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A plant-like mitochondrial carrier family protein facilitates mitochondrial transport of di- and tricarboxylates in Trypanosoma brucei
Accepted Manuscript Title: A plant-like mitochondrial carrier family protein facilitates mitochondrial transport of di- and tricarboxylates in Trypanosoma brucei Authors: Claudia Colasante, Fuli Zheng, Cordula Kemp, Frank Voncken PII: DOI: Reference:
S0166-6851(17)30163-9 https://doi.org/10.1016/j.molbiopara.2018.03.003 MOLBIO 11117
To appear in:
Molecular & Biochemical Parasitology
Received date: Revised date: Accepted date:
1-1-2018 22-2-2018 21-3-2018
Please cite this article as: Colasante Claudia, Zheng Fuli, Kemp Cordula, Voncken Frank.A plant-like mitochondrial carrier family protein facilitates mitochondrial transport of di- and tricarboxylates in Trypanosoma brucei.Molecular and Biochemical Parasitology https://doi.org/10.1016/j.molbiopara.2018.03.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
The T. brucei di- and tricarboxylate transporter TbMCP12
A plant-like mitochondrial carrier family protein facilitates mitochondrial transport of di- and tricarboxylates in Trypanosoma brucei
Running Title: The T. brucei di- and tricarboxylate transporter TbMCP12
Claudia Colasante1#, Fuli Zheng3#, Cordula Kemp2 and Frank Voncken2
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Institute for Anatomy and Cell Biology, Division of Medical Cell Biology, Aulweg 123, University of
Giessen, 35392 Giessen, Germany 3
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Department of Preventive Medicine, School of Public Health, Fujian Medical University, 1 Xue Yuan
Road, Fu Zhou, Fujian, P.R. China 2
Department of Biomedical Sciences, School of Life Sciences, University of Hull, Cottingham Road,
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Hull, HU6 7RX, United Kingdom
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# These authors contributed equally to the paper
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To whom correspondence should be addressed: Claudia Colasante, Institute for Anatomy and Cell Biology, Division of Medical Cell Biology, Aulweg 123, University of Giessen, 35392 Giessen,
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Germany; Tel.: 0049 0641 9947116; Fax: 0049 0641 9947109;
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MCF proteins (MCPs) exchange solutes across the inner mitochondrial membrane The human parasite T. brucei possesses 24 genes encoding MCPs TbMCP12 transports carboxylates across the T. brucei inner mitochondrial membrane Overexpression of TbMCP12 is lethal in procyclic T. brucei The abundance of TbMCP12 regulates NADPH balance and mitochondrial ATP-production
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Highlights
ABSTRACT
The procyclic form of the human parasite Trypanosoma brucei harbors one single, large mitochondrion containing all tricarboxylic acid (TCA) cycle enzymes and respiratory chain complexes
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present also in higher eukaryotes. Metabolite exchange among subcellular compartments such as the cytoplasm, the mitochondrion, and the peroxisomes is crucial for redox homeostasis and for metabolic pathways whose enzymes are dispersed among different organelles. In higher eukaryotes, mitochondrial carrier family (MCF) proteins transport TCA-cycle intermediates across the inner mitochondrial membrane. Previously, we identified several MCF members that are essential for T. brucei survival. Among these, only one MCF protein, TbMCP12, potentially could transport dicarboxylates and tricarboxylates. Here, we conducted phylogenetic and sequence analyses and
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The T. brucei di- and tricarboxylate transporter TbMCP12
functionally characterized TbMCP12 in vivo. Our results suggested that similarly to its homologues in plants, TbMCP12 transports both dicarboxylates and tricarboxylates across the mitochondrial inner membrane. Deleting this carrier in T. brucei was not lethal, while its overexpression was deleterious. Our results suggest that the intracellular abundance of TbMCP12 is an important regulatory element for the NADPH balance and mitochondrial ATP-production. Keywords: mitochondrial transport, Trypanosoma brucei, energy metabolism, MCF-proteins,
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dicarboxylate, tricarboxylate
INTRODUCTION
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The order Kinetoplastida is a member of the Excavata supergroup, which branched from the other supergroups, including Archaeplastida and Opisthokonta, at the beginning of eukaryotic
evolution [1]. Kinetoplastida includes a variety of different flagellated protozoa that can be found in disparate environmental niches: from free-living cells to parasitic forms. Of particular medical,
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veterinary and socio-economic interest is the group of obligate parasites belonging to the
Trypanosomatidae family, especially Leishmania and Trypanosoma spp.. According to current
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estimates infections caused by these parasites affect several millions of people each year
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(http://www.who.int). Trypanosoma brucei subspecies cause African trypanosomiasis, a disease that
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affects both humans and cattle and is transmitted through the bite of the Tsetse fly. T. brucei raises high scientific interest also due to some of its biological peculiarities such as the presence of one single large mitochondrion, the compartmentalisation of glycolysis within specialized
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peroxisomes named glycosomes, and the complex mitochondrial DNA network and mRNA editing [2, 3]. During its parasitic life cycle T. brucei undergoes substantial changes in gene expression, which
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allow its survival within the digestive system of the insect vector and the mammalian bloodstream [47]. The various life cycle forms differ in their cellular morphology, surface protein expression and energy metabolism [8-10]. The energy metabolism of T. brucei involves three subcellular
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compartments: the cytoplasm, the glycosomes and the mitochondrion. In bloodstream trypanosomes, net ATP production exclusively occurs in the cytoplasm by substrate level phosphorylation during glycolysis. This generates incompletely oxidized pyruvate, which the parasites excrete without further metabolisation. Accordingly, bloodstream form mitochondria do not contain all canonical enzymes of
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the tricarboxylic acid cycle or electron transport chain [4, 11]. In contrast, procyclic form T. brucei, which multiply in the Tsetse fly midgut, produce ATP in
the cytoplasm by substrate level phosphorylation as well as inside the elaborate mitochondrion by substrate level and oxidative phosphorylation [4, 8, 12, 13]. The substrates for energy production in this life cycle stage are either pyruvate generated during glycolysis or amino acids like proline and threonine [4, 8, 13-15]. In procyclic mitochondria, all TCA-cycle enzymes are present, but they are not functioning as a complete cycle [16-18].
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The T. brucei di- and tricarboxylate transporter TbMCP12
In addition to energy metabolism, the mitochondria of T. brucei, like those of other eukaryotes, are involved in amino acid catabolism, regulation of reactive oxygen species and the biosynthesis of heme [9]. Substrates for all these pathways need to be imported into the mitochondrion, across its outer and inner membrane [19]. The majority of proteins involved in the translocation of metabolites and other small solutes across the mitochondrial inner membrane belong to the mitochondrial carrier family (MCF). MCF proteins (MCPs) provide a link between cytoplasm and mitochondrion by exchanging metabolic intermediates, maintaining the cellular redox balance, and exerting flux control
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on enzymatic reactions [20-24]. Proteins belonging to the MCF can be found in nearly all eukaryotic cells. Their amino acid
sequence can be highly divergent, but all MCPs retain the same tertiary structure, which consists of six
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transmembrane alpha helices connected by hydrophilic loops, with both C- and N-termini exposed to the intermembrane space [22, 25]. MCP sequences contain three tandemly repeated domains of 100 amino acids harbouring a common signature motif [26]. Using these conserved sequence features we previously identified genes encoding 24 different potential MCPs in the genome of T. brucei
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(TbMCPs) [27]. Amongst them was one with similarity to human dicarboxylate (DIC) and oxoglutarate (OGC) carriers [27]. These carriers catalyse the electron-neutral exchange of
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dicarboxylates (malate, succinate, fumarate, oxoglutarate and oxaloacetate) and inorganic phosphate
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(Pi) across the mitochondrial inner membrane, and are involved in severalexchange mechanisms [28]. The identification and characterisation of kinetoplastid transporters involved in shuttling
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metabolites between the mitochondria, glycosomes and cytosol is necessary to assemble the remarkable puzzle of the compartmentalisation of the energy metabolism in these parasites. Recently
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we provided evidence that TbMCPs play a central role in T. brucei. We previously characterised two TbMCPs and showed that depletion of these proteins was detrimental for the survival of T. brucei and affected cellular and nuclear morphology as well as ATP homeostasis [29, 30]. Here we describe the
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molecular and functional analysis of another mitochondrial carrier of T. brucei, namely TbMCP12. Our results suggest that TbMCP12 displays functional traits of plant DTCs and transports both
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dicarboxylates and tricarboxylates. We also report that though in some eukaryotes there are at least three carriers involved in the transport of carboxylates and tricarboxylates, namely the OGC, DIC and the citrate (CIT) carriers, T. brucei fulfils all these functions with TbMCP12. Further, TbMCP12
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appears to be involved in oxidative stress management. MATERIAL AND METHODS Culture and transfection of Trypanosoma brucei Procyclic-form Trypanosoma brucei strain Lister 427, stably expressing the tetracycline (tet) repressor from the plasmid pHD449, was cultured in standard MEM-PROS medium [31] (supplemented with 10 % (v/v) foetal calf serum (FCS, Sigma-Aldrich). Trypanosomes were
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The T. brucei di- and tricarboxylate transporter TbMCP12
transfected with different plasmids and clonal cell lines were selected using antibiotics as previously described by Biebinger et al. [32]. Next to standard (low-glucose) MEM-PROS medium, also ‘highglucose’ medium (i.e. standard MEM-PROS medium supplemented with 10 mM glucose) was used for the different experiments. Phylogenetic reconstruction and sequence analysis Phylogenetic reconstruction was performed using the “Phylogeny.fr” software available at
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http://www.phylogeny.fr. Multiple sequence alignments were obtained using Psi/TM-Coffee and automatically curated using Gblocks. Maximum likelihood tree was constructed using PhyML and
visualized using TreeDyn. Statistical tests for branch support was assessed by bootstrap re-sampling
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analysis generating 100 reiterated data sets. The resulting bootstrap values, expressed as percentage, were added manually to each node. Only bootstrap values above 50% are shown. The NCBI (http://www.ncbi.nlm.nih.gov) accession numbers for the different protein sequences used for the
phylogenetic analysis are listed in Supplementary Table 1. The multiple sequence alignment shown in
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Figure 2 was performed using ClustalΩ (www.ebi.ac.uk).
RNA isolation, northern blotting and RT-PCR
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Total RNA was isolated from 1 x 107 bloodstream- or procyclic-form T. brucei strain 449 cells
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using TriFast (PeqLab Biotechnology GmbH) according to manufacturer´s protocol. For Northern blotting, total RNA (10 μg) was separated by denaturing (formaldehyde) agarose gel electrophoresis and blotted onto Hybond-N membrane (GE Healthcare). A [32P]-dCTP labelled TbMCP12 ORF (open
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reading frame) DNA probe was generated by PCR using the primer pair 5’GGACGGGTTAACACCATGGCGAAAGAGACAAAGGCGCCCG-3’ (forward) and 5’-
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GCTTGCAGGATCCCTGCTTTGCCTGAAGCTTGGCG-3’ (reverse). Blots were pre-hybridized in
hybridization solution (5x SSC, 5x Denhardt´s reagent and 0.5 % w/v sodium dodecyl sulphate (SDS)) for 1 h at 65 °C after which the [32P]-dCTP labelled DNA probe was added. After over-night
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hybridization at 65 °C the blots were washed at 65 °C in 1x SSC (0.15 M NaCl, 0.015 M sodium citrate) supplemented with 0.1 % w/v SDS, then with 0.1x SSC supplemented with 0.1 % w/v SDS, followed by final exposure to X-ray film (Kodak). RNA from yeast was isolated using the Direct-zol™ RNA MiniPrep kit according to the
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manufacturer´s protocol with some modification. Briefly, yeast cells were treated first with lyticase (10 u/OD) to break down the cell wall and the formed spheroblasts were then lysed using 300 μl TRI reagent. The mixture was transferred into a Zymo-Spin™IIC Column and spun at 13,000 x g for 30 sec followed by a washing step with 400 μl RNA Wash Buffer. After washing, 5 μl DNase I (6 u/μl) in 75 μl DNA Digestion Buffer were added to the column followed by incubation at 25 °C for 15 min. After 2 consecutive washing steps with 400 μl Wash Buffer the column was dried by centrifugation and the RNA eluted with 50 μl of DNase/RNase-free water.
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The T. brucei di- and tricarboxylate transporter TbMCP12
For cDNA synthesis 2 μg yeast total RNA in 10 μl Rnase-free water were mixed with 4 μl 5x M-MuLV reaction buffer, 1 μl RiboLock RNase inhibitor, 2 μl 10 mM dNTP, and 2 μl M-MuLV Reverse transcriptase (cDNA synthesis kit, Thermo). The reaction was incubated at 25 °C for 5 min, followed by 60 min at 37 °C. Then the reaction was terminated by incubation at 70 °C for 5 min. RTPCR for the detection of the TbMPC12 and dicarboxylate carrier (DIC) expression in yeast was performed using 1 μl of synthesised cDNA to set up a 25 μl PCR reaction. For the analysis the primers 3’-GGGGATCCATGGCGAAAGAGACAAAGGCGC-5’ (forward) and 3’-
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GCTCACTGCAGTTATCACTGCTTTGCCTGAAGCTTGGCG-5’ (reverse) were used to amplify
TbMCP12 and the primers 3’-GAGGATCCATGTCAACCAACGCAAAAGAGTCTG-5’ (forward) and 3’-
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CTGCGGCCGCCTACTTGTCTTCCTTTGGCATGC-5’ were used to amplify DIC.
Overexpression of TbMCP12 in procyclic forms
The open reading frame (ORF) of TbMCP12 was PCR-amplified from T. brucei strain Lister 427 genomic DNA using the sense primer 5’-
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GGACGGGTTAACACCATGGCGAAAGAGACAAAGGCGCCCG-3’ and the antisense primer 5’GCTTGCAGGATCCCTGCTTTGCCTGAAGCTTGGCG-3’. The restriction enzyme sites Hpa I (forward
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primer) and Bam HI (reverse primer) used for the subsequent cloning are underlined. The resulting
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PCR product was initially cloned into the pGEM-T Easy TA-cloning vector (Invitrogen) and sequenced (Eurofins Genomics, Wolverhampton, United Kingdom). Comparison of the cloned
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TbMCP12 sequence from T. brucei Lister 427 with the sequence of the corresponding locus Tb927.10.12840 (initially annotated as Tb10.389.0690) (http://www.genedb.org) of T. brucei strain
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927 revealed a few sequence differences at the DNA level but none in the predicted amino acid sequence. The TbMCP12 ORF was subsequently cloned into the trypanosome expression vectors pHD1700 and pHD1701 [27, 29] using the restriction enzyme sites Hpa I and Bam HI introduced
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during PCR. Tetracycline-inducible expression from these vectors results in the expression of the desired protein with a double-myc tag at the carboxy-terminal- (cmyc, pHD1700) or amino-terminal-
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(nmyc, pHD1701) end. The resulting plasmids were used for transfection of procyclic-form T. brucei constitutively expressing the tet-repressor (pHD449, TETR BLE). Trypanosome clones resistant to hygromycin and bearing a tetracycline inducible (ti) and myc-tagged copy of TbMCP12 were isolated by serial dilution and the obtained clonal cell lines analysed by western blotting using an anti-myc
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antibody (Roche Applied Science). The genotype of the resulting cell line is TbMCP12/TbMCP12 TETR BLE TbMCP12-mycti HYG, which will be further referred to as TbMCP12 over-expressing cellline Generation of the conditional TbMCP12 double-knockout cell line The TbMCP12 over-expressing cell line was used as starting point for the generation of a TbMCP12 knockout cell line. Tetracycline (0.5 mg/ml) was added every 24 h to the culture medium to
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The T. brucei di- and tricarboxylate transporter TbMCP12
ensure that the TbMCP12-cmyc was expressed throughout the whole knockout procedure. The two natural TbMCP12 alleles were deleted from the genome of the TbMCP12 over-expressing cell line by successive replacement with the neomycin (NEO) and blasticidin (BSD) antibiotic resistance cassettes, and using the flanking 5’-untranslated region (5’-UTR) and 3’-untranslated region (3’-UTR) of TbMCP12 as target sequences for homologous recombination, as previously described. Sense primer 5’-GCTAGAGCTCCGCGACCTTTGACCGAAGTAACTGCC-3’ and antisense primer 5’GCTAACTAGTTCTTCCTGTCGTTCGATCCTCGAGTAG-3’ were used to PCR-amplify a 500-bp
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genomic fragment located upstream of the TbMCP12 ORF (5’ UTR), and sense primer 5’GCATGGATCCTGTGAATAAATCACTGCGTCACGTTATG-3’ and antisense primer 5’-
CAGTGGGCCCTATATATGAAGTCAAAGTGAGAAAAACAGAG-3’ were used to amplify a 460-bp
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fragment downstream of the TbMCP12 ORF (3’ UTR). The restriction enzyme sites (Sac I, Spe I, Bam HI, and Apa I) included in the PCR-primers and used for subsequent cloning are underlined. As previously described for the construction of other T. brucei double-knockout cell lines [30] using the targeted gene replacement method, the 5’- and 3’-UTRs of TbMCP12 were inserted on either side of
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NEO and BSD genes bearing actin 5’-splice sites and actin 3’-UTRs. The resulting NEO- and BSDbearing double-knockout (NEO-dKO, BSD-dKO) constructs were subsequently used for successive
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transfection of the TbMCP12 over-expressing cell line. After transfection with the NEO-dKO
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construct and selection with 15 mg/ml G418, several procyclic T. brucei cell lines were isolated bearing a single-allele knockout of TbMCP12 with the genotype ∆TbMCP12::NEO/TbMCP12/
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TbMCP12-cmycti. Subsequent transfection of the obtained half-knockout cell line with the BSD-dKO construct and selection with 15 mg/ml G418 and 10 mg/ml blasticidin resulted in the isolation of the
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TbMCP12 double-knockout cell line ∆TbMCP12::NEO/∆TbMCP12::BSD/TbMCP12-cmycti, in this paper further referred to as TbMCP12 knockout. Correct genomic integration of both the NEO-dKO and BSD-dKO constructs and effective replacement of the two natural TbMCP12 alleles were
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confirmed by Southern blot and western blot analysis.
