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• W2939652298 abstract "Article12 April 2019Open Access Transparent process The yeast mitochondrial pyruvate carrier is a hetero-dimer in its functional state Sotiria Tavoulari Corresponding Author Sotiria Tavoulari [email protected] orcid.org/0000-0002-4263-8905 Medical Research Council Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK Search for more papers by this author Chancievan Thangaratnarajah Chancievan Thangaratnarajah Medical Research Council Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK Search for more papers by this author Vasiliki Mavridou Vasiliki Mavridou Medical Research Council Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK Search for more papers by this author Michael E Harbour Michael E Harbour Medical Research Council Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK Search for more papers by this author Jean-Claude Martinou Jean-Claude Martinou Department of Cell Biology, University of Geneva, Genève 4, Switzerland Search for more papers by this author Edmund RS Kunji Corresponding Author Edmund RS Kunji [email protected] orcid.org/0000-0002-0610-4500 Medical Research Council Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK Search for more papers by this author Sotiria Tavoulari Corresponding Author Sotiria Tavoulari [email protected] orcid.org/0000-0002-4263-8905 Medical Research Council Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK Search for more papers by this author Chancievan Thangaratnarajah Chancievan Thangaratnarajah Medical Research Council Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK Search for more papers by this author Vasiliki Mavridou Vasiliki Mavridou Medical Research Council Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK Search for more papers by this author Michael E Harbour Michael E Harbour Medical Research Council Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK Search for more papers by this author Jean-Claude Martinou Jean-Claude Martinou Department of Cell Biology, University of Geneva, Genève 4, Switzerland Search for more papers by this author Edmund RS Kunji Corresponding Author Edmund RS Kunji [email protected] orcid.org/0000-0002-0610-4500 Medical Research Council Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK Search for more papers by this author Author Information Sotiria Tavoulari *,1, Chancievan Thangaratnarajah1,3, Vasiliki Mavridou1, Michael E Harbour1, Jean-Claude Martinou2 and Edmund RS Kunji *,1 1Medical Research Council Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK 2Department of Cell Biology, University of Geneva, Genève 4, Switzerland 3Present address: Groningen Biomolecular Sciences and Biotechnology Institute, Membrane Enzymology, University of Groningen, Groningen, The Netherlands *Corresponding author. Tel: +441223252850; Fax: +441223252875; E-mail: [email protected] *Corresponding author. Tel: +441223252850; Fax: +441223252875; E-mail: [email protected] The EMBO Journal (2019)38:e100785https://doi.org/10.15252/embj.2018100785 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The mitochondrial pyruvate carrier (MPC) is critical for cellular homeostasis, as it is required in central metabolism for transporting pyruvate from the cytosol into the mitochondrial matrix. MPC has been implicated in many diseases and is being investigated as a drug target. A few years ago, small membrane proteins, called MPC1 and MPC2 in mammals and Mpc1, Mpc2 and Mpc3 in yeast, were proposed to form large protein complexes responsible for this function. However, the MPC complexes have never been isolated and their composition, oligomeric state and functional properties have not been defined. Here, we identify the functional unit of MPC from Saccharomyces cerevisiae. In contrast to earlier hypotheses, we demonstrate that MPC is a hetero-dimer, not a multimeric complex. When not engaged in hetero-dimers, the yeast Mpc proteins can also form homo-dimers that are, however, inactive. We show that the earlier described substrate transport properties and inhibitor profiles are embodied by the hetero-dimer. This work provides a foundation for elucidating the structure of the functional complex and the mechanism