Uncoupling proteins 1 and 2 (UCP1 and UCP2) from Arabidopsis thaliana are mitochondrial transporters of aspartate, glutamate, and dicarboxylates

Monné, Magnus; Daddabbo, Lucia; Gagneul, David; Obata, Toshihiro; Hielscher, Björn; Palmieri, Luigi; Miniero, Daniela Valeria; Fernie, Alisdair R.; Weber, Andreas P.M.; Palmieri, Ferdinando

doi:10.1074/jbc.ra117.000771

Cited by 82 publications

(106 citation statements)

References 75 publications

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“…Portulaca oleracea has been classified as a NAD-ME-type species (Lara et al, 2004;Voznesenskaya et al, 2010), implying that most CO 2 incorporated as OAA in this species is converted to aspartate (ASP) in MCs via aspartate aminotransferase (AspAT) activity. To sustain these reactions, ASP and malate movements across mitochondria are facilitated by Uncoupling protein 2 (UCP2), Dicarboxylate transport 1 (DIT1) and Dicarboxylate carrier (DIC) proteins, respectively (Vozza et al, 2014;Monn e et al, 2018;Palmieri et al, 2008). To sustain these reactions, ASP and malate movements across mitochondria are facilitated by Uncoupling protein 2 (UCP2), Dicarboxylate transport 1 (DIT1) and Dicarboxylate carrier (DIC) proteins, respectively (Vozza et al, 2014;Monn e et al, 2018;Palmieri et al, 2008).…”

Section: Using Transcriptomic Data To Identify Ccm-related Genesmentioning

confidence: 99%

“…The ASP formed diffuses to BSCs via plasmodesmata and enters the mitochondria, where AspAT reverts ASP to OAA, and NAD-malate dehydrogenase (MDH) converts OAA to malate, which is decarboxylated by NAD-ME (Kanai & Edwards, 1999). To sustain these reactions, ASP and malate movements across mitochondria are facilitated by Uncoupling protein 2 (UCP2), Dicarboxylate transport 1 (DIT1) and Dicarboxylate carrier (DIC) proteins, respectively (Vozza et al, 2014;Monn e et al, 2018;Palmieri et al, 2008). Transcripts encoding enzymes and transporters characteristic of NAD-MEtype photosynthesis (ASPAT-1E1, NADMDH-2E, NADMDH-3C1a, NADME-1E, NADME-2E.1, UCP-2 and DIC-1.2) were abundant in well-watered leaves and exhibited C 4 -like diel regulation, with peak levels phased to 08:00 h, 2 h into the 12 h light period (Figs 2-5).…”

Section: Using Transcriptomic Data To Identify Ccm-related Genesmentioning

confidence: 99%

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C₄ and crassulacean acid metabolism within a single leaf: deciphering key components behind a rare photosynthetic adaptation

Ferrari¹,

Bittencourt²,

Rodrigues³

et al. 2019

New Phytologist

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Although biochemically related, C 4 and crassulacean acid metabolism (CAM) systems are expected to be incompatible. However, Portulaca species, including P. oleracea, operate C 4 and CAM within a single leaf, and the mechanisms behind this unique photosynthetic arrangement remain largely unknown.Here, we employed RNA-seq to identify candidate genes involved exclusively or shared by C 4 or CAM, and provided an in-depth characterization of their transcript abundance patterns during the drought-induced photosynthetic transitions in P. oleracea. Data revealed fewer candidate CAM-specific genes than those recruited to function in C 4 . The putative CAMspecific genes were predominantly involved in night-time primary carboxylation reactions and malate movement across the tonoplast. Analysis of gene transcript-abundance regulation and photosynthetic physiology indicated that C 4 and CAM coexist within a single P. oleracea leaf under mild drought conditions. Developmental and environmental cues were shown to regulate CAM expression in stems, whereas the shift from C 4 to C 4 -CAM hybrid photosynthesis in leaves was strictly under environmental control. Moreover, efficient starch turnover was identified as part of the metabolic adjustments required for CAM operation in both organs.These findings provide insights into C 4 /CAM connectivity and compatibility, contributing to a deeper understanding of alternative ways to engineer CAM into C 4 crop species.