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Western blot analysis
For each lane, 2 × 106 trypanosomes were pelleted, resuspended in Laemmli buffer and then
denatured for 5 min at 95 °C. Proteins were separated on a denaturing 12 % polyacrylamide gel containing SDS (SDS-PAGE), and were subsequently transferred to a Hybond-P membrane (GE
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Healthcare Life Sciences) in transfer buffer (39 mM glycine, 48 mM Tris-base, 20 % v/v methanol, pH 8.3) for 1 h at 100 V. The membrane was blocked by incubation in Tris–buffered saline containing 0.2 % (v/v) Tween 20 (TBS-Tween) and 7.5 % (w/v) non-fat dry milk for 30 min at room temperature with gentle shaking, and subsequently incubated for 1 h with 7.5 % milk TBS-Tween containing the primary antibody. The membrane was then washed once for 15 min, and twice for 5 min in TBSTween, followed by incubation for 45 min at room temperature with the relevant secondary antibody (GE Healthcare Life Sciences) (dilution 1:1,000 for all used secondary antibodies). Finally, the
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The T. brucei di- and tricarboxylate transporter TbMCP12
membrane was extensively washed once for 15 min, and four times for 5 min in TBS-Tween and processed according to the manufacturers protocol of the ECL detection kit (GE Healthc´are Life Sciences), followed by exposure to ECL-film (GE Health Care Life Sciences). TbMCP12 N-term peptide antibody generation Peptide synthesis and immunisation of rabbits were performed by EZBiolab (USA). The synthesised N-terminal peptide ‘KETKAPANAPLPKVY‘ (amino acid residues 3-17 of TbMCP12)
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was coupled to keyhole limpet hemocyanin (KLH) and used for the immunisation of two rabbits. After collection of 2 ml pre-immune serum, immunisation was initiated by the injection of each rabbit with
1 mg KLH-coupled peptide emulsified in complete Freunds adjuvant. The first injection was followed
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by 3 subsequent boosts in weeks 2, 4 and 7, respectively, with 0.5 mg of KLH-coupled peptide
emulsified in incomplete Freund´s adjuvant. The final TbMCP12 antiserum was collected in week 9, after determination of the TbMCP12 antibody titer: i.e. 1:1,536,000 (determined by EZBiolab, USA). Western blot analysis confirmed the absence of cross-reacting protein bands in the pre-immune sera
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(not shown), whereas the final antiserum detected the single 32 kDa protein band representing
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TbMCP12.
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T. brucei growth analysis
For the growth analysis of procyclic cells, either standard glucose (0.3 mM, “low-glucose
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medium”), glucose supplemented (10 mM, “high-glucose medium”), or glucose depleted MEM-Pros medium were used. For the depletion of the glucose from the medium extensive dialysis (64,000-fold)
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of the FCS using 10,000 Da molecular mass cutoff tubing was performed prior to addition to the incomplete medium. The dialysed FCS was then mixed 1:10 with non-dialysed FCS to preserve a low level of essential cofactors present in FCS, which are required for procyclic cell growth. At the
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beginning of the growth analysis procyclic trypanosomes were diluted to a density of 0.25 x 106 cells/ml and bloodstream trypanosomes to a density of 0.1 x 106 cells/ml; cells were then counted
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every 24 h for a total of 72 h using a Neubauer hemocytometer. Expression of TbMCP12-cmyc in the TbMCP12 over-expressing cell line was induced by the addition of 0.5 mg/ml tetracycline to the culture medium at time point 0. The TbMCP12 knockout cells were grown in constant presence of tetracycline to maintain TbMCP12-cmyc expression. To achieve a non-induced (TbMCP12-depleted)
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condition, TbMCP12 knockout cells were washed three times with excess culture medium for complete withdrawal of tetracycline (TbMCP12-depleted knockout cells) 24 h prior to the start of the growth experiment. Immunofluorescence microscopy Trypanosomes were centrifuged from the culture medium at 2,000 x g and resuspended in phosphate-buffered saline (PBS) containing 4 % (w/v) paraformaldehyde. Fixed cells were allowed to
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The T. brucei di- and tricarboxylate transporter TbMCP12
settle down and attach to poly-L-lysine-coated microscope slides. Immunofluorescent labelling of trypanosomes with 4’,6’-diamidino-2-phenylindole (DAPI), the mitochondrion-specific probe MitoTracker, and the different antibodies was performed as previously described [27]. Cells were examined using a Leica DM RXA digital de-convolution microscope, and images were recorded using a digital camera (Hamamatsu). T. brucei subcellular fractionation
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Trypanosomes were grown to a maximum cell density of 5 x 106 cells/ml. Approximately 9 x 109 cells were harvested by centrifugation for 10 min at 2,000 x g, and washed once in 50 ml of TEDS (25 mM Tris, 1 mM EDTA, 1 mM DTT, 250 mM sucrose, pH 7.8). After centrifugation for 10 min at
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2,000 x g, the cell pellet was resuspended in 2 ml of homogenization medium (250 mM sucrose, 1 mM EDTA, 0.1 % v/v ethanol, 5 mM MOPS, pH 7.2) supplemented with ‘complete EDTA-free’ protease inhibitor cocktail (Roche Applied Science), and grinded in a pre-chilled mortar at room temperature on ice with 1 volume of wet-weight silicon carbide (Crysalon, Norton Company, porous <400 mesh)
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until at least 90 % cell disruption. Cell disruption was checked under the light microscope. The obtained cell lysate was centrifuged at 3,000 x g for 5 min to remove abrasive and nuclei. The
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obtained supernatant was centrifuged at 17,000 x g for 15 min at 4 °C to yield the light-mitochondrial
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fraction, which was subsequently resuspended in 3 ml homogenization buffer. A 32 ml linear 20-40 % (v/v) Optiprep (iodixanol-sucrose) gradient, mounted on top of a 3.5 ml 50 % (v/v) Optiprep cushion,
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was prepared according to the manufacturer´s protocol (Optiprep Application Sheet S9, Axis-shield). Centrifugation was performed at 105,000 x g for 1 h at 4 °C using a Sorvall SW-60 Rotor. Aliquots of
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1 ml were collected from the bottom of the tube by puncture. An equal volume of each fraction was TCA precipitated and the obtained protein pellet was resuspended in denaturing Laemmli SDS-PAGE
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buffer. Proteins were separated on a 12 % SDS-PAGE gel and analysed by western blotting. Measurement of NADP+/NADPH ratios
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NADP+/NADPH ratios were determined in trypanosome cell lysates using the
NADP+/NADPH Assay Kit (Abcam) and according to the manufacturer’s protocol. Briefly, 107 cells were lysed in 800 μl of NADP+/NADPH extraction buffer by performing two freeze/thaw cycles. The lysate was then centrifuged at 13,000 x g for 5 min and the supernatant transferred to a fresh tube to
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obtain the total NADP Sample (NADPt Sample). For the measurement of NADPH, NADP+ was decomposed by heating 200 μl lysate to 60 °C for 30 min (NADPH Sample). From both samples 10 μl were incubated with 100 μl reaction mix (98 μl NADP+ cycling buffer and 2 μl NADP+ cycling enzyme mix) and incubated at room temperature for 5 min to convert NADP+ to NADPH. For background detraction a reaction containing only extraction buffer and reaction mix was set up. NADPH was then determined in all three reactions by addition of 10 μl NADPH developer followed by incubation at room temperature for 1 h and measurement of the OD450. The NADPH amount in
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The T. brucei di- and tricarboxylate transporter TbMCP12
each sample was determined from the obtained OD450 after subtraction of the background using a NADPH standard curve. The NADP+/NADPH ratio was calculated as follows: (NADPt - NADPH)/NADPH
Mitochondrial ATP production assays ATP production assays were performed as previously described by Schneider et al. [33]. Trypanosomes (108) were collected by centrifugation at 1,500 x g for 10 min after 72 h of culture. The
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cell pellet was washed once with SoTE-buffer (20 mM Tris-HCl, 2 mM EDTA, 0.6 M Sorbitol, pH
7.5), and resuspended in 1 ml of SoTE-buffer containing 0.008 % (w/v) digitonin. Permeabilisation of the trypanosome plasma membrane with digitonin was allowed to take place for exactly 5 min on ice,
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followed by immediate centrifugation for 3 min at 8,000 x g and 4 °C. The supernatant was removed and the pellet washed twice with 1 ml SoTE-buffer. The pellet containing the mitochondria-enriched fraction was resuspended in 0.5 ml assay buffer (20 mM Tris-HCl pH 7.4, 15 mM KH2PO4, 0.6 M Sorbitol, 5 mM MgSO4). Mitochondrial ATP production was initiated by the addition of 75 μl assay
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buffer containing 67 μM ADP and 5 mM of the different substrates (i.e. oxoglutarate, succinate,
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malate, citrate, isocitrate or aconitate) to 75 μl of mitochondria-enriched fraction and was allowed to take place for 30 min at 25 °C. The mitochondrial ATP-production was terminated by the addition of
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10 μl TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), followed by denaturation at 100 °C for 3 min. The
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formed protein precipitate was removed by centrifugation for 1 min at 16,000 x g. The ATP concentration in the supernatant was measured using the ATP Bioluminescence Assay Kit CLS II kit
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(Roche Applied Science) and the Junior LB9509 tube luminometer (Berthold Technologies). Yeast functional complementation
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Saccharomyces cerevisiae strains used in this study were the parental strain BY4741 (Mat a his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) and the derived DIC knockout strain YLR348C (ΔDIC1; MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) (EUROSCARF, Frankfurt Germany). The TbMCP12 ORF was
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PCR-amplified from T. brucei strain Lister 427 genomic DNA using the sense primer 5’GGGGATCCATGGCGAAAGAGACAAAGGCGC-3’ and the antisense primer 5’GCTCACTGCAGTTATCACTGCTTTGCCTGAAGCTTGGCG-3’. The ORF of the S. cerevisiae DIC was
amplified from S. cerevisiae cDNA using the sense primer 5´-
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GAGGATCCATGTCAACCAACGCAAAAGAGTCTG-3´ and antisense primer 5´CTGCGGCCGCCTACTTGTCTTCCTTTGGCATGC-3´. The restriction enzyme sites (Bam HI and Pst I
for TbMCP12 and Bam HI and Not I for DIC) included in the PCR-primers and used for subsequent cloning are underlined. The resulting PCR products were initially cloned into the pGEM-T Easy TAcloning vector (Invitrogen) and sequenced (Eurofins Genomics, Wolverhampton, United Kingdom) to confirm that the ORFs were correct. The open reading frames were subsequently cloned into the 2
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The T. brucei di- and tricarboxylate transporter TbMCP12
micron episomal yeast expression vector pCM190 (EUROSCARF, Frankfurt, Germany) using the restriction enzyme sites introduced during PCR. Yeast parental, DIC knockout and derived strains were maintained on standard YPD medium (1 % of yeast extract, 2 % of peptone, 2 % of glucose, and 2 % of agar). In order to test cell growth under different carbon sources the cells were grown in either YPD (containing glucose) or YPG (containing glycerol) medium. Plasmids containing target genes were transfected into the ∆DIC strain according to the lithium acetate/single-stranded carrier DNA method described by Gietz and Woods
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[34]. For clone-selection after transfection, medium without uracil, containing synthetic complete (SC) medium (0.67 % of yeast nitrogen base (w/o amino acids), 1.4 % of drop-out medium supplements (w/o histidine, leucine, tryptophan and uracil), supplemented with 60 mg/L of leucine, 20 mg/L of
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tryptophan, 20 mg/L of histidine, 2 % of agar and 2 % of dextrose) was applied (Sigma). Resulting
yeast clones were maintained on YPD media without uracil with tetracycline. Heterologous protein expression in yeast was induced by tetracycline removal.
For growth experiments a starter culture was inoculated with yeast cells and grown at 28 °C
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overnight. The next day, the absorbance of the cells was measured at 600 nm (OD600) and cultures were diluted to OD600 0.2 in 20 ml medium and grown for 24 h on glucose medium and 96 h on acetate
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medium. RESULTS
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The genome of Trypanosoma brucei contains a plant-like di-tricarboxylate carrier We previously used the conserved sequence and structural features of mitochondrial carriers
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to identify all members of the MCF in the kinetoplastid parasite T. brucei [29]. To find kinetoplastid MCPs involved in the exchange of dicarboxylates, tricarboxylates and oxodicarboxylates, we used the deduced amino acid sequences of the equivalent, functionally characterised metazoan MCPs [28, 35-
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40] (www.ncbi.nlm.nih.gov) as BLASTP queries against protein databases of T. brucei, Leishmania major and Trypanosoma cruzi (www.genedb.org). Searches using either the sequence of the
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tricarboxylate carrier SLC25A1 (CIT) [40] or of the oxodicarboxylate carrier SLC25A21 [41] as query, did not identify kinetoplastid homologues. In contrast, BLASTP mining using the dicarboxylate carrier SLC25A10 (DIC) [42] or the oxoglutarate carrier SLC25A11 (OGC) [37, 39], resulted in the identification of one single gene in the T. brucei genome database: TbMCP12 (Tb927.10.12840). In
A
the T. cruzi genome we found two TcMCP12 genes encoding proteins with 63% identity to TbMCP12 (Table 1), while remarkably, in the L. major genome we found 8, tandemly arranged LmMCP12 genes (55% identity; LmMCP12A-H, Table 1). The presence of multiple genes (up to 3 copies) in the genome of this parasite was previously described for other MCP genes [27]. Reciprocal BLASTP searches of the NCBI gene databases (http://www.ncbi.nlm.nih.gov) using TbMCP12 as sequence query predominantly retrieved oxoglutarate carriers from different eukaryotes, including the functionally characterised mitochondrial OGC (NP_777096) from Bos taurus [37, 39].
10
The T. brucei di- and tricarboxylate transporter TbMCP12
Table 1: TbMCP12 homologues identified in T. cruzi and L. major Accession number
Protein lenght
Conservation
Identity
T. brucei
Tb927.10.12840
304
-
-
T. cruzi
TcCLB.503939.20
326
75%
63%
TcCLB.509805.190
326
75%
63%
LmjF.18.1260
320
71%
56%
LmjF.18.1300
308
71%
LmjF.18.1280
316
70%
LmjF.18.1265
316
LmjF.18.1270
316
LmjF.18.1275
316
LmjF.18.1285
316
LmjF.18.1290
316
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55%
70%
55%
70%
55%
70%
55%
70%
55%
70%
55%
U N
A
56%
M
L. major
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Species
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Since conventional DIC and OGC do not transport tricarboxylates, we wanted to identify the transporter that can take over this function in T. brucei. In vitro reconstitution in liposomes and
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metabolite transport experiments showed that the mitochondrial dicarboxylate-tricarboxylate carrier (DTC) from Arabidopsis thaliana is able to transport both dicarboxylates (such as oxoglutarate, malate and oxaloacetate) and tricarboxylates (such as citrate, isocitrate and cis-aconitate) [38]. So far this type
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of MCF transporter has only been described in plants and Plasmodium falciparum [38, 43], the causative agent of malaria tropica. Interestingly, BLASTP analysis of the T. brucei protein database using the A. thaliana DTC amino acid sequence as query only retrieved TbMCP12 as top hit with significant sequence conservation (48%).
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The BLASTP-predicted function of TbMCP12 as either oxoglutarate/malate or dicarboxylate/tricarboxylate exchanger was further investigated by phylogenetic reconstruction using oxoglutarate, dicarboxylate and dicarboxylate-tricarboxylate carrier amino acid sequences from representative eukaryotes (Figure 1). The phylogenetic tree showed that the carboxylate carriers grouped in well-defined clades depending on their transport functions (Figure 1). TbMCP12 and the homologuous sequences from T. cruzi and L. major branched into a separate clade, which was supported by high bootstrap values (Figure 1). However, the bootstrap values do not support an
11
The T. brucei di- and tricarboxylate transporter TbMCP12
association of TbMCP12 to any of the other carboxylate carrier groups. It is worth noticing that the
A
N
U
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DTC of P. falciparum, in contrast to TbMCP12, clusters within the clade of the plant DTCs.
clade near DTCs and OGCs.
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Figure 1. In the phylogenetic tree TbMCP12 and related MCF proteins cluster in a separate
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The maximum likelihood tree is showing the evolutionary relationship between TbMCP12 and DTCs (green), OGCs (blue) and DICs (yellow) of the MCF. Colors have been applied to the phylogenetic tree to indicate clustering of carriers with the same transport function. The bootstrap values located at
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the nodes represent the percentages obtained after resampling analysis of 100 reiterated data sets. Only significant bootstrap values (≥ 50 %) are shown. The kinetoplastida clade is boxed in red. Asterisks
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indicate MCF proteins that have been already functionally characterised.