of substrate transport and inhibition. Synopsis Yeast mitochondrial pyruvate carrier (MPC) has been proposed to be a multimeric complex containing different combinations of three MPC protomers. Here, heterodimers of Mpc1/Mpc3 and Mpc1/Mpc2 are found to constitute the functional units of MPC complex in respiratory and fermentative conditions, respectively. Mitochondrial pyruvate carrier complexes from Saccharomyces cerevisiae are purified and functionally reconstituted into liposomes. Reconstituted Mpc1/Mpc3 and Mpc1/Mpc2 complexes transport pyruvate in a pH-gradient-dependent manner and are inhibited by known MPC inhibitors. Individual Mpc proteins can form homodimers, which are however not functional. Introduction In recent years, there is an increasing understanding and appreciation that mitochondrial metabolism is involved in major human diseases, such as cancer, neurodegeneration, cardiovascular diseases, metabolic disorders, obesity and diabetes. A key player for the metabolic fate of the cell is the mitochondrial pyruvate carrier (MPC), a protein responsible for the uptake of pyruvate from the cytosol into the mitochondrial matrix (Vanderperre et al, 2015), where it enters the tricarboxylic acid cycle and other biosynthetic pathways. The existence of a membrane protein responsible for pyruvate transport across the mitochondrial inner membrane had been supported by early work on isolated mitochondria, where pyruvate transport had been shown to be saturating and pH-dependent (Papa et al, 1971; Papa & Paradies, 1974). In addition, small-molecule inhibitors had been identified in support of this notion (Halestrap & Denton, 1974; Halestrap, 1975, 1976), but the molecular identity of the protein remained unknown for four decades. Major progress was made in 2012, when mitochondrial pyruvate transport activity was discovered to be associated with two small homologous proteins, MPC1 and MPC2 (Bricker et al, 2012; Herzig et al, 2012). In mammals and Drosophila, the expression of both MPC proteins is necessary for pyruvate transport (Bricker et al, 2012; Herzig et al, 2012). In yeast Saccharomyces cerevisiae, three proteins Mpc1, Mpc2 and Mpc3 are expressed in a carbon source-dependent pattern, forming an Mpc1/Mpc2 complex under fermentative conditions and an Mpc1/Mpc3 under respiratory conditions, called MPCFERM and MPCOX complexes, respectively (Bender et al, 2015; Compan et al, 2015). As the mitochondrial pyruvate carrier is central for cellular homeostasis, the identification of the MPC proteins intensified efforts to understand their role in cancer (Schell et al, 2014, 2017; Yang et al, 2014; Zhong et al, 2015; Li et al, 2016, 2017; Corbet et al, 2018; Ohashi et al, 2018; Bader et al, 2019), diabetes (Colca et al, 2013; Divakaruni et al, 2013; Vigueira et al, 2014; Gray et al, 2015; McCommis et al, 2015, 2016; Vadvalkar et al, 2017) and neurodegeneration (Ghosh et al, 2016; Divakaruni et al, 2017; Quansah et al, 2018). Moreover, pathogenic mutations in the mpc1 gene were found in rare but severe metabolic syndromes, further enhancing the clinical relevance of this transporter (Bricker et al, 2012). Additionally, an increasing number of small-molecule drugs, previously known to have other targets, have now been proposed to inhibit MPC activity (Colca et al, 2013; Divakaruni et al, 2013; Du et al, 2013; Ghosh et al, 2016; Nancolas et al, 2016; Nath et al, 2016; Chen et al, 2018; Corbet et al, 2018). MPC is a newly identified target for the first-generation insulin sensitisers, called thiazolidinediones (TZDs; Colca et al, 2013; Divakaruni et al, 2013), originally known to exert their action on the peroxisome proliferator-activated receptor gamma (PPARγ; Soccio et al, 2014; Nanjan et al, 2018). More recently, a new generation TZD, bypassing PPARγ (Colca et al, 2014) and currently in clinical trials for the treatment of Parkinson's disease, was proposed to exert its action by inhibiting MPC (Ghosh et al, 2016). Despite these major advances, a direct experimental system to measure the interactions of small-molecule drugs with MPC and to study the mechanism of inhibition is not available. Six years after the primary identification of the MPC proteins (Bricker et al, 