Expression and assembly of Arabidopsis thaliana pyruvate dehydrogenase in insect cell cytoplasm

Szurmak, Blanka; Strokovskaya, L. I.; Mooney, Brian; Randall, Douglas D.; Miernyk, Ján A.

doi:10.1016/s1046-5928(02)00712-x

Cited by 8 publications

(5 citation statements)

References 26 publications

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“…2B). Additional immunoblotting of both the purified mitochondria and peroxisome fractions with antibodies to mitochondrial pyruvate dehydrogenase complex protein E1a (Szurmak et al, 2003) confirmed that the peroxisomal fraction was completely free of mitochondria. Given that AtMIA40 proteins reside in the IMS (Hentchel and Escalante-Semerena, 2015), we further fractionated isolated mitochondria into IMS and outer mitochondrial membrane (OMM) and looked for AtSLP2 localization (Fig.…”

Section: Atslp2 and Atmia40 Colocalize To Mitochondriamentioning

confidence: 84%

“…S3 and S4. Mitochondria and peroxisomes were probed using 1:100 antimitochondrial pyruvate dehydrogenase complex E1a (mitochondrial matrix [Szurmak et al, 2003]) and 1:200 anticatalase (peroxisome) IgG (Chuong et al, 2005). Mitochondrial subcompartments were blotted using 1:5000 anti-H protein of Gly decarboxylase complex (anti-GDC-H; mitochondria matrix marker; Agrisera/AS05 074), 1:5000 antivoltage-dependent anion-selective channel protein 1 (anti-VDAC1; OMM marker; Agrisera) and 2 mg/mL anticytochrome c (anticyt c [Sweetlove et al, 2001]) IgG.…”

Section: Western Blottingmentioning

confidence: 99%

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Activation of Mitochondrial Protein Phosphatase SLP2 by MIA40 Regulates Seed Germination

et al. 2016

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Reversible protein phosphorylation catalyzed by protein kinases and phosphatases represents the most prolific and wellcharacterized posttranslational modification known. Here, we demonstrate that Arabidopsis (Arabidopsis thaliana) Shewanella-like protein phosphatase 2 (AtSLP2) is a bona fide Ser/Thr protein phosphatase that is targeted to the mitochondrial intermembrane space (IMS) where it interacts with the mitochondrial oxidoreductase import and assembly protein 40 (AtMIA40), forming a protein complex. Interaction with AtMIA40 is necessary for the phosphatase activity of AtSLP2 and is dependent on the formation of disulfide bridges on AtSLP2. Furthermore, by utilizing atslp2 null mutant, AtSLP2 complemented and AtSLP2 overexpressing plants, we identify a function for the AtSLP2-AtMIA40 complex in negatively regulating gibberellic acid-related processes during seed germination. Results presented here characterize a mitochondrial IMS-localized protein phosphatase identified in photosynthetic eukaryotes as well as a protein phosphatase target of the highly conserved eukaryotic MIA40 IMS oxidoreductase.

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Section: Atslp2 and Atmia40 Colocalize To Mitochondriamentioning

confidence: 84%

Section: Western Blottingmentioning

confidence: 99%

Activation of Mitochondrial Protein Phosphatase SLP2 by MIA40 Regulates Seed Germination

et al. 2016

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“…Shaded ovals highlight the plant proteins predicted to be mitochondrial (left) or plastidic (right). E1a with At5g50850 E1b) has been heterologously expressed, purified, and tested for activity in vitro (Szurmak et al, 2003), a variety of E1 a2b2 heterotetramers may exist in vivo, and these different enzymes may display different catalytic or regulatory properties.…”

Section: Discussionmentioning

confidence: 99%

IAR4, a Gene Required for Auxin Conjugate Sensitivity in Arabidopsis, Encodes a Pyruvate Dehydrogenase E1α Homolog