Residues involved in protein folding and substrate binding are conserved in TbMCP12
A
The deduced amino acid sequence of TbMCP12 consists of 304 amino acid residues, which corresponds to a predicted molecular weight of approximately 33 kDa (http://www.genedb.org). TbMCP12 exhibits all conserved amino acid sequence and structural characteristics of MCPs: it consists of three domains of about 100 amino acids, each containing two transmembrane (TM) alphahelices and the canonical MCP signature sequence ‘Px[D/E]x2[K/R]x[K/R]x20-30[D/E]Gx45[W/F/Y][K/R]G’
(Figure 2A) [22, 27, 28]. The first part of the motif ‘Px[D/E]x2[K/R]x[K/R]’ is
located at the carboxy-terminal end of TM helices H1, H3 and H5, whereas the second part ‘[D/E]Gx4-
12
The T. brucei di- and tricarboxylate transporter TbMCP12
5[W/F/Y][K/R]G’
is located after the amphipathic helices h1-2, h3-4 and h5-6 (Figure 2A). The
conservation of the signature motif in TbMCP12 was analysed in comparison to DTCs from different plants and with metazoan OGCs (Figure 2B). Most amino acids of the first part of the signature motif, indicated as M1a, M2a, and M3a, are conserved including: i) the proline found at the start of M1a, M2a and M3a; ii) the acidic amino acid residue, either an aspartic acid or glutamic acid, at position 3, and iii) the positively charged amino acid, either a lysine or an arginine, at position 6, with exception of M2a, in which this residue is exchanged for a leucine in all analysed sequences (Figure 2B). In M2a
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A
N
U
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also the lysine or arginine at position 8 was exchanged for a leucine.
Figure 2. TbMCP12 displays all the amino acid sequence features of MCF conventional carriers
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and contain functional residues conserved in DTCs and OGCs A: Schematic representation of MCPs. The protein structure consists of six transmembrane helices (H1-6) linked by hydrophilic loops (h1-2, h3-4 and h5-6). M1a, M2a, and M3a indicate the first segment of the signature sequence motif, Px(D/E)xx(K/R)x(K/R), located at the end of the odd-
A
numbered transmembrane helices while M1b, M2b, and M3b indicate the second part of the motif, (D/E)Gxn(K/R)G, located at the end of each hydrophilic loop. B: Multiple sequence alignment of TbMCP12 and different plant DTCs and OGCs. Amino acid sequences were aligned using ClustalΩ. TbMCP12 residues identical to residues in either DTCs or OGCs are shaded in yellow. TbMCP12 residues with conserved substitutions in DTC and OGC are shaded in orange. Conserved contact points are color-coded as follows: CPI, green; CPII, blue; CPIII, red.
13
The T. brucei di- and tricarboxylate transporter TbMCP12
In contrast to the first part of the signature motive, the second part is partially conserved (Figure 2: M1b, M2b, and M3b). The first two amino acids of the motif, i.e. ‘(D/E)G’, are not always present in the T. brucei sequence. The final glycine (‘G’), which allows flexibility in the arm that links the two helices [44], and the aromatic residue at position 7, on the other hand, are highly conserved except in M3b in which the G is missing (Figure 2B). Previous analyses of the structure of the bovine ATP/ADP carrier showed that groups of
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conserved amino acids located downstream of each signature motif participate in substrate discrimination, recognition and binding [45, 46]. From this structural information three conserved substrate-contact points called CPI, CPII and CPIII were extrapolated for all MCPs transporting
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similar substrates [45, 46]. As shown by the amino acid sequence alignments with DTCs and OGCs all three contact points typical for these two carrier types are conserved and present in TbMCP12 (Figure 2B).
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TbMCP12 is predominantly expressed in procyclic-form T. brucei
Previously published northern blot analyses indicated that the expression of some TbMCPs is
N
life-cycle stage dependent [27, 29, 30]. Northern blot analysis using the TbMCP12 ORF as a probe
A
revealed a single cross-hybridizing mRNA band of approximately 2.4 kb in both bloodstream-form and procyclic-form T. brucei (Figure 3A). The observed size-length of this mRNA is similar to the in
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silico predicted TbMCP12 mRNA size of 2.5 kb, when assuming that trans-splicing occurs at position -147 relatively to the ATG and that polyadenylation occurs 1.3 kb downstream of the stop codon TAG
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[47]. Northern blot analysis revealed that the expression of TbMCP12 is higher in procyclic- than in bloodstream-form T. brucei (Figure 3A). Previous publications showed that during the differentiation from bloodstream to procyclic form the amount of TbMCP12 mRNA is increased [48]. The up-
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regulation of the expression of TbMCP12 in the procyclic form was further confirmed at the protein level by western blot analysis (Figure 3B). Antibodies against the N-terminus of TbMCP12 detected a
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single band of the predicted molecular mass of 32 kDa in both bloodstream- and procyclic-form T. brucei (Figure 3B). After quantification using -tubulin as control [49] the results showed, as expected, that TbMCP12 is far more abundant in the procyclic form (Figure 3B). This result corresponds well with previously published proteomic results indicating that the protein is higher
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expressed in the procyclic stage but is already upregulated in the short stumpy form [50, 51]. These results are consistent with the expectation that the transport function of TbMCP12 should be more required in the life cycle stage that displays higher mitochondrial energy metabolism activity.
14
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The T. brucei di- and tricarboxylate transporter TbMCP12
Figure 3. TbMCP12 is differentially expressed in bloodstream and procyclic T. brucei and localised in the mitochondria of procyclic cells
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A: Northern blot analysis of 10 μg total RNA from bloodstream (BS) and procyclic (PF) T. brucei
N
using [a-32P]dCTP-labelled TbMCP12 DNA as hybridization probe. A [⍺-32P]dCTP-labelled signal
A
recognition particle (SRP) hybridization probe was used as loading control B: Western blot analysis of
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2 x 106 wild type BS and PF T. brucei cells using the antibody directed against TbMCP12. An antibody against tubulin [49] was used as a loading control. Antibodies were used in 1:1,000 dilutions. C: Western blot analysis of procyclic T. brucei post-nuclear cell lysate fractions (10 μg/lane) separated
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on an Optiprep (iodixanol-sucrose) gradient. Fractions containing the mitochondria (Mito) were identified using an antibody against LipDH [52], those containing the glycosomes (Gly) with an
PT
antibody directed against PEX11 [53]. TbMCP12 was detected using the TbMCP12 antibody. Fraction 1 indicates the bottom fraction 33 the top of the gradient. D: Immunofluorescence analysis of wild type procyclic form T. brucei. TbMCP12 (green) was visualized using the TbMCP12 antibody (diluted
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1:500) and mitochondria (red) were stained using Mitotracker. Nuclear and kinetoplast DNA (blue) were stained with DAPI.
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TbMCP12 is exclusively located in the mitochondrion of procyclic T. brucei The subcellular localisation of TbMCP12 in procyclic-form T. brucei was determined by cell
fractionation followed by western blotting, and by immunofluorescence microscopy. TbMCP12 cosedimented (fractions 23-27) exclusively with the mitochondrial marker protein dihydrolipoamide dehydrogenase (Figure 3C: gradient fractions 23-29). No TbMCP12 was detected in fractions 9-11, containing the glycosomes (visualized using an antibody directed against the glycosomal marker PEX11). By immunofluorescence microscopy TbMCP12 displayed the typical mitochondrial staining
15
The T. brucei di- and tricarboxylate transporter TbMCP12
pattern, overlapping with the mitochondrial marker MitoTracker (Figure 3D). Analysis of the subcellular localisation of TbMCP12 in bloodstream cells using the antibody directed against TbMCP12 was not possible, due to low expression of the protein TbMCP12 complements the transport function of the yeast dicarboxylate carrier Sequence analysis indicates that of all identified TbMCPs, TbMCP12 is the transporter that displays highest similarity to the yeast dicarboxylate carrier DIC. The function of TbMCP12 as a
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putative dicarboxylate transporter was investigated by functional complementation analysis using a S. cerevisiae DIC knockout strain (DIC). Yeast lacking the dicarboxylate carrier is unable to grow on non-fermentable carbon sources as sole carbon source [54].
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We compared the growth of the S. cerevisiae parental (wild type) and DIC knockout strains after transfection with either the empty pCM190 plasmid (negative control) or the pCM190 plasmid containing the TbMCP12 ORF on glucose and acetate. The absence of DIC in the yeast DIC knockout strain and the successful expression of TbMCP12 after transformation were confirmed by
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RT-PCR (Figure 4A). For DIC yeast transfected with either empty or TbMCP12-containing
N
plasmids, no growth difference was seen in the presence of the fermentable carbon source glucose (Figure 4B). With the non-fermentable carbon source acetate, DIC yeast grew to lower density in the
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same amount of time compared to wild-type yeast (Figure 4C). Growth of DIC yeast could however
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be restored to almost wild type levels by the expression of recombinant TbMCP12 (DIC + TbMCP12) (Figure 4C). These results suggest that the transport function of TbMCP12 overlaps with
ED
the one of the S. cerevisiae dicarboxylate carrier. We were further interested to investigate whether TbMCP12 is involved in the transport of tricarboxylic acids by complementing the corresponding yeast knockout strains. However, the deletion of the yeast citrate carrier (YBR291C) does not cause a
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lethal phenotype [55] and deletion of the citrate-oxoglutarate carrier (YMR241w) causes only a very mild phenotype when the yeast is grown in the presence of hydrogen peroxide [56]. We therefore
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deemed these knockout strains unsuitable for the purpose of further complementation studies.
16
U
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The T. brucei di- and tricarboxylate transporter TbMCP12
Figure 4. Functional complementation studies show that TbMCP12 can complement the function
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of DIC in S. cerevisiae knockout strains
A
A: RT-PCR was used to analyse the expression of TbMCP12 and DIC in the different yeast strains. RNA was isolated from wild type (WT), DIC-deficient strain transfected with empty pCM190 (DIC)
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and DIC transfected with either TbMCP12 (DIC + TbMCP12) or the yeast dicarboxylate carrier (DIC + DIC). As a control 100 ng purified plasmid containing either DIC or TbMCP12 was used as
ED
template. B and C: Growth of the wild type (WT) S. cerevisiae and DIC-deficient yeast strain transfected with either empty pCM190 (DIC) or pCM190 containing TbMCP12 (DIC +
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TbMCP12). Yeast cell lines were isolated and inoculated in medium containing either glucose or acetate and the OD600 was measured after respectively 24 h and 96 h. The results are depicted as
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means of 3 independent experimental replicates and standard deviation is shown.
TbMCP12 is not essential for procyclic form T. brucei but its overexpression causes a
A
growth-defect
To avoid incomplete down-regulation of TbMCP12 following RNAi, we produced a
conditional double TbMCP12 knockout in procyclic T. brucei by generating first a TbMCP12 overexpressing cell line (Figure 5). Western blot analysis of this cell line using a myc-tag antibody revealed a 35 kDa protein band representing the recombinant TbMCP12-cmyc protein after tetracycline induction (Figure 5A). Using the TbMCP12 antibody the endogenous TbMCP12 (32 kDa) was detected in both the non-induced and induced TbMCP12 over-expressing cell line (Figure 5A). Tagging at either the N- or the C-terminus did not affect mitochondrial localisation of the recombinant
17
The T. brucei di- and tricarboxylate transporter TbMCP12
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M
A
N
U
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TbMCP12-cmyc protein (Figure 5B).
Figure 5. Overexpression of TbMCP12 in procyclic T. brucei is detrimental independently of the
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glucose concentration present in the medium A: Expression analysis of endogenous TbMCP12 and TbMCP12cmyc in wild type (WT) and tetracycline (tet)-induced and uninduced TbMCP12 over-expressing (OE) cell line. Each lane contains
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2 x 106 cells. Proteins were detected using 1:1,000 dilutions of antibodies directed against TbMCP12, myc-tag or aldolase (loading control). B: Immunofluorescence analysis of tet-induced TbMCP12 OE cell line. TbMCP12cmyc and TbMCP12nmyc (green) were detected using an anti-myc-tag antibody (1:500) and mitochondria (red) were stained using an antibody directed against LipDH (1:500).
A
Nuclear and kinetoplast DNA were stained with DAPI. C and D: Growth phenotype analysis of the TbMCP12 OE cell line. Shown are growth curves of WT and tet-induced TbMCP12 OE cell line cultured in standard low-glucose (C) and high-glucose (D) medium. Procyclic trypanosome growth was started at 0.25 x 106 cells/ml and cells were counted every 24 h for a total of 72 h. The growth curves represent the means of 6 independent experimental replicates and standard deviation is shown. One-way ANOVA was performed using GraphPad Prism 7. *: p ≤ 0.05 **: p ≤ 0.01; ***p ≤ 0.001. E: Representative western blot analysis of the expression profile of TbMCP12 and TbMCP12cmyc during
18
The T. brucei di- and tricarboxylate transporter TbMCP12
72 h of cell culture of tet-induced TbMCP12 OE cell line cultured in standard low-glucose and highglucose medium. TbMCP12 and TbMCP12cmyc were detected using the TbMCP12 antibody (1:1,000) and aldolase (1:1,000) was used as loading control.
Following growth analysis we found that in the presence of tetracycline, the growth-rate of the TbMCP12 over-expressing cell line was significantly lower than that of wild type cells, independently
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of the growth medium (Figure 5C and D). These cells express both the endogenous and the inducible genes: quantification of Figure 5A suggested a 5-fold TbMCP12 over-expression compared to wild type cells grown on either high or low glucose medium. Over-expression of TbMCP12 therefore
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appeared to inhibit growth.
To obtain the TbMCP12 knockout cell-line the endogenous TbMCP12 in the TbMCP12 overexpressing cell line was replaced by homologous recombination with neomycin (NEO) and blasticidin (BSD) resistance cassettes. The successful replacement of both natural TbMCP12 alleles in the
U
TbMCP12 knockout cell line was confirmed by Southern blot (not shown). Western blot analysis showed that in the presence of tetracycline the TbMCP12 knockout cell-line expressed TbMCP12-
N
cmyc (Figure 6A). After the removal of tetracycline TbMCP12-cmyc was fully depleted and we
A
obtained the TbMCP12-depleted knockout (Figure 6A). TbMCP12-cmyc was only detected in the
A
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ED
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mitochondrion of procyclic cells exclusively in the cells grown with tetracycline (Figure 6B and C).
19
N
U
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The T. brucei di- and tricarboxylate transporter TbMCP12
Figure 6. The depletion of TbMCP12 by conditional knockout is not lethal for procyclic or
A
bloodstream form T. brucei
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A: Endogenous TbMCP12 and TbMCP12cmyc expression analysis in wild type (WT), and tetracycline (tet)-induced and uninduced TbMCP12 knockout cells (KO). In each lane 2 x 106 cells were loaded.
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Proteins were detected using 1:1,000 dilutions of antibodies directed against TbMCP12, myc-tag or aldolase (loading control). B and C: Immunofluorescence analysis of TbMCP12 knockout cells after 24 h of tet removal. The depletion of the endogenous TbMCP12 (B, green) was detected using the
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TbMCP12 antibody (1:500) while TbMCP12cmyc (C, green) was detected using an antibody directed against the myc-tag (1:500). Mitochondria (B and C, red) were stained using Mitotracker. Nuclear and
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kinetoplast DNA (B and C, blue) were stained with DAPI. D and E: Growth phenotype analysis of TbMCP12 KO cells (tet-induced) and TbMCP12-depleted KO cells (after 24 h of tet removal). Cells were cultured in standard low-glucose (D) and high-glucose (E) medium. Procyclic trypanosome growth was started at 0.25 x 106 cells/ml and cells were counted every 24 h for a total of 72 h. The
A
growth curves represent the means of 6 independent experimental replicates and standard deviation is shown. F: Representative western blot analysis (F) of the expression profile of TbMCP12 and TbMCP12cmyc during 72 h of cell culture of WT and TbMCP12-depleted KO cells cultured in standard low glucose and high glucose medium. TbMCP12 and TbMCP12cmyc were detected using the TbMCP12 antibody (1:1,000) and aldolase (1:1,000) was used as loading control.
20
The T. brucei di- and tricarboxylate transporter TbMCP12
The growth of the TbMCP12-depleted knockout cells was assessed in low- (Figure 6D) and high-glucose medium (Figure 6E). All analysed cells grew at similar rates independently of the medium used (Figure 6D and E). The lack of growth defect in the TbMCP12-depleted knockout was unexpected and suggested that the carrier is not essential for the survival of procyclic form T. brucei under the applied culture conditions. The absence of TbMCP12 in the knockout cell line was confirmed throughout the growth period (Figure 6F). Since the depletion of TbMCP12 was not lethal we cannot fully exclude the presence of another carrier with similar transport function or the presence
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of alternative metabolic pathways compensating its absence. TbMCP12 transports metabolic intermediates that are metabolised to ATP inside the
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mitochondrion.
Results so far suggested a role for TbMCP12 as either OGC or DTC (Figure 1, 2 and 4). To test this, we measured the mitochondrial ATP production from different TCA-cycle intermediates [33], using mitochondria isolated from T. brucei grown in either high- or low-glucose medium.
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We first analysed the mitochondrial ATP-production in mitochondria-enriched fractions from wild type procyclic T. brucei. The results revealed the highest ATP production when oxoglutarate or
N
succinate where used as substrates (~4.7 nmol ATP), followed by aconitate (~3 nmol ATP) and citrate
A
(~1.3 nmol ATP), irrespective of growth medium (Figure 7A). The lowest mitochondrial ATP production was found with malate and isocitrate, but for both, about 3 times more ATP was produced
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by mitochondria derived from trypanosomes grown on low-glucose medium (Figure 7A). No ATP was produced on any of the substrates when the mitochondrial ADP/ATP carrier (TbMCP5, [30]) inhibitor
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carboxyatractyloside (CAT) was included confirming that the measured ATP derived exclusively from
A
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the isolated mitochondria (not shown).
