Molecular identification and functional characterization of a novel glutamate transporter in yeast and plant mitochondria

Porcelli, Vito; Vozza, Angelo; Calcagnile, Valeria Mariajolanda; Gorgoglione, Ruggiero; Arrigoni, Roberto; Fontanesi, Flavia; Marobbio, C; Castegna, Alessandra; Palmieri, Ferdinando; Palmieri, Luigi

doi:10.1016/j.bbabio.2018.08.001

Cited by 38 publications

(44 citation statements)

References 68 publications

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“…5E). Such a decrease was unlikely to be contributed by a reduction in peroxisomal citrate synthase because labelled acetyl-CoA cannot be directly exported from mitochondria (53), and the existence of a carnitine/acylcarnitine carrier in plant mitochondria is questionable since its closest homolog BOU has recently been reported to transport glutamate (54). On the other hand, the citrate pool remained stable in the dark in dic2-1 (Fig.…”

Section: Resultsmentioning

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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Section: Resultsmentioning

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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“…Recently, analyses of mitochondrial proteome content, complexome composition, post-translational modifications, energy metabolism, OXPHOS complex formation and function, protein translocation, and metabolite shuttles have been conducted to further our understanding of mitochondrial metabolism in Arabidopsis (König et al, 2014; Fromm et al, 2016; De Col et al, 2017; Rao et al, 2017; Senkler et al, 2017; Porcelli et al, 2018; Hu et al, 2019; Kolli et al, 2019; Meyer et al, 2019; Nickel et al, 2019). Many of these analyses required the isolation of intact mitochondria.…”

Section: Discussionmentioning

confidence: 99%

“…The BOU protein was recently assigned the function of a glutamate transporter (Porcelli et al, 2018). 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).…”

Section: Discussionmentioning

confidence: 99%

“…These data indicate that BOU performs other functions, in addition to glutamate transport in vivo . We exclude its function as a mitochondrial glutamate/glutamine shuttle, as BOU shows no glutamine uptake activity (Porcelli et al, 2018). However, it is possible that BOU is involved in folate metabolism, as folate biosynthesis occurs only in mitochondria (Hanson and Gregory, 2011).…”

Section: Discussionmentioning

confidence: 99%

“…Because BOU is co-expressed with genes encoding components of the GDC complex, and the bou-2 mutant shows a similar metabolic phenotype as the shm1 mutant, it was hypothesized that BOU transports a metabolite necessary for the proper functioning of GDC (Voll et al, 2006; Eisenhut et al, 2013). Recently, it was demonstrated that heterologously expressed BOU functions as a glutamate transporter (Porcelli et al, 2018). Glutamate is neither a substrate nor a product of the reaction catalyzed by GDC.…”

Section: Introductionmentioning

confidence: 99%

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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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Mitochondrial carriers of Ustilago maydis and Aspergillus terreus involved in itaconate production: same physiological role but different biochemical features

et al. 2019

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Itaconic acid (IA) is a naturally occurring dicarboxylic acid with applications in the manufacture of polymers. IA can be produced by fermentation using the fungi Aspergillus terreus or Ustilago maydis as biocatalysts. Indirect evidence has suggested that the mitochondrial carriers U. maydis Um_Mtt1 and A. terreus At_MttA export mitochondrially synthesized cis‐aconitate to the cytosol for IA synthesis using malate as a countersubstrate. Here, by assaying the transport features of recombinant Um_Mtt1 and At_MttA in reconstituted liposomes, we find that both proteins efficiently transport cis‐aconitate, but malate is well transported only by Um_Mtt1 and 2‐oxoglutarate only by At_MttA. Bioinformatic analysis shows that Um_Mtt1 and At_MttA form a distinctive mitochondrial carrier subfamily. Our data show that although fulfilling the same physiological task, Um_Mtt1 and At_MttA have different biochemical features.

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Molecular identification and functional characterization of a novel glutamate transporter in yeast and plant mitochondria

Cited by 38 publications

References 68 publications

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

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

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

Mitochondrial carriers of Ustilago maydis and Aspergillus terreus involved in itaconate production: same physiological role but different biochemical features

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