Identification of 14 new phosphoproteins involved in important plant mitochondrial processes

Bykova, Natalia V.; Egsgaard, Helge; Møller, Ian Max

doi:10.1016/s0014-5793(03)00250-3

Cited by 119 publications

(83 citation statements)

References 30 publications

(47 reference statements)

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“…These include several complex I subunits, enzymes involved in fatty acid metabolism, and the gamma subunit of the F 1 -ATPase (γF 1 ). MnSOD has been shown to be phosphorylated in potato mitochondria (19) using radiolabeled ATP, but we have not found evidence of this phosphorylation described in mammalian systems. The Pro-Q Diamondstained gel shows 4 distinct spots that were each identified as MnSOD using MS/MS, which have similar molecular weight but different isoelectric points, consistent with multiple phosphorylation states.…”

Section: Resultscontrasting

confidence: 76%

Mitochondrial Matrix Phosphoproteome: Effect of Extra Mitochondrial Calcium

et al. 2006

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Post-translational modification of mitochondrial proteins by phosphorylation or dephosphorylation plays an essential role in numerous cell signaling pathways involved in regulating energy metabolism and in mitochondria-induced apoptosis. Here we present a phosphoproteomic screen of the mitochondria matrix proteins and begin to establish the protein phosphorylations acutely associated with calcium ions (Ca 2+ ) signaling in porcine heart mitochondria. Forty-five phosphorylated proteins were detected by gel electrophoresis/mass spectrometry of Pro-Q Diamond staining while many more Pro-Q Diamond stained proteins were below mass spectrometry detection. Time dependent 32 P incorporation in intact mitochondria confirmed the extensive matrix protein phosphoryation and revealed the dynamic nature of this process. Classes of proteins detected included all of the mitochondrial respiratory chain complexes, as well as enzymes involved in intermediary metabolism, such as pyruvate dehydrogenase (PDH), citrate synthase and acyl-CoA dehydrogenases. These data demonstrate that the phosphoproteome of the mitochondria matrix is extensive and dynamic. Ca 2+ has previously been shown to activate various dehydrogenases, promote reactive oxygen species (ROS) generation, and initiate apoptosis via cytochrome c release. To evaluate the Ca 2+ signaling network, the effects of a Ca 2+ challenge sufficient to release cytochrome c were evaluated on the mitochondrial phosphoproteome. Novel Ca 2+ -induced dephosphorylation was observed in manganese superoxide dismutase (MnSOD) as well as the previously characterized PDH. A Ca 2+ dose dependent dephosphorylation of MnSOD was associated with a ∼2-fold maximum increase in activity; neither the dephosphorylation nor activity changes were induced by ROS production in the absence of Ca 2+ . These data demonstrate the use of a phosphoproteome screen in determining mitochondrial signaling pathways and reveal new pathways for Ca 2+ modification of mitochondrial function at the level of MnSOD.Mitochondria are thought to be the result of an early interaction of two lines of cellular life, the bacterium and eukaryotic cell (1;2). At this point in time, mitochondria play a critical role in energy metabolism, apoptosis and cell signaling pathways in the cell. However, the acute and chronic regulatory mechanisms of this organelle remain poorly defined. One approach to assessing the function and regulation of the mitochondrion is an evaluation of the mitochondrial Address correspondence to: Robert S. Balaban, Laboratory of Cardiac Energetics, National Heart Lung and Blood Institute, National Institutes of Health, 10 Center Drive Room B1D416, Bethesda, MD 20892-1061. Tel. 301 496-3658; Fax. 301 402-2389; E-mail: rsb@nih.gov. proteome. Estimates predict up to 3000 proteins (3;4) in mitochondria, however, recent largescale screening studies by Taylor (5) and Mootha (6) identified only about 600 distinct mitochondrial proteins. Many have used proteomic approaches to evaluate differential protein expr...

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Section: Resultscontrasting

confidence: 76%

Mitochondrial Matrix Phosphoproteome: Effect of Extra Mitochondrial Calcium

et al. 2006

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“…Whereas there is evidence that a number of TCA cycle enzymes, including m-acon, are phosphoproteins, there are few examples where the consequences of phosphorylation are clearly understood (29). A well defined example is the case of E. coli isocitrate dehydrogenase (30), where phosphorylation at the active site residue S113 inactivates the enzyme when acetate is the sole carbon source and isocitrate flux through the TCA cycle is decreased to provide for increased flux through the glyoxylate cycle.…”

Section: Discussionmentioning

confidence: 99%

Selective inhibition of the citrate-to-isocitrate reaction of cytosolic aconitase by phosphomimetic mutation of serine-711