21
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PT
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M
A
N
U
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The T. brucei di- and tricarboxylate transporter TbMCP12
Figure 7. Procyclic T. brucei depleted of TbMCP12 cannot produce mitochondrial ATP on citrate
A: ATP production of mitochondria derived from 1 x 107 procyclic cells grown on either low or high
A
glucose medium. ATP production was measured after incubation of the isolated mitochondria with ADP and different substrates. B and C: ATP production of mitochondria derived from procyclic T. brucei overexpressing (OE) or depleted (KO) of TbMCP12 grown on either low (B) or high glucose medium (C). The values are indicated as percentage of the ATP production of wild type cells. Grey dashed lines indicate wild type as 100 %. The significance is related to the concentrations measured in wild type cells. Box-plots were derived from at least five independent experiments. Statistical significance was determined by one-way ANOVA using GraphPad Prism 7: *: p ≤ 0.05 **: p ≤ 0.01;
22
The T. brucei di- and tricarboxylate transporter TbMCP12
***p ≤ 0.001.
Mitochondria isolated from the TbMCP12 over-expressing cells, produced significantly more (~2 times) ATP than wild type cells using tricarboxylates as substrate when they were cultured in low, but not in high glucose medium (Figures 7B and C). In contrast, ATP production on malate was completely abolished by TbMCP12 over-expression, while no significant changes were observed for
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oxoglutarate and succinate (Figures 7B and C). Strikingly, mitochondria depleted of TbMCP12 (TbMCP12-depleted knockout cells) showed no ATP-production when either citrate or isocitrate were used as substrates (Figures 7B and C). ATP
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production from aconitate was significantly reduced in low glucose medium and completely abolished in high glucose medium (Figures 7B and C). ATP production from oxoglutarate and succinate was
only reduced in mitochondria from cells grown on low glucose medium (Figures 7B and C). Together, the results indicate that TbMCP12 is involved in the transport of citrate, isocitrate and aconitate, and
U
that its function affects the metabolism of succinate and malate and, to a minor extent, oxoglutarate.
N
The NADP+/NADPH balance is disturbed in TbMCP12-overexpressing cell lines
A
In T. brucei TCA-cycle-derived malate can be exported from the mitochondrion and converted to pyruvate by cytosolic malic enzyme. Since this reaction generates NADPH it contributes to the
M
maintenance of the intracellular redox balance [57, 58]. To investigate the involvement of TbMCP12 in this system, we measured the NADP+/NADPH ratio in wild type, TbMCP12 overexpressing and
ED
depleted knockout cells, cultured in either low-glucose or high-glucose medium (Table 2). While no variation of the NADP+/NADPH ratio was measured when cells were grown on low glucose medium, we observed a significant (p = 0.009) reduction of the NADP+/NADPH ratio in cells overexpressing
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TbMCP12 and grown in high glucose medium compared to wild type cells (Table 2). This was caused by a 5-fold reduction in the concentration of NADP+ compared to wild type cells (p = 0.00022) (Table
A
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2).
23
The T. brucei di- and tricarboxylate transporter TbMCP12
NADP+ (pmol)
NADP+/NADPH
WT
67.9 +/- 15.1
214.3 +/- 54.3
3.1+/- 0.9
KO
48.0 +/- 13.4
142.0 +/- 21.4
2.9 +/- 1.1
OE
80.9 +/- 33.6
175.1 +/- 32.4
2.1 +/- 1.2
WT
71.0 +/- 20.7
211.8 +/- 50.3
2.9 +/- 1.6
KO
122.5 +/- 29.8
164.6 +/- 39.1
1.3 +/- 0.5
OE
90.0 +/- 29.2
42.5 +/- 7.5 p = 0.00022 0.5 +/- 0.1 p = 0.009
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High glucose
NADPH (pmol)
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Low glucose
Cells
Table 2: pmol of NADP+ and NADPH measured in 1.25 x 105 cells. The results are the means of 3 independent experimental replicates and standard deviations are indicated. One-way ANOVA was performed using GraphPad Prism 7 to determine statistical significance. Statistical significance in
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comparison to WT values was determined by one-way ANOVA using GraphPad Prism 7. Only significant p-values are indicated. WT: wild type; KO: TbMCP12 depleted knockout; OE: TbMCP12
M
A
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overexpression
The expression of TbMCP12 is increased in the presence of hydrogen peroxide, glucose and haemin
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Our results show an unbalance in the T. brucei NADP+/NADPH ratio in response to the increased expression of TbMCP12. We next tested if the abundance of TbMCP12 was also affected
PT
when the cells were subjected to oxidative stress by adding hydrogen peroxide (H2O2), haemin or high glucose. High concentrations of porphyrins such as Haemin have cytotoxic effects on tissues and cells due to the generation of reactive oxygen species like oxygen radicals, lipid peroxides and H2O2 [59,
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60]. High amounts of glucose can also result in elevated cellular oxidative stress because of the accumulation of glycolytic intermediates, which are diverted to and metabolised through ROSgenerating pathways [61]. When we treated procyclic T. brucei with 10 μM H2O2 the abundance of TbMCP12 was doubled (Figure 8A). Further, the addition of 11 mM Haemin or 10 mM glucose to
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medium containing 5 mM proline as carbon source, induced 3.5-fold and 1.75-fold increments, respectively, in the TbMCP12 abundance compared to medium containing proline only (without glucose and Haemin) (Figure 8B). The highest expression of TbMCP12 (5-fold increment) compared to medium containing neither glucose nor Haemin was found when T. brucei was grown on medium containing 10 mM glucose and 11 mM Haemin (Figure 8B). In all analysed conditions the TbMCP12 expression, however, was never as high as the one obtained in the TbMCP12 overexpressing cell-line in which the TbMCP12 abundance was 5-fold higher than the one measured in high or low glucose
24
The T. brucei di- and tricarboxylate transporter TbMCP12
medium containing Haemin. No significant reduction of the growth rate of procyclic T. brucei was observed in haemin-depleted medium (not shown). Furthermore, no difference was observed in the expression of the TbMCP12 at the mRNA level suggesting that the abundance of TbMCP12 is
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A
N
U
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regulated at the level of translation or protein stability (not shown).
Figure 8. Increased TbMCP12 expression in the presence of oxidative stress
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A: Representative western blot analysis of the protein abundance of TbMCP12 in the presence of oxidative stress caused by 5 μM and 10 μM H2O2. The western blot was labeled with antibodies against TbMCP12 or tubulin (diluted 1:1,000). Results were quantified using imageJ, normalised against tubulin and expressed as percentage of the TbMCP12 abundance present in cells that were not
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subjected to oxidative stress. B: Analysis of the protein abundance of TbMCP12 after the removal of glucose and haemin from the growth medium. Cells were cultivated in either the presence or absence of glucose or haemin and analysed by western blotting using antibodies against TbMCP12 and tubulin as loading control (both antibodies were diluted 1:1,000). Results were quantified using imageJ, normalised against tubulin and expressed as percentage of the TbMCP12 abundance present in cells grown in standard MEM-Pros medium containing 0.3 mM glucose and haemin C: Effect of the overexpression (OE) or knockdown (KO) of TbMCP12 on the growth of procyclic T. brucei in the
25
The T. brucei di- and tricarboxylate transporter TbMCP12
presence of oxidative stress. Cells overexpressing (OE) or depleted (KO) of TbMCP12 were grown for 24 h after tetracycline induction/depletion and then treated with H2O2 for 6 h. The survival rate is expressed as percentage of the survival rate of the same cell-line grown without the addition of H2O2. The results are depicted as means of 3 independent experimental replicates and standard deviation is shown.
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To investigate whether the overexpression of TbMCP12 protects the cells from oxidative stress we analysed T. brucei cell growth after the addition of 10 μM H202. Indeed, T. brucei
overexpressing TbMCP12 survived better after exposure to oxidative stress compared to wild type
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cells (Figure 8C). Accordingly, the removal of TbMCP12 decreased the cell´s resistance to H202. DISCUSSION
Using Blast searches we identified TbMCP12 as putative mitochondrial carboxylic acid
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transporter (this paper and [27]). TbMCP12 displayed high sequence similarity to metazoan DICs and OGCs and to plant DTCs (This paper and [27]). Since plant DTCs are the only carriers of the MCF
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that catalyse the mitochondrial import of both dicarboxylates and tricarboxylates we speculated that
A
TbMCP12 has a similar substrate spectrum as the plant carriers. Horizontal gene transfer and the phylogenetic relationship between kinetoplastida and plants, algae or cyanobacteria have been
M
previously discussed in the literature [62-64]. Though these parasites do not possess plastids, they controversial [63, 65-67].
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contain a number of proteins bearing “plant-like” traits, but how these genes have been acquired is still Much information is available on the substrates that MCPs can transport in vitro (for a review see [19]), but relatively little is known on their function in vivo. Based on their in vitro transport
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activity mitochondrial carboxylate transporter were proposed to regulate the cytosolic and mitochondrial concentration of TCA cycle intermediates in dependence of nutrient demand and
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oxidative stress levels [19, 33, 68]. In S. cerevisiae the dicarboxylate carrier is essential for sustaining the import of succinate produced during the glyoxylate cycle, and is necessary for the mitochondrial ATP generation on non-fermentable carbon sources [54]. In the absence of DIC only the supplementation of the growth medium with TCA cycle intermediates can maintain normal growth of
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yeast on acetate [54]. The expression of TbMCP12 successfully restored the growth defect indicating that the carrier transports dicarboxylates into the mitochondrial matrix. However, wild type growth rate could not be fully reached probably because TbMCP12 is targeted to yeast mitochondria with lower efficiency or has different transport activity rates than the yeast DIC. According to the currently available model for the T. brucei energy metabolism [16, 69, 70] the TCA cycle branch going from citrate to oxoglutarate (Figure 9, enzymes number 1 and 2) is not involved in mitochondrial ATP production in procyclic cells [16, 17, 70-72]. Supporting this
26
The T. brucei di- and tricarboxylate transporter TbMCP12
hypothesis are knockout experiments, which showed that aconitase (Figure 9, enzyme number 1) is not essential for procyclic trypanosome survival [17]. Further, the activity of isocitrate dehydrogenase (Figure 9, enzyme number 2) is very low and NADP+-dependant and therefore not linked to mitochondrial ATP production via oxidative phosphorylation [2, 13, 73]. We showed here that ATP is synthesised in isolated procyclic mitochondria using citrate, isocitrate or aconitate as substrates. The amount of ATP was, however, very low compared to the one produced on oxoglutarate and succinate suggesting that the activity of this TCA cycle branch is negligible for T. brucei ATP production and
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survival.
Figure 9. Schematic representation illustrating the potential functions of TbMCP12 in the
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context of the procyclic T. brucei mitochondrial energy metabolism The illustration depicts the key enzymatic steps that are required for the ATP synthesis from the carbon sources glucose and proline and for the generation of the end-products succinate and acetate. TbMCP12 is highlighted in yellow; “a”, “b” and “c” indicate the putative TbMCP12 functions,
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homologue to the mammalian dicarboxylate carrier (“a”), oxoglutarate carrier (“b”) and tricarboxylate carrier (“c”). The aconitase (enzyme 1) reaction produces isocitrate from citrate with cis-aconitate as enzyme-bond intermediate. Aconitase can also use cis-aconitate as substrate regenerating either isocitrate or citrate. Based on the results obtained from the mitochondrial ATP production of wild type cells (Fig. 7A) we have postulated that aconitase is active in procyclic T. brucei cultured in either high or low glucose MEM-Pros. Red arrows: direction of the enzymatic step in high-glucose medium. Dashed red arrows: direction of the enzymatic step in low-glucose medium. Black arrows: direction of
27
The T. brucei di- and tricarboxylate transporter TbMCP12
the enzymatic step in either low- or high-glucose medium. Numbers I-IV indicate respiratory chain complexes. The numbers on the arrows indicate the respective enzyme as follows: 1 – aconitase, 2 – isocitrate dehydrogenase, 3 – oxoglutarate dehydrogenase complex, 4 – succinyl-CoA synthetase, 5 – fumarate reductase, 6 - fumarase, 7 – mitochondrial malate dehydrogenase, 8 – citrate synthase, 9 – pyruvate dehydrogenase complex, 10 - acetate:succinate CoA-transferase, 11 - succinyl CoA synthetase, 12 – mitochondrial malic enzyme, 13 – cytosolic malic enzyme, 14 – L-alanine
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aminotransferase, 15 – ATP-synthase, 16 acetyl-CoA thioesterase.
Depending on the availability of the carbon sources, the reactions of the T. brucei TCA-cycle can work in alternating directions [66]. As shown in Figure 9, the TCA-cycle branch that involves
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succinate dehydrogenase, fumarase and malate dehydrogenase is metabolically flexible and produces malate when glucose is scarce while it consumes malate when glucose is highly abundant. Our
findings support this metabolic model. The ATP production on malate was lower when mitochondria were “primed” on high glucose medium. When glucose is highly abundant, malate is converted within
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a reversed branch of the TCA cycle to fumarate and subsequently to succinate, a reaction that does not
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produce either ATP or redox equivalents (Figure 9, enzyme number 5 and 6) [69, 70]. On low glucose medium, malate is either exported from the mitochondrion or is metabolised by a mitochondrial malic
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enzyme (Figure 9, enzyme number 12) [57]. This reaction produces NADPH and pyruvate, which is
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then metabolised to acetate and ATP (Figure link between functional mitochondrial ATP metabolism and TbMCP12 is highlighted when changes to the carrier´s abundance are made. In isolated mitochondria depletion of TbMCP12 drastically reduced while its overexpression significantly
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augmented the mitochondrial ATP synthesis on citrate, isocitrate and aconitate indicating that the carrier is involved in the transport of tricarboxylates. The detrimental effect of the TbMCP12
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overexpression might be ascribed to alterations of the TCA-cycle flux that cause a depletion of metabolic intermediates or redox equivalents. Whether an increased mitochondrial import or export of dicarboxylates and tricarboxylates is influencing the TCA-cycle flux in response to TbMCP12
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abundance is unclear at the moment, but this protein might represent an attractive “metabolic switch”. NADPH is the central electron source for the synthesis of fatty acids, gluconeogenesis, and the
regeneration of enzymes belonging to the oxidative stress defence system [74]. Differently to other
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eukaryotes, the T. brucei redox metabolism is not based on peroxisomal catalase or the molecule gluthathione but on a trypanothione peroxidase system that is regenerated in a NADPH-dependent manner [75, 76]. In T. brucei there are two main sources of cytosolic NADPH: cytosolic malic enzyme (Figure 9, enzyme number 13) and the pentose phosphate pathway [77]. The two systems coexist and independently sustain the production of NADPH [77]. Cytosolic malic enzyme produces pyruvate and NADPH through the decarboxylation of malate and requires the cooperation with mitochondrial carboxylate carriers for the maintenance of the cytosolic NADPH balance (Figure 11, TbMCP12 “a”).
28
The T. brucei di- and tricarboxylate transporter TbMCP12
In mammals, the dicarboxylate, oxoglutarate and citrate carriers are involved in the so-called citrate shuttle. The citrate shuttle exports mitochondrial citrate to the cytosol where it is converted to malate. The existence of this shuttle in T. brucei was not supported by previous studies [78]. The provision of cytosolic malate must therefore occur exclusively through its direct export from the mitochondrion possibly through TbMCP12 (Figure 9, TbMCP12 “a”). Our results show that TbMCP12 is upregulated under oxidative stress conditions and that the 2-fold overexpression of TbMCP12 slightly improved while the knockout of TbMCP12 reduced the survival of T. brucei under the influence of oxidative
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stress. Our results suggest that the abundance of TbMCP12 might contribute to the regulation of the NAPDH/NADP+ balance. It is possible that increasing the abundance of TbMCP12 acts as a
“metabolic switch” redirecting malate from the mitochondrial (Figure 9, enzyme number 12) to the
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cytosolic malic enzyme (Figure 9, enzyme number 13). Such a system would be more required in
procyclic cells since bloodstream form trypanosomes do not rely on malic enzyme [79] for NADPH production but on the pentose phosphate pathway [57, 58]. The involvement of TbMCP12 in the cellular maintenance of the NAPDH/NADP+ balance, however, requires further elucidation.
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Considering the results presented in this article we propose that TbMCP12 transports dicarboxylates and tricarboxylates across the mitochondrial inner member and is involved in cellular
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oxidative stress defence mechanisms and maintenance of the cytosolic NADPH homeostasis. Further,
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the abundance of TbMCP12 appears to be a critical regulatory element of the mitochondrial ATP-
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AUTHOR CONTRIBUTION
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production.
CC designed and performed all the experiments related with the knockdown and the overexpression in T. brucei, ATP and NADP/NADPH measurements and phylogenetic and sequence analyses (Figures
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1, 2, 3, 5, 6, 7, 9, Table 1 and 2). FZ, CK and FV designed and performed all the experiments related with the yeast complementation and the treatment of T. brucei with hydrogen peroxide and haemin
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(Figures 4 and 8). All authors reviewed the results and contributed equally to the drafting and correction of the manuscript as well as to the design of Figure 9. The final version of the manuscript was approved by all authors.
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CONFLICT OF INTEREST The authors declare that they have no conflicts of interest with the contents of this article. AKNOWLEDGEMENT We thank Christine Clayton for proof-reading the manuscript. This work was funded by the BBSRC, grant number BB/G00448X/1.
29
The T. brucei di- and tricarboxylate transporter TbMCP12
FOOTNOTES MCF: mitochondrial carrier family; TCA: tricarboxylic acid cycle; TbMCP: T. brucei MCF protein; CIT: citrate carrier; DIC: dicarboxylate carrier; OGC: oxoglutarate carrier; DTC: di-tricarboxylate carrier; UCP: uncoupling proteins; CAT: carboxyatractyloside; ROS: reactive oxygen species; NEO:
A
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A
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neomycin; BSD: blasticidin; UTR: untranslated region
30
The T. brucei di- and tricarboxylate transporter TbMCP12
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A.E. Leroux, D.A. Maugeri, J.J. Cazzulo, C. Nowicki, Functional characterization of NADP-
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Z. Verner, S. Basu, C. Benz, S. Dixit, E. Dobáková, D. Faktorová, H. Hashimi, E. Horáková, Z. Huang, Z. Paris, P. Peña-Diaz, L. Ridlon, J. Týč, D. Wildridge, A. Zíková, J. Lukeš, Malleable mitochondrion of Trypanosoma brucei. Int. Rev. Cell Mol. Biol. 315 (2015) 73–151
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P.O. Durieux, P. Schütz, R. Brun, P. Köhler, Alterations in Krebs cycle enzyme activities and carbohydrate catabolism in two strains of Trypanosoma brucei during in vitro differentiation of
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Franconi, P.A.M Michels, J. Portais, M. Boshart, Cytosolic NADPH homeostasis in glucose-
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starved procyclic Trypanosoma brucei relies on malic enzyme and the pentose phosphate pathway fed by gluconeogenic flux J. Biol. Chem. 288 (2013) 18494–18505 L. Rivière, P. Moreau, S. Allmann, M. Hahn, M. Biran, N. Plazolles, J.M. Franconi, M.