2004

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The formation and hydrolysis of indole-3-acetic acid (IAA) conjugates represent a potentially important means for plants to regulate IAA levels and thereby auxin responses. The identification and characterization of mutants defective in these processes is advancing the understanding of auxin regulation and response. Here we report the isolation and characterization of the Arabidopsis iar4 mutant, which has reduced sensitivity to several IAA-amino acid conjugates. iar4 is less sensitive to a synthetic auxin and low concentrations of an ethylene precursor but responds to free IAA and other hormones tested similarly to wild type. The gene defective in iar4 encodes a homolog of the E1a-subunit of mitochondrial pyruvate dehydrogenase, which converts pyruvate to acetyl-coenzyme A. We did not detect glycolysis or Krebs-cycle-related defects in the iar4 mutant, and a T-DNA insertion in the IAR4 coding sequence conferred similar phenotypes as the originally identified missense allele. In contrast, we found that disruption of the previously described mitochondrial pyruvate dehydrogenase E1a-subunit does not alter IAA-Ala responsiveness or confer any obvious phenotypes. It is possible that IAR4 acts in the conversion of indole-3-pyruvate to indole-3-acetyl-coenzyme A, which is a potential precursor of IAA and IAA conjugates.Auxins affect virtually every aspect of plant development, including phototropism, gravitropism, cell expansion, apical dominance, root growth, fruit development, vascular development, and senescence (Davies, 1995). By understanding how plants regulate levels of free indole-3-acetic acid (IAA), the active form of the most abundant naturally occurring auxin, we can gain insight into how plants develop and respond to environmental stimuli.The IAA in Arabidopsis is found in three basic forms. The free acid is the active form of IAA but constitutes only a small fraction of the total IAA in Arabidopsis. The majority of IAA is found conjugated to peptides or amino acids via amide linkages or to sugars via ester linkages Ljung et al., 2002). This high proportion of conjugates suggests that auxin homeostasis may be regulated through formation and hydrolysis of conjugates in addition to regulation through de novo synthesis, transport, degradation, and interconversion between IAA and indole-3-butyric acid Ljung et al., 2002). IAA conjugates may function as storage, inactivation, or transport forms of IAA (Hangarter et al., 1980;Nowacki and Bandurski, 1980;Slovin, 1997) and some conjugates may have roles independent of hydrolysis (Hangarter et al., 1980;Magnus et al., 1992a).The amide conjugates IAA-Asp, IAA-Glu, IAA-Ala, and IAA-Leu have been identified in Arabidopsis seedlings (Tam et al., 2000;Kowalczyk and Sandberg, 2001), and an IAA-peptide is abundant in Arabidopsis seeds (Walz et al., 2002). Certain endogenous IAA conjugates can elicit auxin responses in bioassays (Hangarter et al., 1980;Hangarter and Good, 1981;Bialek et al., 1983;Magnus et al., 1992b;Davies et al., 1999;LeClere et al., 2002), and conjugat...

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“…When individually expressed at high levels neither subunit was soluble, suggesting that correct folding and assembly of large amounts of the heteromeric enzyme require the simultaneous presence of both subunits. Complexity in the folding and assembly pathway was not unexpected based upon previous reports [23, 24, 37, 38] and the reported participation of the GroE chaperonins [39–41]. …”

Section: Discussionmentioning

confidence: 59%

Asp295 Stabilizes the Active-Site Loop Structure of Pyruvate Dehydrogenase, Facilitating Phosphorylation of Ser292 by Pyruvate Dehydrogenase-Kinase

et al. 2011

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We have developed an in vitro system for detailed analysis of reversible phosphorylation of the plant mitochondrial pyruvate dehydrogenase complex, comprising recombinant Arabidopsis thalianaα2β2-heterotetrameric pyruvate dehydrogenase (E1) plus A. thaliana E1-kinase (AtPDK). Upon addition of MgATP, Ser292, which is located within the active-site loop structure of E1α, is phosphorylated. In addition to Ser292, Asp295 and Gly297 are highly conserved in the E1α active-site loop sequences. Mutation of Asp295 to Ala, Asn, or Leu greatly reduced phosphorylation of Ser292, while mutation of Gly297 had relatively little effect. Quantitative two-hybrid analysis was used to show that mutation of Asp295 did not substantially affect binding of AtPDK to E1α. When using pyruvate as a variable substrate, the Asp295 mutant proteins had modest changes in kcat, Km, and kcat/Km values. Therefore, we propose that Asp295 plays an important role in stabilizing the active-site loop structure, facilitating transfer of the γ-phosphate from ATP to the Ser residue at regulatory site one of E1α.

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Expression and assembly of Arabidopsis thaliana pyruvate dehydrogenase in insect cell cytoplasm

Cited by 8 publications

References 26 publications

Activation of Mitochondrial Protein Phosphatase SLP2 by MIA40 Regulates Seed Germination

Activation of Mitochondrial Protein Phosphatase SLP2 by MIA40 Regulates Seed Germination

IAR4, a Gene Required for Auxin Conjugate Sensitivity in Arabidopsis, Encodes a Pyruvate Dehydrogenase E1α Homolog

Asp295 Stabilizes the Active-Site Loop Structure of Pyruvate Dehydrogenase, Facilitating Phosphorylation of Ser292 by Pyruvate Dehydrogenase-Kinase

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