Enzymes of glycolysis are functionally associated with the mitochondrion in Arabidopsis cells - PubMed (original) (raw)

Enzymes of glycolysis are functionally associated with the mitochondrion in Arabidopsis cells

Philippe Giegé et al. Plant Cell. 2003 Sep.

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 (13)C-glucose to (13)C-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.

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Figures

Figure 1.

Figure 1.

Proteins of the IMS and OMM Separated by Two-Dimensional Gel Electrophoresis. An IMS/OMM fraction was prepared from isolated Arabidopsis mitochondria by osmotic shock and centrifugation (for details, see Methods). Proteins were separated by isoelectric focusing on 3 to 10 nonlinear immobilized pH gradients for the first dimension and by SDS-PAGE for the second dimension. For clarity, only the relevant section of the gel is shown. The numbered spots are those that have been identified by MS/MS (see Table 2).

Figure 2.

Figure 2.

Localization of Aldolase and Enolase in Arabidopsis Protoplasts. Arabidopsis protoplasts were transformed with constructs for the expression of an aldolase-YFP fusion, an enolase-YFP fusion, and YFP alone (for more details, see Methods). YFP fluorescence was visualized using a confocal microscope and compared with the distribution of the mitochondrion-specific probe Mitotracker Red (Molecular Probes). (A) to (C) YFP fluorescence. (D) to (F) Mitotracker Red fluorescence. (A) and (D) show a protoplast expressing aldolase::YFP, (B) and (E) show a protoplast expressing enolase::YFP, and (C) and (F) show a protoplast expressing YFP alone as a control.

Figure 3.

Figure 3.

Submitochondrial Localization of Aldolase Is Revealed by a Protease Protection Assay. The protease thermolysin was added to isolated mitochondria in the presence or absence of Triton X-100 to rupture the mitochondrial membranes. The reaction was terminated by the addition of EGTA, and 50 μg of the protein sample was fractionated by SDS-PAGE before being transferred to a nitrocellulose membrane. The membrane was probed with antisera against aldolase and fumarase.

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References

    1. Ali, M., Rellos, P., and Cox, T.M. (1998). Hereditary fructose intolerance. J. Med. Genet. 35, 353–365. - PMC - PubMed
    1. Anderson, L.E., Wang, X., and Gibbons, J.T. (1995). Three enzymes of carbon metabolism or their antigenic analogs in pea leaf nuclei. Plant Physiol. 108, 659–667. - PMC - PubMed
    1. Arabidopsis Genome Initiative (2000). Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408, 796–815. - PubMed
    1. Azama, K., Abe, S., Sugimoto, H., and Davies, E. (2003). Lysine-containing proteins in maize endosperm: A major contribution from cytoskeleton-associated carbohydrate-metabolizing enzymes. Planta, in press. - PubMed
    1. Balk, J., and Leaver, C.J. (2001). The PET1-CMS mitochondrial mutation in sunflower is associated with premature programmed cell death and cytochrome c release. Plant Cell 13, 1803–1818. - PMC - PubMed

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