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S0166-6851(17)30163-9 https://doi.org/10.1016/j.molbiopara.2018.03.003 MOLBIO 11117
To appear in:
Molecular & Biochemical Parasitology
Received date: Revised date: Accepted date:
1-1-2018 22-2-2018 21-3-2018
Please cite this article as: Colasante Claudia, Zheng Fuli, Kemp Cordula, Voncken Frank.A plant-like mitochondrial carrier family protein facilitates mitochondrial transport of di- and tricarboxylates in Trypanosoma brucei.Molecular and Biochemical Parasitology https://doi.org/10.1016/j.molbiopara.2018.03.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
The T. brucei di- and tricarboxylate transporter TbMCP12
A plant-like mitochondrial carrier family protein facilitates mitochondrial transport of di- and tricarboxylates in Trypanosoma brucei
Running Title: The T. brucei di- and tricarboxylate transporter TbMCP12
Claudia Colasante1#, Fuli Zheng3#, Cordula Kemp2 and Frank Voncken2
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1
Institute for Anatomy and Cell Biology, Division of Medical Cell Biology, Aulweg 123, University of
Giessen, 35392 Giessen, Germany 3
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Department of Preventive Medicine, School of Public Health, Fujian Medical University, 1 Xue Yuan
Road, Fu Zhou, Fujian, P.R. China 2
Department of Biomedical Sciences, School of Life Sciences, University of Hull, Cottingham Road,
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Hull, HU6 7RX, United Kingdom
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# These authors contributed equally to the paper
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To whom correspondence should be addressed: Claudia Colasante, Institute for Anatomy and Cell Biology, Division of Medical Cell Biology, Aulweg 123, University of Giessen, 35392 Giessen,
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Germany; Tel.: 0049 0641 9947116; Fax: 0049 0641 9947109;
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MCF proteins (MCPs) exchange solutes across the inner mitochondrial membrane The human parasite T. brucei possesses 24 genes encoding MCPs TbMCP12 transports carboxylates across the T. brucei inner mitochondrial membrane Overexpression of TbMCP12 is lethal in procyclic T. brucei The abundance of TbMCP12 regulates NADPH balance and mitochondrial ATP-production
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Highlights
ABSTRACT
The procyclic form of the human parasite Trypanosoma brucei harbors one single, large mitochondrion containing all tricarboxylic acid (TCA) cycle enzymes and respiratory chain complexes
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present also in higher eukaryotes. Metabolite exchange among subcellular compartments such as the cytoplasm, the mitochondrion, and the peroxisomes is crucial for redox homeostasis and for metabolic pathways whose enzymes are dispersed among different organelles. In higher eukaryotes, mitochondrial carrier family (MCF) proteins transport TCA-cycle intermediates across the inner mitochondrial membrane. Previously, we identified several MCF members that are essential for T. brucei survival. Among these, only one MCF protein, TbMCP12, potentially could transport dicarboxylates and tricarboxylates. Here, we conducted phylogenetic and sequence analyses and
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The T. brucei di- and tricarboxylate transporter TbMCP12
functionally characterized TbMCP12 in vivo. Our results suggested that similarly to its homologues in plants, TbMCP12 transports both dicarboxylates and tricarboxylates across the mitochondrial inner membrane. Deleting this carrier in T. brucei was not lethal, while its overexpression was deleterious. Our results suggest that the intracellular abundance of TbMCP12 is an important regulatory element for the NADPH balance and mitochondrial ATP-production. Keywords: mitochondrial transport, Trypanosoma brucei, energy metabolism, MCF-proteins,
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dicarboxylate, tricarboxylate
INTRODUCTION
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The order Kinetoplastida is a member of the Excavata supergroup, which branched from the other supergroups, including Archaeplastida and Opisthokonta, at the beginning of eukaryotic
evolution [1]. Kinetoplastida includes a variety of different flagellated protozoa that can be found in disparate environmental niches: from free-living cells to parasitic forms. Of particular medical,
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veterinary and socio-economic interest is the group of obligate parasites belonging to the
Trypanosomatidae family, especially Leishmania and Trypanosoma spp.. According to current
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estimates infections caused by these parasites affect several millions of people each year
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(http://www.who.int). Trypanosoma brucei subspecies cause African trypanosomiasis, a disease that
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affects both humans and cattle and is transmitted through the bite of the Tsetse fly. T. brucei raises high scientific interest also due to some of its biological peculiarities such as the presence of one single large mitochondrion, the compartmentalisation of glycolysis within specialized
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peroxisomes named glycosomes, and the complex mitochondrial DNA network and mRNA editing [2, 3]. During its parasitic life cycle T. brucei undergoes substantial changes in gene expression, which
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allow its survival within the digestive system of the insect vector and the mammalian bloodstream [47]. The various life cycle forms differ in their cellular morphology, surface protein expression and energy metabolism [8-10]. The energy metabolism of T. brucei involves three subcellular
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compartments: the cytoplasm, the glycosomes and the mitochondrion. In bloodstream trypanosomes, net ATP production exclusively occurs in the cytoplasm by substrate level phosphorylation during glycolysis. This generates incompletely oxidized pyruvate, which the parasites excrete without further metabolisation. Accordingly, bloodstream form mitochondria do not contain all canonical enzymes of
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the tricarboxylic acid cycle or electron transport chain [4, 11]. In contrast, procyclic form T. brucei, which multiply in the Tsetse fly midgut, produce ATP in
the cytoplasm by substrate level phosphorylation as well as inside the elaborate mitochondrion by substrate level and oxidative phosphorylation [4, 8, 12, 13]. The substrates for energy production in this life cycle stage are either pyruvate generated during glycolysis or amino acids like proline and threonine [4, 8, 13-15]. In procyclic mitochondria, all TCA-cycle enzymes are present, but they are not functioning as a complete cycle [16-18].
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The T. brucei di- and tricarboxylate transporter TbMCP12
In addition to energy metabolism, the mitochondria of T. brucei, like those of other eukaryotes, are involved in amino acid catabolism, regulation of reactive oxygen species and the biosynthesis of heme [9]. Substrates for all these pathways need to be imported into the mitochondrion, across its outer and inner membrane [19]. The majority of proteins involved in the translocation of metabolites and other small solutes across the mitochondrial inner membrane belong to the mitochondrial carrier family (MCF). MCF proteins (MCPs) provide a link between cytoplasm and mitochondrion by exchanging metabolic intermediates, maintaining the cellular redox balance, and exerting flux control
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on enzymatic reactions [20-24]. Proteins belonging to the MCF can be found in nearly all eukaryotic cells. Their amino acid
sequence can be highly divergent, but all MCPs retain the same tertiary structure, which consists of six
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transmembrane alpha helices connected by hydrophilic loops, with both C- and N-termini exposed to the intermembrane space [22, 25]. MCP sequences contain three tandemly repeated domains of 100 amino acids harbouring a common signature motif [26]. Using these conserved sequence features we previously identified genes encoding 24 different potential MCPs in the genome of T. brucei
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(TbMCPs) [27]. Amongst them was one with similarity to human dicarboxylate (DIC) and oxoglutarate (OGC) carriers [27]. These carriers catalyse the electron-neutral exchange of
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dicarboxylates (malate, succinate, fumarate, oxoglutarate and oxaloacetate) and inorganic phosphate
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(Pi) across the mitochondrial inner membrane, and are involved in severalexchange mechanisms [28]. The identification and characterisation of kinetoplastid transporters involved in shuttling
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metabolites between the mitochondria, glycosomes and cytosol is necessary to assemble the remarkable puzzle of the compartmentalisation of the energy metabolism in these parasites. Recently
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we provided evidence that TbMCPs play a central role in T. brucei. We previously characterised two TbMCPs and showed that depletion of these proteins was detrimental for the survival of T. brucei and affected cellular and nuclear morphology as well as ATP homeostasis [29, 30]. Here we describe the
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molecular and functional analysis of another mitochondrial carrier of T. brucei, namely TbMCP12. Our results suggest that TbMCP12 displays functional traits of plant DTCs and transports both
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dicarboxylates and tricarboxylates. We also report that though in some eukaryotes there are at least three carriers involved in the transport of carboxylates and tricarboxylates, namely the OGC, DIC and the citrate (CIT) carriers, T. brucei fulfils all these functions with TbMCP12. Further, TbMCP12
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appears to be involved in oxidative stress management. MATERIAL AND METHODS Culture and transfection of Trypanosoma brucei Procyclic-form Trypanosoma brucei strain Lister 427, stably expressing the tetracycline (tet) repressor from the plasmid pHD449, was cultured in standard MEM-PROS medium [31] (supplemented with 10 % (v/v) foetal calf serum (FCS, Sigma-Aldrich). Trypanosomes were
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The T. brucei di- and tricarboxylate transporter TbMCP12
transfected with different plasmids and clonal cell lines were selected using antibiotics as previously described by Biebinger et al. [32]. Next to standard (low-glucose) MEM-PROS medium, also ‘highglucose’ medium (i.e. standard MEM-PROS medium supplemented with 10 mM glucose) was used for the different experiments. Phylogenetic reconstruction and sequence analysis Phylogenetic reconstruction was performed using the “Phylogeny.fr” software available at
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http://www.phylogeny.fr. Multiple sequence alignments were obtained using Psi/TM-Coffee and automatically curated using Gblocks. Maximum likelihood tree was constructed using PhyML and
visualized using TreeDyn. Statistical tests for branch support was assessed by bootstrap re-sampling
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analysis generating 100 reiterated data sets. The resulting bootstrap values, expressed as percentage, were added manually to each node. Only bootstrap values above 50% are shown. The NCBI (http://www.ncbi.nlm.nih.gov) accession numbers for the different protein sequences used for the
phylogenetic analysis are listed in Supplementary Table 1. The multiple sequence alignment shown in
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Figure 2 was performed using ClustalΩ (www.ebi.ac.uk).
RNA isolation, northern blotting and RT-PCR
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Total RNA was isolated from 1 x 107 bloodstream- or procyclic-form T. brucei strain 449 cells
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using TriFast (PeqLab Biotechnology GmbH) according to manufacturer´s protocol. For Northern blotting, total RNA (10 μg) was separated by denaturing (formaldehyde) agarose gel electrophoresis and blotted onto Hybond-N membrane (GE Healthcare). A [32P]-dCTP labelled TbMCP12 ORF (open
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reading frame) DNA probe was generated by PCR using the primer pair 5’GGACGGGTTAACACCATGGCGAAAGAGACAAAGGCGCCCG-3’ (forward) and 5’-
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GCTTGCAGGATCCCTGCTTTGCCTGAAGCTTGGCG-3’ (reverse). Blots were pre-hybridized in
hybridization solution (5x SSC, 5x Denhardt´s reagent and 0.5 % w/v sodium dodecyl sulphate (SDS)) for 1 h at 65 °C after which the [32P]-dCTP labelled DNA probe was added. After over-night
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hybridization at 65 °C the blots were washed at 65 °C in 1x SSC (0.15 M NaCl, 0.015 M sodium citrate) supplemented with 0.1 % w/v SDS, then with 0.1x SSC supplemented with 0.1 % w/v SDS, followed by final exposure to X-ray film (Kodak). RNA from yeast was isolated using the Direct-zol™ RNA MiniPrep kit according to the
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manufacturer´s protocol with some modification. Briefly, yeast cells were treated first with lyticase (10 u/OD) to break down the cell wall and the formed spheroblasts were then lysed using 300 μl TRI reagent. The mixture was transferred into a Zymo-Spin™IIC Column and spun at 13,000 x g for 30 sec followed by a washing step with 400 μl RNA Wash Buffer. After washing, 5 μl DNase I (6 u/μl) in 75 μl DNA Digestion Buffer were added to the column followed by incubation at 25 °C for 15 min. After 2 consecutive washing steps with 400 μl Wash Buffer the column was dried by centrifugation and the RNA eluted with 50 μl of DNase/RNase-free water.
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The T. brucei di- and tricarboxylate transporter TbMCP12
For cDNA synthesis 2 μg yeast total RNA in 10 μl Rnase-free water were mixed with 4 μl 5x M-MuLV reaction buffer, 1 μl RiboLock RNase inhibitor, 2 μl 10 mM dNTP, and 2 μl M-MuLV Reverse transcriptase (cDNA synthesis kit, Thermo). The reaction was incubated at 25 °C for 5 min, followed by 60 min at 37 °C. Then the reaction was terminated by incubation at 70 °C for 5 min. RTPCR for the detection of the TbMPC12 and dicarboxylate carrier (DIC) expression in yeast was performed using 1 μl of synthesised cDNA to set up a 25 μl PCR reaction. For the analysis the primers 3’-GGGGATCCATGGCGAAAGAGACAAAGGCGC-5’ (forward) and 3’-
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GCTCACTGCAGTTATCACTGCTTTGCCTGAAGCTTGGCG-5’ (reverse) were used to amplify
TbMCP12 and the primers 3’-GAGGATCCATGTCAACCAACGCAAAAGAGTCTG-5’ (forward) and 3’-
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CTGCGGCCGCCTACTTGTCTTCCTTTGGCATGC-5’ were used to amplify DIC.
Overexpression of TbMCP12 in procyclic forms
The open reading frame (ORF) of TbMCP12 was PCR-amplified from T. brucei strain Lister 427 genomic DNA using the sense primer 5’-
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GGACGGGTTAACACCATGGCGAAAGAGACAAAGGCGCCCG-3’ and the antisense primer 5’GCTTGCAGGATCCCTGCTTTGCCTGAAGCTTGGCG-3’. The restriction enzyme sites Hpa I (forward
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primer) and Bam HI (reverse primer) used for the subsequent cloning are underlined. The resulting
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PCR product was initially cloned into the pGEM-T Easy TA-cloning vector (Invitrogen) and sequenced (Eurofins Genomics, Wolverhampton, United Kingdom). Comparison of the cloned
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TbMCP12 sequence from T. brucei Lister 427 with the sequence of the corresponding locus Tb927.10.12840 (initially annotated as Tb10.389.0690) (http://www.genedb.org) of T. brucei strain
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927 revealed a few sequence differences at the DNA level but none in the predicted amino acid sequence. The TbMCP12 ORF was subsequently cloned into the trypanosome expression vectors pHD1700 and pHD1701 [27, 29] using the restriction enzyme sites Hpa I and Bam HI introduced
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during PCR. Tetracycline-inducible expression from these vectors results in the expression of the desired protein with a double-myc tag at the carboxy-terminal- (cmyc, pHD1700) or amino-terminal-
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(nmyc, pHD1701) end. The resulting plasmids were used for transfection of procyclic-form T. brucei constitutively expressing the tet-repressor (pHD449, TETR BLE). Trypanosome clones resistant to hygromycin and bearing a tetracycline inducible (ti) and myc-tagged copy of TbMCP12 were isolated by serial dilution and the obtained clonal cell lines analysed by western blotting using an anti-myc
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antibody (Roche Applied Science). The genotype of the resulting cell line is TbMCP12/TbMCP12 TETR BLE TbMCP12-mycti HYG, which will be further referred to as TbMCP12 over-expressing cellline Generation of the conditional TbMCP12 double-knockout cell line The TbMCP12 over-expressing cell line was used as starting point for the generation of a TbMCP12 knockout cell line. Tetracycline (0.5 mg/ml) was added every 24 h to the culture medium to
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The T. brucei di- and tricarboxylate transporter TbMCP12
ensure that the TbMCP12-cmyc was expressed throughout the whole knockout procedure. The two natural TbMCP12 alleles were deleted from the genome of the TbMCP12 over-expressing cell line by successive replacement with the neomycin (NEO) and blasticidin (BSD) antibiotic resistance cassettes, and using the flanking 5’-untranslated region (5’-UTR) and 3’-untranslated region (3’-UTR) of TbMCP12 as target sequences for homologous recombination, as previously described. Sense primer 5’-GCTAGAGCTCCGCGACCTTTGACCGAAGTAACTGCC-3’ and antisense primer 5’GCTAACTAGTTCTTCCTGTCGTTCGATCCTCGAGTAG-3’ were used to PCR-amplify a 500-bp
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genomic fragment located upstream of the TbMCP12 ORF (5’ UTR), and sense primer 5’GCATGGATCCTGTGAATAAATCACTGCGTCACGTTATG-3’ and antisense primer 5’-
CAGTGGGCCCTATATATGAAGTCAAAGTGAGAAAAACAGAG-3’ were used to amplify a 460-bp
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fragment downstream of the TbMCP12 ORF (3’ UTR). The restriction enzyme sites (Sac I, Spe I, Bam HI, and Apa I) included in the PCR-primers and used for subsequent cloning are underlined. As previously described for the construction of other T. brucei double-knockout cell lines [30] using the targeted gene replacement method, the 5’- and 3’-UTRs of TbMCP12 were inserted on either side of
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NEO and BSD genes bearing actin 5’-splice sites and actin 3’-UTRs. The resulting NEO- and BSDbearing double-knockout (NEO-dKO, BSD-dKO) constructs were subsequently used for successive
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transfection of the TbMCP12 over-expressing cell line. After transfection with the NEO-dKO
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construct and selection with 15 mg/ml G418, several procyclic T. brucei cell lines were isolated bearing a single-allele knockout of TbMCP12 with the genotype ∆TbMCP12::NEO/TbMCP12/
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TbMCP12-cmycti. Subsequent transfection of the obtained half-knockout cell line with the BSD-dKO construct and selection with 15 mg/ml G418 and 10 mg/ml blasticidin resulted in the isolation of the
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TbMCP12 double-knockout cell line ∆TbMCP12::NEO/∆TbMCP12::BSD/TbMCP12-cmycti, in this paper further referred to as TbMCP12 knockout. Correct genomic integration of both the NEO-dKO and BSD-dKO constructs and effective replacement of the two natural TbMCP12 alleles were
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confirmed by Southern blot and western blot analysis.