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Deck

Clarke

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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Iron-regulatory protein 1 (IRP1) is a dual-function protein with mutually exclusive roles as a posttranscriptional regulator of animal-cell iron metabolism or as the cytosolic isoform of the ironsulfur enzyme aconitase (c-acon). Much effort has focused on the role of IRP1 in posttranscriptional gene regulation and in factors that influence its interconversion with c-acon, but little is known about the metabolic function and regulation of c-acon. The role of PKC-dependent phosphorylation of S711 on IRP1͞c-acon function was examined. Phosphorylation state-specific antibodies revealed that S711 is phosphorylated by PKC in vitro and in human embryonic kidney cells treated with a PKC activator. In aco1 yeast, the phosphomimetic mutants S711D and S711E exhibited severely impaired aconitase function, whereas S711A and S711T were unaffected relative to the WT protein. Aconitase activity in yeast extracts displayed a similar pattern when assayed for capacity to convert citrate to isocitrate: WT, S711A, and S711T were active, but S711D and S711E activity was undetectable. In contrast, when measured by the conversion of isocitrate to cis-aconitate, S711D and S711E displayed substantial activity, indicating that phosphorylation impairs the citrate but not isocitrate mode of aconitase function. This possibility was confirmed in vivo by demonstrating that S711D and S711E specifically antagonized the requirement for isocitrate in two metabolic scenarios. Iron-responsive element RNA-binding affinity was unaffected by S711 mutations. Our results show that S711 is a target of phosphorylation capable of conferring distinct effects on c-acon function potentially dictating changes in cytosolic citrate͞isocitrate metabolism. I t is critical that organisms balance the metabolic requirement for iron with its potential toxicity. Vertebrates possess an elegant system to sense cellular iron status, regulate the uptake and metabolic fate of iron, and thereby maintain iron homeostasis. Iron-regulatory protein 1 (IRP1) and IRP2 are sensory components of this critical regulatory network that promote increased iron uptake when cells are iron depleted and storage of iron when cells are iron overloaded (1, 2). IRPs control mRNA fate by binding to iron-responsive elements (IRE) in the untranslated regions of mRNA encoding proteins that control iron homeostasis. The RNA-binding activity of IRP can be regulated by multiple factors. Iron promotes the loss of IRP1 RNA-binding activity through insertion of a [4Fe-4S] cluster into the protein, converting it to the cytosolic isoform of the tricarboxylic acid (TCA) cycle enzyme aconitase (c-acon). Solvent accessibility of the Fe-S cluster in aconitases allows for perturbants to promote loss of the cluster from c-acon, converting it to IRP1 (1, 2). Consequently, reactive species such as NO and H 2 O 2 are able to promote removal of the Fe-S cluster, favoring generation of IRP1 from c-acon, although H 2 O 2 may do so through direct and indirect mechanisms (reviewed in refs. 1 and 2).Whereas much is known a...

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“…There also is extensive discussion of reactive oxygen species as signal molecules from mitochondria to other parts of the cell (Dutilleul et al, 2003) but little information on the pathways involved. Likewise, the presence of phosphorylated proteins in plant mitochondria has been documented (Bykova et al, 2003a(Bykova et al, , 2003b, but again, there is little information on the kinase/phosphatases involved or any associated signaling components that may regulate signaling through phosphorylation/dephosphorylation. Despite the importance of calcium in regulating mammalian mitochondrial function (Smaili et al, 2000), there is no direct evidence for such a role in plants.…”

Section: Cellular Signaling Componentsmentioning

confidence: 99%

Experimental Analysis of the Arabidopsis Mitochondrial Proteome Highlights Signaling and Regulatory Components, Provides Assessment of Targeting Prediction Programs, and Indicates Plant-Specific Mitochondrial Proteins [W]

et al. 2004

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A novel insight into Arabidopsis mitochondrial function was revealed from a large experimental proteome derived by liquid chromatography-tandem mass spectrometry. Within the experimental set of 416 identified proteins, a significant number of low-abundance proteins involved in DNA synthesis, transcriptional regulation, protein complex assembly, and cellular signaling were discovered. Nearly 20% of the experimentally identified proteins are of unknown function, suggesting a wealth of undiscovered mitochondrial functions in plants. Only approximately half of the experimental set is predicted to be mitochondrial by targeting prediction programs, allowing an assessment of the benefits and limitations of these programs in determining plant mitochondrial proteomes. Maps of putative orthology networks between yeast, human, and Arabidopsis mitochondrial proteomes and the Rickettsia prowazekii proteome provide detailed insights into the divergence of the plant mitochondrial proteome from those of other eukaryotes. These show a clear set of putative cross-species orthologs in the core metabolic functions of mitochondria, whereas considerable diversity exists in many signaling and regulatory functions.

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Identification of 14 new phosphoproteins involved in important plant mitochondrial processes

Cited by 119 publications

References 30 publications

Mitochondrial Matrix Phosphoproteome: Effect of Extra Mitochondrial Calcium

Mitochondrial Matrix Phosphoproteome: Effect of Extra Mitochondrial Calcium

Selective inhibition of the citrate-to-isocitrate reaction of cytosolic aconitase by phosphomimetic mutation of serine-711

Experimental Analysis of the Arabidopsis Mitochondrial Proteome Highlights Signaling and Regulatory Components, Provides Assessment of Targeting Prediction Programs, and Indicates Plant-Specific Mitochondrial Proteins [W]

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