2012; Herzig et al, 2012), there has been no report of a successful purification and functional reconstitution of an MPC hetero-complex. Consequently, the composition of the MPC complexes, the oligomeric state and the protomer stoichiometry remain controversial (Bricker et al, 2012; Bender et al, 2015; Nagampalli et al, 2018) and their involvement in pyruvate transport has been questioned (Halestrap, 2012). The yeast MPC hetero-complexes migrate at 150 kDa (Bricker et al, 2012; Bender et al, 2015) or at even higher molecular weights (Bender et al, 2015) in blue native gel electrophoresis, leading to proposals that the complexes are multimeric and might even include additional, yet unidentified proteins (Bricker et al, 2012; Halestrap, 2012). The application of chemical cross-linking on MPC proteins has produced bands corresponding to monomers, dimers and higher oligomers (Bender et al, 2015; Nagampalli et al, 2018). In the only published attempt to purify an MPC hetero-complex, it was only possible to purify individual MPC protomers (Nagampalli et al, 2018). It has also been proposed that the individual human MPC2 protein can form high-order multi-species capable of transporting pyruvate (Nagampalli et al, 2018), raising more questions regarding the functional unit of the mitochondrial pyruvate carrier. Here, we report the first successful purification and characterisation of MPC hetero-complexes from yeast, providing a model system for future structural and mechanistic studies. We demonstrate that the natural state of yeast MPC is a hetero-dimer capable of transporting pyruvate. In the absence of other protomers, MPC proteins can form homo-dimers, but they do not transport pyruvate. Results Expression and purification of the yeast Mpc proteins Although it has been established that the MPC proteins constitute the mitochondrial pyruvate carrier (Bricker et al, 2012; Herzig et al, 2012), there are still many outstanding questions regarding the composition and the oligomeric state of the functional complex. The most straightforward way to settle these issues is through the purification and reconstitution of the individual components and putative complexes to determine whether they have pyruvate transport activity. Here, we have used the Mpc proteins from S. cerevisiae (Fig EV1) as a model system to study the composition and functional properties of the mitochondrial pyruvate carrier. We decided to concentrate on the Mpc1/Mpc3 complex, as it is the principle pyruvate carrier in oxidative phosphorylation (MPCOX; Bender et al, 2015). It is also most related to MPCs of mammals and other organisms, whereas the Mpc1/Mpc2 complex (MPCFERM), expressed under fermentative conditions, only exists in some fungi. The principal approach was to purify the Mpc1/Mpc3 hetero-complex by tagging one of the two protomers with a Factor Xa cleavage site and an eight-histidine tag on the C-terminus (Fig 1A). To achieve co-expression of the protein pair, we used an inducible bidirectional vector (Miller et al, 1998) for expression in mitochondria of the mpc triple deletion strain SHY15 (Herzig et al, 2012). We also expressed the C-terminally tagged Mpc1 and Mpc3 proteins individually in yeast mitochondria. Click here to expand this figure. Figure EV1. Topology of yeast Mpc1, Mpc2 and Mpc3The alignment was generated by Clustal Omega and by manual curation (Sievers et al, 2011). The aligned residues are coloured by the ZAPPO colour scheme in which aliphatic, polar, aromatic, positively charged, negatively charged, Pro/Gly and Cys are coloured pink, green, orange, blue, red, magenta and yellow, respectively. The asterisks indicate identical residues and the colon conserved substitutions. Also indicated are putative transmembrane helices, loop regions and the N-terminal amphipathic helix. The secondary structure elements were assigned based on PSIPRED (Buchan et al, 2013), MEMSAT3 (Jones et al, 1994) and conservation analysis. Download figure Download PowerPoint Figure 1. Purification and stability analysis of Mpc proteins Strategy for purification of the Mpc1/Mpc3 hetero-complex by nickel-affinity chromatography. Expression of Mpc proteins in mitochondria assessed by SDS–PAGE and immunoblot analysis of