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Western blot analysis
For each lane, 2 × 106 trypanosomes were pelleted, resuspended in Laemmli buffer and then
denatured for 5 min at 95 °C. Proteins were separated on a denaturing 12 % polyacrylamide gel containing SDS (SDS-PAGE), and were subsequently transferred to a Hybond-P membrane (GE
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Healthcare Life Sciences) in transfer buffer (39 mM glycine, 48 mM Tris-base, 20 % v/v methanol, pH 8.3) for 1 h at 100 V. The membrane was blocked by incubation in Tris–buffered saline containing 0.2 % (v/v) Tween 20 (TBS-Tween) and 7.5 % (w/v) non-fat dry milk for 30 min at room temperature with gentle shaking, and subsequently incubated for 1 h with 7.5 % milk TBS-Tween containing the primary antibody. The membrane was then washed once for 15 min, and twice for 5 min in TBSTween, followed by incubation for 45 min at room temperature with the relevant secondary antibody (GE Healthcare Life Sciences) (dilution 1:1,000 for all used secondary antibodies). Finally, the
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The T. brucei di- and tricarboxylate transporter TbMCP12
membrane was extensively washed once for 15 min, and four times for 5 min in TBS-Tween and processed according to the manufacturers protocol of the ECL detection kit (GE Healthc´are Life Sciences), followed by exposure to ECL-film (GE Health Care Life Sciences). TbMCP12 N-term peptide antibody generation Peptide synthesis and immunisation of rabbits were performed by EZBiolab (USA). The synthesised N-terminal peptide ‘KETKAPANAPLPKVY‘ (amino acid residues 3-17 of TbMCP12)
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was coupled to keyhole limpet hemocyanin (KLH) and used for the immunisation of two rabbits. After collection of 2 ml pre-immune serum, immunisation was initiated by the injection of each rabbit with
1 mg KLH-coupled peptide emulsified in complete Freunds adjuvant. The first injection was followed
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by 3 subsequent boosts in weeks 2, 4 and 7, respectively, with 0.5 mg of KLH-coupled peptide
emulsified in incomplete Freund´s adjuvant. The final TbMCP12 antiserum was collected in week 9, after determination of the TbMCP12 antibody titer: i.e. 1:1,536,000 (determined by EZBiolab, USA). Western blot analysis confirmed the absence of cross-reacting protein bands in the pre-immune sera
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(not shown), whereas the final antiserum detected the single 32 kDa protein band representing
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TbMCP12.
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T. brucei growth analysis
For the growth analysis of procyclic cells, either standard glucose (0.3 mM, “low-glucose
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medium”), glucose supplemented (10 mM, “high-glucose medium”), or glucose depleted MEM-Pros medium were used. For the depletion of the glucose from the medium extensive dialysis (64,000-fold)
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of the FCS using 10,000 Da molecular mass cutoff tubing was performed prior to addition to the incomplete medium. The dialysed FCS was then mixed 1:10 with non-dialysed FCS to preserve a low level of essential cofactors present in FCS, which are required for procyclic cell growth. At the
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beginning of the growth analysis procyclic trypanosomes were diluted to a density of 0.25 x 106 cells/ml and bloodstream trypanosomes to a density of 0.1 x 106 cells/ml; cells were then counted
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every 24 h for a total of 72 h using a Neubauer hemocytometer. Expression of TbMCP12-cmyc in the TbMCP12 over-expressing cell line was induced by the addition of 0.5 mg/ml tetracycline to the culture medium at time point 0. The TbMCP12 knockout cells were grown in constant presence of tetracycline to maintain TbMCP12-cmyc expression. To achieve a non-induced (TbMCP12-depleted)
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condition, TbMCP12 knockout cells were washed three times with excess culture medium for complete withdrawal of tetracycline (TbMCP12-depleted knockout cells) 24 h prior to the start of the growth experiment. Immunofluorescence microscopy Trypanosomes were centrifuged from the culture medium at 2,000 x g and resuspended in phosphate-buffered saline (PBS) containing 4 % (w/v) paraformaldehyde. Fixed cells were allowed to
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The T. brucei di- and tricarboxylate transporter TbMCP12
settle down and attach to poly-L-lysine-coated microscope slides. Immunofluorescent labelling of trypanosomes with 4’,6’-diamidino-2-phenylindole (DAPI), the mitochondrion-specific probe MitoTracker, and the different antibodies was performed as previously described [27]. Cells were examined using a Leica DM RXA digital de-convolution microscope, and images were recorded using a digital camera (Hamamatsu). T. brucei subcellular fractionation
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Trypanosomes were grown to a maximum cell density of 5 x 106 cells/ml. Approximately 9 x 109 cells were harvested by centrifugation for 10 min at 2,000 x g, and washed once in 50 ml of TEDS (25 mM Tris, 1 mM EDTA, 1 mM DTT, 250 mM sucrose, pH 7.8). After centrifugation for 10 min at
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2,000 x g, the cell pellet was resuspended in 2 ml of homogenization medium (250 mM sucrose, 1 mM EDTA, 0.1 % v/v ethanol, 5 mM MOPS, pH 7.2) supplemented with ‘complete EDTA-free’ protease inhibitor cocktail (Roche Applied Science), and grinded in a pre-chilled mortar at room temperature on ice with 1 volume of wet-weight silicon carbide (Crysalon, Norton Company, porous <400 mesh)
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until at least 90 % cell disruption. Cell disruption was checked under the light microscope. The obtained cell lysate was centrifuged at 3,000 x g for 5 min to remove abrasive and nuclei. The
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obtained supernatant was centrifuged at 17,000 x g for 15 min at 4 °C to yield the light-mitochondrial
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fraction, which was subsequently resuspended in 3 ml homogenization buffer. A 32 ml linear 20-40 % (v/v) Optiprep (iodixanol-sucrose) gradient, mounted on top of a 3.5 ml 50 % (v/v) Optiprep cushion,
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was prepared according to the manufacturer´s protocol (Optiprep Application Sheet S9, Axis-shield). Centrifugation was performed at 105,000 x g for 1 h at 4 °C using a Sorvall SW-60 Rotor. Aliquots of
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1 ml were collected from the bottom of the tube by puncture. An equal volume of each fraction was TCA precipitated and the obtained protein pellet was resuspended in denaturing Laemmli SDS-PAGE
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buffer. Proteins were separated on a 12 % SDS-PAGE gel and analysed by western blotting. Measurement of NADP+/NADPH ratios
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NADP+/NADPH ratios were determined in trypanosome cell lysates using the
NADP+/NADPH Assay Kit (Abcam) and according to the manufacturer’s protocol. Briefly, 107 cells were lysed in 800 μl of NADP+/NADPH extraction buffer by performing two freeze/thaw cycles. The lysate was then centrifuged at 13,000 x g for 5 min and the supernatant transferred to a fresh tube to
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obtain the total NADP Sample (NADPt Sample). For the measurement of NADPH, NADP+ was decomposed by heating 200 μl lysate to 60 °C for 30 min (NADPH Sample). From both samples 10 μl were incubated with 100 μl reaction mix (98 μl NADP+ cycling buffer and 2 μl NADP+ cycling enzyme mix) and incubated at room temperature for 5 min to convert NADP+ to NADPH. For background detraction a reaction containing only extraction buffer and reaction mix was set up. NADPH was then determined in all three reactions by addition of 10 μl NADPH developer followed by incubation at room temperature for 1 h and measurement of the OD450. The NADPH amount in
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The T. brucei di- and tricarboxylate transporter TbMCP12
each sample was determined from the obtained OD450 after subtraction of the background using a NADPH standard curve. The NADP+/NADPH ratio was calculated as follows: (NADPt - NADPH)/NADPH
Mitochondrial ATP production assays ATP production assays were performed as previously described by Schneider et al. [33]. Trypanosomes (108) were collected by centrifugation at 1,500 x g for 10 min after 72 h of culture. The
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cell pellet was washed once with SoTE-buffer (20 mM Tris-HCl, 2 mM EDTA, 0.6 M Sorbitol, pH
7.5), and resuspended in 1 ml of SoTE-buffer containing 0.008 % (w/v) digitonin. Permeabilisation of the trypanosome plasma membrane with digitonin was allowed to take place for exactly 5 min on ice,
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followed by immediate centrifugation for 3 min at 8,000 x g and 4 °C. The supernatant was removed and the pellet washed twice with 1 ml SoTE-buffer. The pellet containing the mitochondria-enriched fraction was resuspended in 0.5 ml assay buffer (20 mM Tris-HCl pH 7.4, 15 mM KH2PO4, 0.6 M Sorbitol, 5 mM MgSO4). Mitochondrial ATP production was initiated by the addition of 75 μl assay
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buffer containing 67 μM ADP and 5 mM of the different substrates (i.e. oxoglutarate, succinate,
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malate, citrate, isocitrate or aconitate) to 75 μl of mitochondria-enriched fraction and was allowed to take place for 30 min at 25 °C. The mitochondrial ATP-production was terminated by the addition of
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10 μl TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), followed by denaturation at 100 °C for 3 min. The
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formed protein precipitate was removed by centrifugation for 1 min at 16,000 x g. The ATP concentration in the supernatant was measured using the ATP Bioluminescence Assay Kit CLS II kit
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(Roche Applied Science) and the Junior LB9509 tube luminometer (Berthold Technologies). Yeast functional complementation
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Saccharomyces cerevisiae strains used in this study were the parental strain BY4741 (Mat a his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) and the derived DIC knockout strain YLR348C (ΔDIC1; MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) (EUROSCARF, Frankfurt Germany). The TbMCP12 ORF was
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PCR-amplified from T. brucei strain Lister 427 genomic DNA using the sense primer 5’GGGGATCCATGGCGAAAGAGACAAAGGCGC-3’ and the antisense primer 5’GCTCACTGCAGTTATCACTGCTTTGCCTGAAGCTTGGCG-3’. The ORF of the S. cerevisiae DIC was
amplified from S. cerevisiae cDNA using the sense primer 5´-
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GAGGATCCATGTCAACCAACGCAAAAGAGTCTG-3´ and antisense primer 5´CTGCGGCCGCCTACTTGTCTTCCTTTGGCATGC-3´. The restriction enzyme sites (Bam HI and Pst I
for TbMCP12 and Bam HI and Not I for DIC) included in the PCR-primers and used for subsequent cloning are underlined. The resulting PCR products were initially cloned into the pGEM-T Easy TAcloning vector (Invitrogen) and sequenced (Eurofins Genomics, Wolverhampton, United Kingdom) to confirm that the ORFs were correct. The open reading frames were subsequently cloned into the 2
9
The T. brucei di- and tricarboxylate transporter TbMCP12
micron episomal yeast expression vector pCM190 (EUROSCARF, Frankfurt, Germany) using the restriction enzyme sites introduced during PCR. Yeast parental, DIC knockout and derived strains were maintained on standard YPD medium (1 % of yeast extract, 2 % of peptone, 2 % of glucose, and 2 % of agar). In order to test cell growth under different carbon sources the cells were grown in either YPD (containing glucose) or YPG (containing glycerol) medium. Plasmids containing target genes were transfected into the ∆DIC strain according to the lithium acetate/single-stranded carrier DNA method described by Gietz and Woods
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[34]. For clone-selection after transfection, medium without uracil, containing synthetic complete (SC) medium (0.67 % of yeast nitrogen base (w/o amino acids), 1.4 % of drop-out medium supplements (w/o histidine, leucine, tryptophan and uracil), supplemented with 60 mg/L of leucine, 20 mg/L of
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tryptophan, 20 mg/L of histidine, 2 % of agar and 2 % of dextrose) was applied (Sigma). Resulting
yeast clones were maintained on YPD media without uracil with tetracycline. Heterologous protein expression in yeast was induced by tetracycline removal.
For growth experiments a starter culture was inoculated with yeast cells and grown at 28 °C
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overnight. The next day, the absorbance of the cells was measured at 600 nm (OD600) and cultures were diluted to OD600 0.2 in 20 ml medium and grown for 24 h on glucose medium and 96 h on acetate
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medium. RESULTS
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The genome of Trypanosoma brucei contains a plant-like di-tricarboxylate carrier We previously used the conserved sequence and structural features of mitochondrial carriers
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to identify all members of the MCF in the kinetoplastid parasite T. brucei [29]. To find kinetoplastid MCPs involved in the exchange of dicarboxylates, tricarboxylates and oxodicarboxylates, we used the deduced amino acid sequences of the equivalent, functionally characterised metazoan MCPs [28, 35-
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40] (www.ncbi.nlm.nih.gov) as BLASTP queries against protein databases of T. brucei, Leishmania major and Trypanosoma cruzi (www.genedb.org). Searches using either the sequence of the
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tricarboxylate carrier SLC25A1 (CIT) [40] or of the oxodicarboxylate carrier SLC25A21 [41] as query, did not identify kinetoplastid homologues. In contrast, BLASTP mining using the dicarboxylate carrier SLC25A10 (DIC) [42] or the oxoglutarate carrier SLC25A11 (OGC) [37, 39], resulted in the identification of one single gene in the T. brucei genome database: TbMCP12 (Tb927.10.12840). In
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the T. cruzi genome we found two TcMCP12 genes encoding proteins with 63% identity to TbMCP12 (Table 1), while remarkably, in the L. major genome we found 8, tandemly arranged LmMCP12 genes (55% identity; LmMCP12A-H, Table 1). The presence of multiple genes (up to 3 copies) in the genome of this parasite was previously described for other MCP genes [27]. Reciprocal BLASTP searches of the NCBI gene databases (http://www.ncbi.nlm.nih.gov) using TbMCP12 as sequence query predominantly retrieved oxoglutarate carriers from different eukaryotes, including the functionally characterised mitochondrial OGC (NP_777096) from Bos taurus [37, 39].
10
The T. brucei di- and tricarboxylate transporter TbMCP12
Table 1: TbMCP12 homologues identified in T. cruzi and L. major Accession number
Protein lenght
Conservation
Identity
T. brucei
Tb927.10.12840
304
-
-
T. cruzi
TcCLB.503939.20
326
75%
63%
TcCLB.509805.190
326
75%
63%
LmjF.18.1260
320
71%
56%
LmjF.18.1300
308
71%
LmjF.18.1280
316
70%
LmjF.18.1265
316
LmjF.18.1270
316
LmjF.18.1275
316
LmjF.18.1285
316
LmjF.18.1290
316
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55%
70%
55%
70%
55%
70%
55%
70%
55%
70%
55%
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56%
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L. major
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Species
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Since conventional DIC and OGC do not transport tricarboxylates, we wanted to identify the transporter that can take over this function in T. brucei. In vitro reconstitution in liposomes and
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metabolite transport experiments showed that the mitochondrial dicarboxylate-tricarboxylate carrier (DTC) from Arabidopsis thaliana is able to transport both dicarboxylates (such as oxoglutarate, malate and oxaloacetate) and tricarboxylates (such as citrate, isocitrate and cis-aconitate) [38]. So far this type
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of MCF transporter has only been described in plants and Plasmodium falciparum [38, 43], the causative agent of malaria tropica. Interestingly, BLASTP analysis of the T. brucei protein database using the A. thaliana DTC amino acid sequence as query only retrieved TbMCP12 as top hit with significant sequence conservation (48%).
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The BLASTP-predicted function of TbMCP12 as either oxoglutarate/malate or dicarboxylate/tricarboxylate exchanger was further investigated by phylogenetic reconstruction using oxoglutarate, dicarboxylate and dicarboxylate-tricarboxylate carrier amino acid sequences from representative eukaryotes (Figure 1). The phylogenetic tree showed that the carboxylate carriers grouped in well-defined clades depending on their transport functions (Figure 1). TbMCP12 and the homologuous sequences from T. cruzi and L. major branched into a separate clade, which was supported by high bootstrap values (Figure 1). However, the bootstrap values do not support an
11
The T. brucei di- and tricarboxylate transporter TbMCP12
association of TbMCP12 to any of the other carboxylate carrier groups. It is worth noticing that the
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DTC of P. falciparum, in contrast to TbMCP12, clusters within the clade of the plant DTCs.
clade near DTCs and OGCs.
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Figure 1. In the phylogenetic tree TbMCP12 and related MCF proteins cluster in a separate
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The maximum likelihood tree is showing the evolutionary relationship between TbMCP12 and DTCs (green), OGCs (blue) and DICs (yellow) of the MCF. Colors have been applied to the phylogenetic tree to indicate clustering of carriers with the same transport function. The bootstrap values located at
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the nodes represent the percentages obtained after resampling analysis of 100 reiterated data sets. Only significant bootstrap values (≥ 50 %) are shown. The kinetoplastida clade is boxed in red. Asterisks
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indicate MCF proteins that have been already functionally characterised.
