriate cofactors, isolated, intact mitochondria were capable of the metabolism of 13C-glucose to 13C-labeled intermediates of the trichloroacetic acid cycle, suggesting that the complete glycolytic sequence is present and active in this subcellular fraction. On the basis of these data, we propose that the entire glycolytic pathway is associated with plant mitochondria by attachment to the cytosolic face of the outer mitochondrial membrane and that this microcompartmentation of glycolysis allows pyruvate to be provided directly to the mitochondrion, where it is used as a respiratory substrate.Keywords:
Intermembrane space
Phosphoglycerate mutase
Phosphoglycerate kinase
Yeast strains carrying recessive mutations representing four different loci that cause defects in pyruvate kinase, pyruvate decarboxylase, 3-phosphoglycerate kinase, and 3-phosphoglycerate mutase were isolated and partially characterized. Cells carrying these mutations were unable to use glucose as a carbon source as measured in turbidimetric growth experiments. Tetrad analysis indicated that these mutations were not linked to each other; one of the mutations, that affecting phosphoglycerate kinase, was located on chromosome III.
Phosphoglycerate kinase
Phosphoglycerate mutase
Pyruvate decarboxylase
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Phosphoglycerate mutases (PGAMs) participate in both the glycolytic and the gluconeogenic pathways in reversible isomerization of 3‐phosphoglycerate and 2‐phosphoglycerate. PGAMs are members of two distinct protein families: enzymes that are dependent on or independent of the 2,3‐bisphosphoglycerate cofactor. We determined the X‐ray structure of the monomeric Trypanosoma brucei independent PGAM ( Tb iPGAM) in its apoenzyme form, and confirmed this observation by small angle X‐ray scattering data. Comparing the Tb iPGAM structure with the Leishmania mexicana independent PGAM structure, previously reported with a phosphoglycerate molecule bound to the active site, revealed the domain movement resulting from active site occupation. The structure reported here shows the interaction between Asp319 and the metal bound to the active site, and its contribution to the domain movement. Substitution of the metal‐binding residue Asp319 by Ala resulted in complete loss of independent PGAM activity, and showed for the first time its involvement in the enzyme’s function. As Tb iPGAM is an attractive molecular target for drug development, the apoenzyme conformation described here provides opportunities for its use in structure‐based drug design approaches. Database Structural data for the Trypanosoma brucei 2,3‐bisphosphoglycerate‐independent phosphoglycerate mutase (iPGAM) has been deposited with the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank under code 3NVL . Structured digital abstract TbiPGAM and TbiPGAM bind by x‐raycrystallography ( View interaction )
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Phosphoglycerate kinase
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Extracts of embryonic mouse tissues (skeletal, cardiac and smooth muscle, and brain) were analysed by Cellogel electrophoresis for their isoenzymic distributions of three enzymes, creatine phosphokinase, aldolase and phosphoglycerate mutase. Embryonic tissues from the 12th day to the end of gestation were examined for isoenzyme transitions, and it was found that the adult forms of these enzymes appeared during gestation. Extracts from cloned teratocarcinoma cells were similarly examined in order to determine their degree of bio-chemical differentiation. Undifferentiated embryonal carcinoma cells contained only the early embryonic forms of all three enzymes, while differentiated cells formed in vivo, and in some cases in vitro, started to express the adult types of creatine phosphokinase and aldolase. Thus, biochemical parallels have been demonstrated between developing embryonic tissues and teratocarcinoma cells differentiating in vitro.
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Creatine kinase
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Teratocarcinoma
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2-Phosphotartronate has been synthesized by permanganate oxidation of glycerol 2-phosphate and has been tested as an inhibitor of five glycolytic enzymes that bind phosphoglycerate or phosphoglycollate. Competitive inhibition of rabbit muscle phosphoglycerate mutase, enolase and pyruvate kinase was observed. Triose phosphate isomerase and 3-phosphoglycerate kinase were not inhibited.
Phosphoglycerate mutase
Phosphoglycerate kinase
Triosephosphate isomerase
DHAP
Dihydroxyacetone phosphate
Phosphotransferases
Glycerol kinase
Enolase
Glucose-6-phosphate isomerase
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The glycolytic enzyme phosphoglycerate mutase exists in two evolutionarily unrelated forms. Vertebrates have only the 2,3‐bisphosphoglycerate‐dependent enzyme (dPGM), whilst higher plants have only the cofactor‐independent enzyme (iPGM). Certain eubacteria possess genes encoding both enzymes, and their respective metabolic roles and activities are unclear. We have over‐expressed, purified and characterised the two PGMs of Escherichia coli . Both are expressed at high levels, but dPGM has a 10‐fold higher specific activity than iPGM. Differential inhibition by vanadate was observed. The presence of an integral manganese ion in iPGM was confirmed by EPR spectroscopy.
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Phosphoglycerate kinase
Vanadate
Mutase
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Phosphoglycerate kinase
Phosphoglycerate mutase
Glucose-6-phosphate isomerase
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The phosphonomethyl analogue of 3-phosphoglycerate (2-hydroxy-4-phosphonobutanoate) is a potent competitive inhibitor of cofactor-dependent phosphoglycerate mutase from yeast and of cofactor-independent phosphoglycerate mutase from wheat germ. For the yeast enzyme Ki is 1.3 mM (Km for substrate is 0.71 mM); for the wheatgerm enzyme Ki is 18 mM (Km for substrate is 0.86 mM). This analogue should be a useful tool for n.m.r. spectroscopic studies on the mechanism of action of the two mutases. The arsonomethyl analogue of 3-phosphoglycerate (4-arsono-2-hydroxybutanoate) was a relatively poor inhibitor.
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Phosphoglycerate kinase
Phosphofructokinase 2
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Phosphoglycerate kinase
Phosphoglycerate mutase
Phosphotransferases
Phosphoglucomutase
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The subcellular localization of the muscle aldolase (aldolase A) in cardiomyocytes was determined immunocytochemically by light and electron microscopy. The enzyme was localized in the cytoplasm and also in cardiomyocyte nuclei. Inside the nuclei it was preferentially localized in the heterochromatin region. The nuclear localization was confirmed by the measurement of aldolase activity in subcellular fractions of a heart muscle, and in isolated nuclei of cardiomyocytes. There was no detectable aldolase activity in isolated cardiomyocyte nuclei fractions if the fraction was not preincubated with a solution containing Triton X-100 and KCl. The calculated concentration of aldolase in the nucleus was about 0.6 micro M. This paper is the first report on the localization of aldolase A inside cardiomyocyte nuclei.
Aldolase B
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Phosphoglycerate mutase
Phosphoglycerate kinase
Mechanism of Action
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