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Section: Using Transcriptomic Data To Identify Ccm-related Genesmentioning

confidence: 99%

Section: Using Transcriptomic Data To Identify Ccm-related Genesmentioning

confidence: 99%

C₄ and crassulacean acid metabolism within a single leaf: deciphering key components behind a rare photosynthetic adaptation

Ferrari¹,

Bittencourt²,

Rodrigues³

et al. 2019

New Phytologist

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“…Glutamate is indirectly linked to photorespiration, as it is needed for the polyglutamylation of THF, which increases its stability and promotes the activity of THF-dependent enzymes (Hanson and Gregory, 2011). However, in addition to BOU, mitochondria-localized uncoupling proteins 1 and 2 show glutamate uptake activity in Arabidopsis (Monné et al, 2018). This raises the question why knockout of the BOU gene leads to a photorespiratory phenotype in young tissues, if BOU is not the only glutamate transporter of mitochondria.…”

Section: Discussionmentioning

confidence: 99%

“…Moreover, THF-dependent enzymes generally prefer polyglutamylated folates over monoglutamylated folates as a substrate (Suh et al, 2001). However, because (1) BOU is not the only glutamate transporter in mitochondria, and (2) glutamylation of folates is not restricted to mitochondria, the exact physiological function of BOU remains unclear (Hanson and Gregory, 2011; Monné et al, 2018). Notably, a glutamate/glutamine shuttle across the mitochondrial membrane was previously suggested to support the reclamation of ammonia released during photorespiration (Linka and Weber, 2005).…”

Section: Introductionmentioning

confidence: 99%

Rapid single-step affinity purification of HA-tagged mitochondria from Arabidopsis thaliana

Kuhnert

Stefanski

Overbeck

et al. 2019

Preprint

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Photosynthesis in plant cells would not be possible without the supportive role of mitochondria. However, isolation of mitochondria from plant cells, for physiological and biochemical analyses, is a lengthy and tedious process. Established isolation protocols require multiple centrifugation steps and substantial amounts of starting material. To overcome these limitations, we tagged mitochondria in Arabidopsis thaliana with a triple haemagglutinin-tag for rapid purification via a single affinity purification step. This protocol yields a substantial quantity of highly pure mitochondria from 1 g of Arabidopsis seedlings. The purified mitochondria were suitable for enzyme activity analyses and yielded sufficient amounts of proteins for deep proteomic profiling. We applied this method for the proteomic analysis of the Arabidopsis bou-2 mutant deficient in the mitochondrial glutamate transporter À bout de souffle (BOU) and identified 27 differentially expressed mitochondrial proteins compared with transgenic Col-0 controls. Our work also sets the stage for the development of advanced mitochondria isolation protocols for distinct cell types.One-sentence summaryAffinity-tagging of mitochondria in plant cells with a triple hemagglutinin-tag enables single-step affinity purification of mitochondria in less than 20 min.

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“…By identifying homologues of yeast and mammalian carriers in Arabidopsis thaliana , several possible candidates that may contribute to mitochondrial malate transport in plants were identified (24–26). In contrast to the historical, well-established models that mitochondrial metabolite transporters are discrete and highly specific (27–29), these carriers appeared to lack substrate specificities under in vitro conditions – for example, dicarboxylate carrier (DIC) isoforms in proteoliposomes can rapidly exchange sulfate with phosphate, malate, OAA and succinate, with an apparent low exchange activity in the presence of citrate, 2-oxoglutarate and fumarate (24).…”

Section: Introductionmentioning

confidence: 99%

The Arabidopsis mitochondrial dicarboxylate carrier 2 maintains leaf metabolic homeostasis by uniting malate import and citrate export