Residues involved in protein folding and substrate binding are conserved in TbMCP12
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The deduced amino acid sequence of TbMCP12 consists of 304 amino acid residues, which corresponds to a predicted molecular weight of approximately 33 kDa (http://www.genedb.org). TbMCP12 exhibits all conserved amino acid sequence and structural characteristics of MCPs: it consists of three domains of about 100 amino acids, each containing two transmembrane (TM) alphahelices and the canonical MCP signature sequence ‘Px[D/E]x2[K/R]x[K/R]x20-30[D/E]Gx45[W/F/Y][K/R]G’
(Figure 2A) [22, 27, 28]. The first part of the motif ‘Px[D/E]x2[K/R]x[K/R]’ is
located at the carboxy-terminal end of TM helices H1, H3 and H5, whereas the second part ‘[D/E]Gx4-
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The T. brucei di- and tricarboxylate transporter TbMCP12
5[W/F/Y][K/R]G’
is located after the amphipathic helices h1-2, h3-4 and h5-6 (Figure 2A). The
conservation of the signature motif in TbMCP12 was analysed in comparison to DTCs from different plants and with metazoan OGCs (Figure 2B). Most amino acids of the first part of the signature motif, indicated as M1a, M2a, and M3a, are conserved including: i) the proline found at the start of M1a, M2a and M3a; ii) the acidic amino acid residue, either an aspartic acid or glutamic acid, at position 3, and iii) the positively charged amino acid, either a lysine or an arginine, at position 6, with exception of M2a, in which this residue is exchanged for a leucine in all analysed sequences (Figure 2B). In M2a
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also the lysine or arginine at position 8 was exchanged for a leucine.
Figure 2. TbMCP12 displays all the amino acid sequence features of MCF conventional carriers
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and contain functional residues conserved in DTCs and OGCs A: Schematic representation of MCPs. The protein structure consists of six transmembrane helices (H1-6) linked by hydrophilic loops (h1-2, h3-4 and h5-6). M1a, M2a, and M3a indicate the first segment of the signature sequence motif, Px(D/E)xx(K/R)x(K/R), located at the end of the odd-
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numbered transmembrane helices while M1b, M2b, and M3b indicate the second part of the motif, (D/E)Gxn(K/R)G, located at the end of each hydrophilic loop. B: Multiple sequence alignment of TbMCP12 and different plant DTCs and OGCs. Amino acid sequences were aligned using ClustalΩ. TbMCP12 residues identical to residues in either DTCs or OGCs are shaded in yellow. TbMCP12 residues with conserved substitutions in DTC and OGC are shaded in orange. Conserved contact points are color-coded as follows: CPI, green; CPII, blue; CPIII, red.
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The T. brucei di- and tricarboxylate transporter TbMCP12
In contrast to the first part of the signature motive, the second part is partially conserved (Figure 2: M1b, M2b, and M3b). The first two amino acids of the motif, i.e. ‘(D/E)G’, are not always present in the T. brucei sequence. The final glycine (‘G’), which allows flexibility in the arm that links the two helices [44], and the aromatic residue at position 7, on the other hand, are highly conserved except in M3b in which the G is missing (Figure 2B). Previous analyses of the structure of the bovine ATP/ADP carrier showed that groups of
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conserved amino acids located downstream of each signature motif participate in substrate discrimination, recognition and binding [45, 46]. From this structural information three conserved substrate-contact points called CPI, CPII and CPIII were extrapolated for all MCPs transporting
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similar substrates [45, 46]. As shown by the amino acid sequence alignments with DTCs and OGCs all three contact points typical for these two carrier types are conserved and present in TbMCP12 (Figure 2B).
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TbMCP12 is predominantly expressed in procyclic-form T. brucei
Previously published northern blot analyses indicated that the expression of some TbMCPs is
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life-cycle stage dependent [27, 29, 30]. Northern blot analysis using the TbMCP12 ORF as a probe
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revealed a single cross-hybridizing mRNA band of approximately 2.4 kb in both bloodstream-form and procyclic-form T. brucei (Figure 3A). The observed size-length of this mRNA is similar to the in
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silico predicted TbMCP12 mRNA size of 2.5 kb, when assuming that trans-splicing occurs at position -147 relatively to the ATG and that polyadenylation occurs 1.3 kb downstream of the stop codon TAG
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[47]. Northern blot analysis revealed that the expression of TbMCP12 is higher in procyclic- than in bloodstream-form T. brucei (Figure 3A). Previous publications showed that during the differentiation from bloodstream to procyclic form the amount of TbMCP12 mRNA is increased [48]. The up-
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regulation of the expression of TbMCP12 in the procyclic form was further confirmed at the protein level by western blot analysis (Figure 3B). Antibodies against the N-terminus of TbMCP12 detected a
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single band of the predicted molecular mass of 32 kDa in both bloodstream- and procyclic-form T. brucei (Figure 3B). After quantification using -tubulin as control [49] the results showed, as expected, that TbMCP12 is far more abundant in the procyclic form (Figure 3B). This result corresponds well with previously published proteomic results indicating that the protein is higher
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expressed in the procyclic stage but is already upregulated in the short stumpy form [50, 51]. These results are consistent with the expectation that the transport function of TbMCP12 should be more required in the life cycle stage that displays higher mitochondrial energy metabolism activity.
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The T. brucei di- and tricarboxylate transporter TbMCP12
Figure 3. TbMCP12 is differentially expressed in bloodstream and procyclic T. brucei and localised in the mitochondria of procyclic cells
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A: Northern blot analysis of 10 μg total RNA from bloodstream (BS) and procyclic (PF) T. brucei
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using [a-32P]dCTP-labelled TbMCP12 DNA as hybridization probe. A [⍺-32P]dCTP-labelled signal
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recognition particle (SRP) hybridization probe was used as loading control B: Western blot analysis of
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2 x 106 wild type BS and PF T. brucei cells using the antibody directed against TbMCP12. An antibody against tubulin [49] was used as a loading control. Antibodies were used in 1:1,000 dilutions. C: Western blot analysis of procyclic T. brucei post-nuclear cell lysate fractions (10 μg/lane) separated
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on an Optiprep (iodixanol-sucrose) gradient. Fractions containing the mitochondria (Mito) were identified using an antibody against LipDH [52], those containing the glycosomes (Gly) with an
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antibody directed against PEX11 [53]. TbMCP12 was detected using the TbMCP12 antibody. Fraction 1 indicates the bottom fraction 33 the top of the gradient. D: Immunofluorescence analysis of wild type procyclic form T. brucei. TbMCP12 (green) was visualized using the TbMCP12 antibody (diluted
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1:500) and mitochondria (red) were stained using Mitotracker. Nuclear and kinetoplast DNA (blue) were stained with DAPI.
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TbMCP12 is exclusively located in the mitochondrion of procyclic T. brucei The subcellular localisation of TbMCP12 in procyclic-form T. brucei was determined by cell
fractionation followed by western blotting, and by immunofluorescence microscopy. TbMCP12 cosedimented (fractions 23-27) exclusively with the mitochondrial marker protein dihydrolipoamide dehydrogenase (Figure 3C: gradient fractions 23-29). No TbMCP12 was detected in fractions 9-11, containing the glycosomes (visualized using an antibody directed against the glycosomal marker PEX11). By immunofluorescence microscopy TbMCP12 displayed the typical mitochondrial staining
15
The T. brucei di- and tricarboxylate transporter TbMCP12
pattern, overlapping with the mitochondrial marker MitoTracker (Figure 3D). Analysis of the subcellular localisation of TbMCP12 in bloodstream cells using the antibody directed against TbMCP12 was not possible, due to low expression of the protein TbMCP12 complements the transport function of the yeast dicarboxylate carrier Sequence analysis indicates that of all identified TbMCPs, TbMCP12 is the transporter that displays highest similarity to the yeast dicarboxylate carrier DIC. The function of TbMCP12 as a
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putative dicarboxylate transporter was investigated by functional complementation analysis using a S. cerevisiae DIC knockout strain (DIC). Yeast lacking the dicarboxylate carrier is unable to grow on non-fermentable carbon sources as sole carbon source [54].
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We compared the growth of the S. cerevisiae parental (wild type) and DIC knockout strains after transfection with either the empty pCM190 plasmid (negative control) or the pCM190 plasmid containing the TbMCP12 ORF on glucose and acetate. The absence of DIC in the yeast DIC knockout strain and the successful expression of TbMCP12 after transformation were confirmed by
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RT-PCR (Figure 4A). For DIC yeast transfected with either empty or TbMCP12-containing
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plasmids, no growth difference was seen in the presence of the fermentable carbon source glucose (Figure 4B). With the non-fermentable carbon source acetate, DIC yeast grew to lower density in the
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same amount of time compared to wild-type yeast (Figure 4C). Growth of DIC yeast could however
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be restored to almost wild type levels by the expression of recombinant TbMCP12 (DIC + TbMCP12) (Figure 4C). These results suggest that the transport function of TbMCP12 overlaps with
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the one of the S. cerevisiae dicarboxylate carrier. We were further interested to investigate whether TbMCP12 is involved in the transport of tricarboxylic acids by complementing the corresponding yeast knockout strains. However, the deletion of the yeast citrate carrier (YBR291C) does not cause a
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lethal phenotype [55] and deletion of the citrate-oxoglutarate carrier (YMR241w) causes only a very mild phenotype when the yeast is grown in the presence of hydrogen peroxide [56]. We therefore
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deemed these knockout strains unsuitable for the purpose of further complementation studies.
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The T. brucei di- and tricarboxylate transporter TbMCP12
Figure 4. Functional complementation studies show that TbMCP12 can complement the function
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of DIC in S. cerevisiae knockout strains
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A: RT-PCR was used to analyse the expression of TbMCP12 and DIC in the different yeast strains. RNA was isolated from wild type (WT), DIC-deficient strain transfected with empty pCM190 (DIC)
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and DIC transfected with either TbMCP12 (DIC + TbMCP12) or the yeast dicarboxylate carrier (DIC + DIC). As a control 100 ng purified plasmid containing either DIC or TbMCP12 was used as
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template. B and C: Growth of the wild type (WT) S. cerevisiae and DIC-deficient yeast strain transfected with either empty pCM190 (DIC) or pCM190 containing TbMCP12 (DIC +
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TbMCP12). Yeast cell lines were isolated and inoculated in medium containing either glucose or acetate and the OD600 was measured after respectively 24 h and 96 h. The results are depicted as
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means of 3 independent experimental replicates and standard deviation is shown.
TbMCP12 is not essential for procyclic form T. brucei but its overexpression causes a
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growth-defect
To avoid incomplete down-regulation of TbMCP12 following RNAi, we produced a
conditional double TbMCP12 knockout in procyclic T. brucei by generating first a TbMCP12 overexpressing cell line (Figure 5). Western blot analysis of this cell line using a myc-tag antibody revealed a 35 kDa protein band representing the recombinant TbMCP12-cmyc protein after tetracycline induction (Figure 5A). Using the TbMCP12 antibody the endogenous TbMCP12 (32 kDa) was detected in both the non-induced and induced TbMCP12 over-expressing cell line (Figure 5A). Tagging at either the N- or the C-terminus did not affect mitochondrial localisation of the recombinant
17
The T. brucei di- and tricarboxylate transporter TbMCP12
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TbMCP12-cmyc protein (Figure 5B).
Figure 5. Overexpression of TbMCP12 in procyclic T. brucei is detrimental independently of the
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glucose concentration present in the medium A: Expression analysis of endogenous TbMCP12 and TbMCP12cmyc in wild type (WT) and tetracycline (tet)-induced and uninduced TbMCP12 over-expressing (OE) cell line. Each lane contains
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2 x 106 cells. Proteins were detected using 1:1,000 dilutions of antibodies directed against TbMCP12, myc-tag or aldolase (loading control). B: Immunofluorescence analysis of tet-induced TbMCP12 OE cell line. TbMCP12cmyc and TbMCP12nmyc (green) were detected using an anti-myc-tag antibody (1:500) and mitochondria (red) were stained using an antibody directed against LipDH (1:500).
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Nuclear and kinetoplast DNA were stained with DAPI. C and D: Growth phenotype analysis of the TbMCP12 OE cell line. Shown are growth curves of WT and tet-induced TbMCP12 OE cell line cultured in standard low-glucose (C) and high-glucose (D) medium. Procyclic trypanosome growth was started at 0.25 x 106 cells/ml and cells were counted every 24 h for a total of 72 h. The growth curves represent the means of 6 independent experimental replicates and standard deviation is shown. One-way ANOVA was performed using GraphPad Prism 7. *: p ≤ 0.05 **: p ≤ 0.01; ***p ≤ 0.001. E: Representative western blot analysis of the expression profile of TbMCP12 and TbMCP12cmyc during
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The T. brucei di- and tricarboxylate transporter TbMCP12
72 h of cell culture of tet-induced TbMCP12 OE cell line cultured in standard low-glucose and highglucose medium. TbMCP12 and TbMCP12cmyc were detected using the TbMCP12 antibody (1:1,000) and aldolase (1:1,000) was used as loading control.
Following growth analysis we found that in the presence of tetracycline, the growth-rate of the TbMCP12 over-expressing cell line was significantly lower than that of wild type cells, independently
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of the growth medium (Figure 5C and D). These cells express both the endogenous and the inducible genes: quantification of Figure 5A suggested a 5-fold TbMCP12 over-expression compared to wild type cells grown on either high or low glucose medium. Over-expression of TbMCP12 therefore
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appeared to inhibit growth.
To obtain the TbMCP12 knockout cell-line the endogenous TbMCP12 in the TbMCP12 overexpressing cell line was replaced by homologous recombination with neomycin (NEO) and blasticidin (BSD) resistance cassettes. The successful replacement of both natural TbMCP12 alleles in the
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TbMCP12 knockout cell line was confirmed by Southern blot (not shown). Western blot analysis showed that in the presence of tetracycline the TbMCP12 knockout cell-line expressed TbMCP12-
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cmyc (Figure 6A). After the removal of tetracycline TbMCP12-cmyc was fully depleted and we
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obtained the TbMCP12-depleted knockout (Figure 6A). TbMCP12-cmyc was only detected in the
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mitochondrion of procyclic cells exclusively in the cells grown with tetracycline (Figure 6B and C).
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The T. brucei di- and tricarboxylate transporter TbMCP12
Figure 6. The depletion of TbMCP12 by conditional knockout is not lethal for procyclic or
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bloodstream form T. brucei
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A: Endogenous TbMCP12 and TbMCP12cmyc expression analysis in wild type (WT), and tetracycline (tet)-induced and uninduced TbMCP12 knockout cells (KO). In each lane 2 x 106 cells were loaded.
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Proteins were detected using 1:1,000 dilutions of antibodies directed against TbMCP12, myc-tag or aldolase (loading control). B and C: Immunofluorescence analysis of TbMCP12 knockout cells after 24 h of tet removal. The depletion of the endogenous TbMCP12 (B, green) was detected using the
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TbMCP12 antibody (1:500) while TbMCP12cmyc (C, green) was detected using an antibody directed against the myc-tag (1:500). Mitochondria (B and C, red) were stained using Mitotracker. Nuclear and
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kinetoplast DNA (B and C, blue) were stained with DAPI. D and E: Growth phenotype analysis of TbMCP12 KO cells (tet-induced) and TbMCP12-depleted KO cells (after 24 h of tet removal). Cells were cultured in standard low-glucose (D) and high-glucose (E) medium. Procyclic trypanosome growth was started at 0.25 x 106 cells/ml and cells were counted every 24 h for a total of 72 h. The
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growth curves represent the means of 6 independent experimental replicates and standard deviation is shown. F: Representative western blot analysis (F) of the expression profile of TbMCP12 and TbMCP12cmyc during 72 h of cell culture of WT and TbMCP12-depleted KO cells cultured in standard low glucose and high glucose medium. TbMCP12 and TbMCP12cmyc were detected using the TbMCP12 antibody (1:1,000) and aldolase (1:1,000) was used as loading control.
20
The T. brucei di- and tricarboxylate transporter TbMCP12
The growth of the TbMCP12-depleted knockout cells was assessed in low- (Figure 6D) and high-glucose medium (Figure 6E). All analysed cells grew at similar rates independently of the medium used (Figure 6D and E). The lack of growth defect in the TbMCP12-depleted knockout was unexpected and suggested that the carrier is not essential for the survival of procyclic form T. brucei under the applied culture conditions. The absence of TbMCP12 in the knockout cell line was confirmed throughout the growth period (Figure 6F). Since the depletion of TbMCP12 was not lethal we cannot fully exclude the presence of another carrier with similar transport function or the presence
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of alternative metabolic pathways compensating its absence. TbMCP12 transports metabolic intermediates that are metabolised to ATP inside the
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mitochondrion.
Results so far suggested a role for TbMCP12 as either OGC or DTC (Figure 1, 2 and 4). To test this, we measured the mitochondrial ATP production from different TCA-cycle intermediates [33], using mitochondria isolated from T. brucei grown in either high- or low-glucose medium.
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We first analysed the mitochondrial ATP-production in mitochondria-enriched fractions from wild type procyclic T. brucei. The results revealed the highest ATP production when oxoglutarate or
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succinate where used as substrates (~4.7 nmol ATP), followed by aconitate (~3 nmol ATP) and citrate
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(~1.3 nmol ATP), irrespective of growth medium (Figure 7A). The lowest mitochondrial ATP production was found with malate and isocitrate, but for both, about 3 times more ATP was produced
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by mitochondria derived from trypanosomes grown on low-glucose medium (Figure 7A). No ATP was produced on any of the substrates when the mitochondrial ADP/ATP carrier (TbMCP5, [30]) inhibitor
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carboxyatractyloside (CAT) was included confirming that the measured ATP derived exclusively from
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the isolated mitochondria (not shown).
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The T. brucei di- and tricarboxylate transporter TbMCP12
Figure 7. Procyclic T. brucei depleted of TbMCP12 cannot produce mitochondrial ATP on citrate
A: ATP production of mitochondria derived from 1 x 107 procyclic cells grown on either low or high
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glucose medium. ATP production was measured after incubation of the isolated mitochondria with ADP and different substrates. B and C: ATP production of mitochondria derived from procyclic T. brucei overexpressing (OE) or depleted (KO) of TbMCP12 grown on either low (B) or high glucose medium (C). The values are indicated as percentage of the ATP production of wild type cells. Grey dashed lines indicate wild type as 100 %. The significance is related to the concentrations measured in wild type cells. Box-plots were derived from at least five independent experiments. Statistical significance was determined by one-way ANOVA using GraphPad Prism 7: *: p ≤ 0.05 **: p ≤ 0.01;
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The T. brucei di- and tricarboxylate transporter TbMCP12
***p ≤ 0.001.
Mitochondria isolated from the TbMCP12 over-expressing cells, produced significantly more (~2 times) ATP than wild type cells using tricarboxylates as substrate when they were cultured in low, but not in high glucose medium (Figures 7B and C). In contrast, ATP production on malate was completely abolished by TbMCP12 over-expression, while no significant changes were observed for
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oxoglutarate and succinate (Figures 7B and C). Strikingly, mitochondria depleted of TbMCP12 (TbMCP12-depleted knockout cells) showed no ATP-production when either citrate or isocitrate were used as substrates (Figures 7B and C). ATP
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production from aconitate was significantly reduced in low glucose medium and completely abolished in high glucose medium (Figures 7B and C). ATP production from oxoglutarate and succinate was
only reduced in mitochondria from cells grown on low glucose medium (Figures 7B and C). Together, the results indicate that TbMCP12 is involved in the transport of citrate, isocitrate and aconitate, and
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that its function affects the metabolism of succinate and malate and, to a minor extent, oxoglutarate.
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The NADP+/NADPH balance is disturbed in TbMCP12-overexpressing cell lines
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In T. brucei TCA-cycle-derived malate can be exported from the mitochondrion and converted to pyruvate by cytosolic malic enzyme. Since this reaction generates NADPH it contributes to the
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maintenance of the intracellular redox balance [57, 58]. To investigate the involvement of TbMCP12 in this system, we measured the NADP+/NADPH ratio in wild type, TbMCP12 overexpressing and
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depleted knockout cells, cultured in either low-glucose or high-glucose medium (Table 2). While no variation of the NADP+/NADPH ratio was measured when cells were grown on low glucose medium, we observed a significant (p = 0.009) reduction of the NADP+/NADPH ratio in cells overexpressing
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TbMCP12 and grown in high glucose medium compared to wild type cells (Table 2). This was caused by a 5-fold reduction in the concentration of NADP+ compared to wild type cells (p = 0.00022) (Table
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2).
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The T. brucei di- and tricarboxylate transporter TbMCP12
NADP+ (pmol)
NADP+/NADPH
WT
67.9 +/- 15.1
214.3 +/- 54.3
3.1+/- 0.9
KO
48.0 +/- 13.4
142.0 +/- 21.4
2.9 +/- 1.1
OE
80.9 +/- 33.6
175.1 +/- 32.4
2.1 +/- 1.2
WT
71.0 +/- 20.7
211.8 +/- 50.3
2.9 +/- 1.6
KO
122.5 +/- 29.8
164.6 +/- 39.1
1.3 +/- 0.5
OE
90.0 +/- 29.2
42.5 +/- 7.5 p = 0.00022 0.5 +/- 0.1 p = 0.009
SC R
High glucose
NADPH (pmol)
IP T
Low glucose
Cells
Table 2: pmol of NADP+ and NADPH measured in 1.25 x 105 cells. The results are the means of 3 independent experimental replicates and standard deviations are indicated. One-way ANOVA was performed using GraphPad Prism 7 to determine statistical significance. Statistical significance in
U
comparison to WT values was determined by one-way ANOVA using GraphPad Prism 7. Only significant p-values are indicated. WT: wild type; KO: TbMCP12 depleted knockout; OE: TbMCP12
M
A
N
overexpression
The expression of TbMCP12 is increased in the presence of hydrogen peroxide, glucose and haemin
ED
Our results show an unbalance in the T. brucei NADP+/NADPH ratio in response to the increased expression of TbMCP12. We next tested if the abundance of TbMCP12 was also affected
PT
when the cells were subjected to oxidative stress by adding hydrogen peroxide (H2O2), haemin or high glucose. High concentrations of porphyrins such as Haemin have cytotoxic effects on tissues and cells due to the generation of reactive oxygen species like oxygen radicals, lipid peroxides and H2O2 [59,
CC E
60]. High amounts of glucose can also result in elevated cellular oxidative stress because of the accumulation of glycolytic intermediates, which are diverted to and metabolised through ROSgenerating pathways [61]. When we treated procyclic T. brucei with 10 μM H2O2 the abundance of TbMCP12 was doubled (Figure 8A). Further, the addition of 11 mM Haemin or 10 mM glucose to
A
medium containing 5 mM proline as carbon source, induced 3.5-fold and 1.75-fold increments, respectively, in the TbMCP12 abundance compared to medium containing proline only (without glucose and Haemin) (Figure 8B). The highest expression of TbMCP12 (5-fold increment) compared to medium containing neither glucose nor Haemin was found when T. brucei was grown on medium containing 10 mM glucose and 11 mM Haemin (Figure 8B). In all analysed conditions the TbMCP12 expression, however, was never as high as the one obtained in the TbMCP12 overexpressing cell-line in which the TbMCP12 abundance was 5-fold higher than the one measured in high or low glucose
24
The T. brucei di- and tricarboxylate transporter TbMCP12
medium containing Haemin. No significant reduction of the growth rate of procyclic T. brucei was observed in haemin-depleted medium (not shown). Furthermore, no difference was observed in the expression of the TbMCP12 at the mRNA level suggesting that the abundance of TbMCP12 is
PT
ED
M
A
N
U
SC R
IP T
regulated at the level of translation or protein stability (not shown).
Figure 8. Increased TbMCP12 expression in the presence of oxidative stress
CC E
A: Representative western blot analysis of the protein abundance of TbMCP12 in the presence of oxidative stress caused by 5 μM and 10 μM H2O2. The western blot was labeled with antibodies against TbMCP12 or tubulin (diluted 1:1,000). Results were quantified using imageJ, normalised against tubulin and expressed as percentage of the TbMCP12 abundance present in cells that were not
A
subjected to oxidative stress. B: Analysis of the protein abundance of TbMCP12 after the removal of glucose and haemin from the growth medium. Cells were cultivated in either the presence or absence of glucose or haemin and analysed by western blotting using antibodies against TbMCP12 and tubulin as loading control (both antibodies were diluted 1:1,000). Results were quantified using imageJ, normalised against tubulin and expressed as percentage of the TbMCP12 abundance present in cells grown in standard MEM-Pros medium containing 0.3 mM glucose and haemin C: Effect of the overexpression (OE) or knockdown (KO) of TbMCP12 on the growth of procyclic T. brucei in the
25
The T. brucei di- and tricarboxylate transporter TbMCP12
presence of oxidative stress. Cells overexpressing (OE) or depleted (KO) of TbMCP12 were grown for 24 h after tetracycline induction/depletion and then treated with H2O2 for 6 h. The survival rate is expressed as percentage of the survival rate of the same cell-line grown without the addition of H2O2. The results are depicted as means of 3 independent experimental replicates and standard deviation is shown.
IP T
To investigate whether the overexpression of TbMCP12 protects the cells from oxidative stress we analysed T. brucei cell growth after the addition of 10 μM H202. Indeed, T. brucei
overexpressing TbMCP12 survived better after exposure to oxidative stress compared to wild type
SC R
cells (Figure 8C). Accordingly, the removal of TbMCP12 decreased the cell´s resistance to H202. DISCUSSION
Using Blast searches we identified TbMCP12 as putative mitochondrial carboxylic acid
U
transporter (this paper and [27]). TbMCP12 displayed high sequence similarity to metazoan DICs and OGCs and to plant DTCs (This paper and [27]). Since plant DTCs are the only carriers of the MCF
N
that catalyse the mitochondrial import of both dicarboxylates and tricarboxylates we speculated that
A
TbMCP12 has a similar substrate spectrum as the plant carriers. Horizontal gene transfer and the phylogenetic relationship between kinetoplastida and plants, algae or cyanobacteria have been
M
previously discussed in the literature [62-64]. Though these parasites do not possess plastids, they controversial [63, 65-67].
ED
contain a number of proteins bearing “plant-like” traits, but how these genes have been acquired is still Much information is available on the substrates that MCPs can transport in vitro (for a review see [19]), but relatively little is known on their function in vivo. Based on their in vitro transport
PT
activity mitochondrial carboxylate transporter were proposed to regulate the cytosolic and mitochondrial concentration of TCA cycle intermediates in dependence of nutrient demand and
CC E
oxidative stress levels [19, 33, 68]. In S. cerevisiae the dicarboxylate carrier is essential for sustaining the import of succinate produced during the glyoxylate cycle, and is necessary for the mitochondrial ATP generation on non-fermentable carbon sources [54]. In the absence of DIC only the supplementation of the growth medium with TCA cycle intermediates can maintain normal growth of
A
yeast on acetate [54]. The expression of TbMCP12 successfully restored the growth defect indicating that the carrier transports dicarboxylates into the mitochondrial matrix. However, wild type growth rate could not be fully reached probably because TbMCP12 is targeted to yeast mitochondria with lower efficiency or has different transport activity rates than the yeast DIC. According to the currently available model for the T. brucei energy metabolism [16, 69, 70] the TCA cycle branch going from citrate to oxoglutarate (Figure 9, enzymes number 1 and 2) is not involved in mitochondrial ATP production in procyclic cells [16, 17, 70-72]. Supporting this
26
The T. brucei di- and tricarboxylate transporter TbMCP12
hypothesis are knockout experiments, which showed that aconitase (Figure 9, enzyme number 1) is not essential for procyclic trypanosome survival [17]. Further, the activity of isocitrate dehydrogenase (Figure 9, enzyme number 2) is very low and NADP+-dependant and therefore not linked to mitochondrial ATP production via oxidative phosphorylation [2, 13, 73]. We showed here that ATP is synthesised in isolated procyclic mitochondria using citrate, isocitrate or aconitate as substrates. The amount of ATP was, however, very low compared to the one produced on oxoglutarate and succinate suggesting that the activity of this TCA cycle branch is negligible for T. brucei ATP production and
PT
ED
M
A
N
U
SC R
IP T
survival.
Figure 9. Schematic representation illustrating the potential functions of TbMCP12 in the
CC E
context of the procyclic T. brucei mitochondrial energy metabolism The illustration depicts the key enzymatic steps that are required for the ATP synthesis from the carbon sources glucose and proline and for the generation of the end-products succinate and acetate. TbMCP12 is highlighted in yellow; “a”, “b” and “c” indicate the putative TbMCP12 functions,
A
homologue to the mammalian dicarboxylate carrier (“a”), oxoglutarate carrier (“b”) and tricarboxylate carrier (“c”). The aconitase (enzyme 1) reaction produces isocitrate from citrate with cis-aconitate as enzyme-bond intermediate. Aconitase can also use cis-aconitate as substrate regenerating either isocitrate or citrate. Based on the results obtained from the mitochondrial ATP production of wild type cells (Fig. 7A) we have postulated that aconitase is active in procyclic T. brucei cultured in either high or low glucose MEM-Pros. Red arrows: direction of the enzymatic step in high-glucose medium. Dashed red arrows: direction of the enzymatic step in low-glucose medium. Black arrows: direction of
27
The T. brucei di- and tricarboxylate transporter TbMCP12
the enzymatic step in either low- or high-glucose medium. Numbers I-IV indicate respiratory chain complexes. The numbers on the arrows indicate the respective enzyme as follows: 1 – aconitase, 2 – isocitrate dehydrogenase, 3 – oxoglutarate dehydrogenase complex, 4 – succinyl-CoA synthetase, 5 – fumarate reductase, 6 - fumarase, 7 – mitochondrial malate dehydrogenase, 8 – citrate synthase, 9 – pyruvate dehydrogenase complex, 10 - acetate:succinate CoA-transferase, 11 - succinyl CoA synthetase, 12 – mitochondrial malic enzyme, 13 – cytosolic malic enzyme, 14 – L-alanine
IP T
aminotransferase, 15 – ATP-synthase, 16 acetyl-CoA thioesterase.
Depending on the availability of the carbon sources, the reactions of the T. brucei TCA-cycle can work in alternating directions [66]. As shown in Figure 9, the TCA-cycle branch that involves
SC R
succinate dehydrogenase, fumarase and malate dehydrogenase is metabolically flexible and produces malate when glucose is scarce while it consumes malate when glucose is highly abundant. Our
findings support this metabolic model. The ATP production on malate was lower when mitochondria were “primed” on high glucose medium. When glucose is highly abundant, malate is converted within
U
a reversed branch of the TCA cycle to fumarate and subsequently to succinate, a reaction that does not
N
produce either ATP or redox equivalents (Figure 9, enzyme number 5 and 6) [69, 70]. On low glucose medium, malate is either exported from the mitochondrion or is metabolised by a mitochondrial malic
A
enzyme (Figure 9, enzyme number 12) [57]. This reaction produces NADPH and pyruvate, which is
M
then metabolised to acetate and ATP (Figure link between functional mitochondrial ATP metabolism and TbMCP12 is highlighted when changes to the carrier´s abundance are made. In isolated mitochondria depletion of TbMCP12 drastically reduced while its overexpression significantly
ED
augmented the mitochondrial ATP synthesis on citrate, isocitrate and aconitate indicating that the carrier is involved in the transport of tricarboxylates. The detrimental effect of the TbMCP12
PT
overexpression might be ascribed to alterations of the TCA-cycle flux that cause a depletion of metabolic intermediates or redox equivalents. Whether an increased mitochondrial import or export of dicarboxylates and tricarboxylates is influencing the TCA-cycle flux in response to TbMCP12
CC E
abundance is unclear at the moment, but this protein might represent an attractive “metabolic switch”. NADPH is the central electron source for the synthesis of fatty acids, gluconeogenesis, and the
regeneration of enzymes belonging to the oxidative stress defence system [74]. Differently to other
A
eukaryotes, the T. brucei redox metabolism is not based on peroxisomal catalase or the molecule gluthathione but on a trypanothione peroxidase system that is regenerated in a NADPH-dependent manner [75, 76]. In T. brucei there are two main sources of cytosolic NADPH: cytosolic malic enzyme (Figure 9, enzyme number 13) and the pentose phosphate pathway [77]. The two systems coexist and independently sustain the production of NADPH [77]. Cytosolic malic enzyme produces pyruvate and NADPH through the decarboxylation of malate and requires the cooperation with mitochondrial carboxylate carriers for the maintenance of the cytosolic NADPH balance (Figure 11, TbMCP12 “a”).
28
The T. brucei di- and tricarboxylate transporter TbMCP12
In mammals, the dicarboxylate, oxoglutarate and citrate carriers are involved in the so-called citrate shuttle. The citrate shuttle exports mitochondrial citrate to the cytosol where it is converted to malate. The existence of this shuttle in T. brucei was not supported by previous studies [78]. The provision of cytosolic malate must therefore occur exclusively through its direct export from the mitochondrion possibly through TbMCP12 (Figure 9, TbMCP12 “a”). Our results show that TbMCP12 is upregulated under oxidative stress conditions and that the 2-fold overexpression of TbMCP12 slightly improved while the knockout of TbMCP12 reduced the survival of T. brucei under the influence of oxidative
IP T
stress. Our results suggest that the abundance of TbMCP12 might contribute to the regulation of the NAPDH/NADP+ balance. It is possible that increasing the abundance of TbMCP12 acts as a
“metabolic switch” redirecting malate from the mitochondrial (Figure 9, enzyme number 12) to the
SC R
cytosolic malic enzyme (Figure 9, enzyme number 13). Such a system would be more required in
procyclic cells since bloodstream form trypanosomes do not rely on malic enzyme [79] for NADPH production but on the pentose phosphate pathway [57, 58]. The involvement of TbMCP12 in the cellular maintenance of the NAPDH/NADP+ balance, however, requires further elucidation.
U
Considering the results presented in this article we propose that TbMCP12 transports dicarboxylates and tricarboxylates across the mitochondrial inner member and is involved in cellular
N
oxidative stress defence mechanisms and maintenance of the cytosolic NADPH homeostasis. Further,
A
the abundance of TbMCP12 appears to be a critical regulatory element of the mitochondrial ATP-
ED
AUTHOR CONTRIBUTION
M
production.
CC designed and performed all the experiments related with the knockdown and the overexpression in T. brucei, ATP and NADP/NADPH measurements and phylogenetic and sequence analyses (Figures
PT
1, 2, 3, 5, 6, 7, 9, Table 1 and 2). FZ, CK and FV designed and performed all the experiments related with the yeast complementation and the treatment of T. brucei with hydrogen peroxide and haemin
CC E
(Figures 4 and 8). All authors reviewed the results and contributed equally to the drafting and correction of the manuscript as well as to the design of Figure 9. The final version of the manuscript was approved by all authors.
A
CONFLICT OF INTEREST The authors declare that they have no conflicts of interest with the contents of this article. AKNOWLEDGEMENT We thank Christine Clayton for proof-reading the manuscript. This work was funded by the BBSRC, grant number BB/G00448X/1.
29
The T. brucei di- and tricarboxylate transporter TbMCP12
FOOTNOTES MCF: mitochondrial carrier family; TCA: tricarboxylic acid cycle; TbMCP: T. brucei MCF protein; CIT: citrate carrier; DIC: dicarboxylate carrier; OGC: oxoglutarate carrier; DTC: di-tricarboxylate carrier; UCP: uncoupling proteins; CAT: carboxyatractyloside; ROS: reactive oxygen species; NEO:
A
CC E
PT
ED
M
A
N
U
SC R
IP T
neomycin; BSD: blasticidin; UTR: untranslated region
30
The T. brucei di- and tricarboxylate transporter TbMCP12
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