Lee

Elsässer

Fuchs

et al. 2020

Preprint

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Author Contributions: CPL performed most of the experiments in this manuscript and undertook data 22 analysis. ME and PF conducted experiments with Peredox-mCherry lines and carried out data analysis. 23 RF undertook MRM development and analysis. CPL, MS and AHM developed the experimental plan. CPL 24 and AHM wrote the manuscript and all authors revised the manuscript. 25 26 2 ABSTRACT 27Malate is the major substrate for respiratory oxidative phosphorylation in illuminated leaves. In the 28 mitochondria malate is converted to citrate either for replenishing tricarboxylic acid (TCA) cycle with 29 carbon, or to be exported as substrate for cytosolic biosynthetic pathways or for storage in the 30 vacuole. In this study, we show that DIC2 functions as a mitochondrial malate/citrate carrier in vivo in 31Arabidopsis. DIC2 knockout (dic2-1) results in growth retardation that can only be restored by 32 expressing DIC2 but not its closest homologs DIC1 or DIC3, indicating that their substrate preferences 33 are not identical. Malate uptake by non-energised dic2-1 mitochondria is reduced but can be restored 34 in fully energised mitochondria by altering fumarate and pyruvate/oxaloacetate transport. A reduced 35 citrate export but an increased citrate accumulation in substrate-fed, energised dic2-1 mitochondria 36 suggest that DIC2 facilitates the export of citrate from the matrix. Consistent with this, metabolic 37 defects in response to a sudden dark shift or prolonged darkness could be observed in dic2-1 leaves, 38including altered malate, citrate and 2-oxoglutarate utilisation. There was no alteration in TCA cycle 39 metabolite pools and NAD redox state at night; however, isotopic glucose tracing reveals a reduction 40 in citrate labelling in dic2-1 which resulted in a diversion of flux towards glutamine, as well as the 41 removal of excess malate via asparagine and threonine synthesis. Overall, these observations 42 indicate that DIC2 is responsible in vivo for mitochondrial malate import and citrate export which 43 coordinate carbon metabolism between the mitochondrial matrix and the other cell compartments. 44 45 SIGNIFICANCE STATEMENT 46Mitochondria are pivotal for plant metabolism. One of their central functions is to provide carbon 47 57Malate is a prominent metabolite that occupies a pivotal node in the regulation of plant carbon 58 metabolism. It is the mainstay of leaf respiration and metabolic redox shuttling between organelles 59(1). Early studies with isolated plant mitochondria demonstrated that exogenous malate can be 60 translocated by an unknown mechanism into the mitochondrial matrix where it is then rapidly oxidized 61 by both mitochondrial malate dehydrogenase (mMDH) and malic enzyme (NAD-ME), generating 62 oxaloacetate (OAA) and pyruvate as products (2, 3). OAA inhibits mitochondrial respiration by direct 63 inhibition of succinate dehydrogenase and due to the fact that the chemical equilibrium of the mMDH 64 reaction highly favours OAA conversion to malate, thereby diverting NADH away from the elect...

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Uncoupling proteins 1 and 2 (UCP1 and UCP2) from Arabidopsis thaliana are mitochondrial transporters of aspartate, glutamate, and dicarboxylates

Cited by 82 publications

References 75 publications

C₄ and crassulacean acid metabolism within a single leaf: deciphering key components behind a rare photosynthetic adaptation

C₄ and crassulacean acid metabolism within a single leaf: deciphering key components behind a rare photosynthetic adaptation

Rapid single-step affinity purification of HA-tagged mitochondria from Arabidopsis thaliana

The Arabidopsis mitochondrial dicarboxylate carrier 2 maintains leaf metabolic homeostasis by uniting malate import and citrate export

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Uncoupling proteins 1 and 2 (UCP1 and UCP2) from Arabidopsis thaliana are mitochondrial transporters of aspartate, glutamate, and dicarboxylates

Cited by 82 publications

References 75 publications

C4 and crassulacean acid metabolism within a single leaf: deciphering key components behind a rare photosynthetic adaptation

C4 and crassulacean acid metabolism within a single leaf: deciphering key components behind a rare photosynthetic adaptation

Rapid single-step affinity purification of HA-tagged mitochondria from Arabidopsis thaliana

The Arabidopsis mitochondrial dicarboxylate carrier 2 maintains leaf metabolic homeostasis by uniting malate import and citrate export

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C₄ and crassulacean acid metabolism within a single leaf: deciphering key components behind a rare photosynthetic adaptation

C₄ and crassulacean acid metabolism within a single leaf: deciphering key components behind a rare photosynthetic adaptation