crude mitochondrial preparations. The individual untagged Mpc1 (Mpc1), histidine-tagged Mpc1 (Mpc1his), histidine-tagged Mpc3 (Mpc3his) or the Mpc1/Mpc3 hetero-complex (Mpc1/Mpc3his) were detected with antibodies raised against Mpc1 (left panel) or Mpc3 (right panel) and are shown with dashed arrows. Five micrograms of each affinity-purified Mpc protein were analysed by SDS–PAGE, and the bands were visualised by Coomassie Blue stain. Peptide mass finger printing was used to identify the major protein bands (Table EV1). The stability of the purified proteins was assessed via thermal denaturation by fluorescent CPM-adduct formation. The thermal denaturation profiles (left) were used to calculate the first derivative (right), which provides the apparent melting temperature, indicated with the same colour coding. The stability of the same samples, as in panel (D), was assessed by NanoDSF. The changes in the 330 nm/350 nm ratio with temperature (left) were used to calculate the first derivative (right). Colour coding is as in panel (D). Download figure Download PowerPoint All proteins expressed well, both in the presence and in the absence of their proposed complex partner (Fig 1B). It is notable though that in expression trials of Mpc1 alone an additional prominent band appeared, approximately corresponding to the molecular weight of an SDS-resistant dimer, as detected by immunoblotting of crude yeast mitochondrial preparations (Fig 1B, left panel). However, this band was not detected in mitochondria expressing the untagged Mpc1 protein (Fig 1B, left panel). For these reasons, we chose to co-express an unmodified Mpc1 together with a tagged Mpc3 for hetero-complex formation. With this strategy, the purification of the Mpc1/Mpc3 hetero-complex was successful and both proteins were present in a 1:1 ratio (Fig 1C). The highest protein yield (~1 mg protein per g of mitochondria) was achieved with the detergent lauryl maltose neopentyl glycol (LMNG), supplemented with tetraoleoyl cardiolipin. However, the stoichiometric Mpc1/Mpc3 complex could also be purified in Triton X-100, decyl maltose neopentyl glycol (DMNG) or n-dodecyl β-D-maltoside (DDM), each supplemented with tetraoleoyl cardiolipin, albeit with lower purification yields (Fig EV2). In LMNG and tetraoleoyl cardiolipin, the Mpc1/Mpc3 complex eluted as a single peak by size-exclusion chromatography, indicating that the complex was monodisperse (Fig EV3). Moreover, analysis of the peak fractions showed that the protomers remained associated during purification, consistent with a stable complex (Fig EV3A, inset). When we purified histidine-tagged Mpc1 or Mpc3 on their own in LMNG and tetraoleoyl cardiolipin (Fig 1C), the yields were at least three times lower, despite similar expression levels in mitochondria in the presence or absence of their partner (Fig 1B), indicating stability issues. After purification and histidine-tag cleavage, Mpc1 contained again an SDS-resistant dimer (Fig 1C), as detected by peptide mass fingerprinting (Table EV1). Click here to expand this figure. Figure EV2. The Mpc1/Mpc3 hetero-complex purified in different detergentsThe Mpc1/Mpc3p hetero-complex was purified in n-dodecyl β-D-maltoside (DDM), decyl maltose neopentyl glycol (DMNG) or Triton X-100. The solubilisation of mitochondria was performed as under Materials and Methods but in buffer containing 2% (w/v) DDM or DMNG or 1% (w/v) Triton X-100. For affinity purification, the nickel Sepharose columns were washed with wash buffers containing 0.1% (w/v) DDM, DMNG or 0.1% (w/v) Triton X-100 supplemented with 0.1 mg/ml tetraoleoyl cardiolipin (TOCL). The samples are the solubilisate of mitochondria (Sol), proteins remaining on the resin after Factor Xa cleavage (B) and flow-through from the resin (FT). Asterisks indicate the Mpc1 and Mpc3 proteins. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Size-exclusion chromatography Size-exclusion chromatography of 500 μg nickel-affinity-purified Mpc1/Mpc3 hetero-complex. In the A280 profiles, the Mpc1/Mpc3 hetero-complex was present in a symmetrical peak. Size-exclusion chromatography of 150 μg nickel-affinity-purified Mpc3. Mpc3 produced a peak similar to that of Mpc1/Mpc3. Data information: Insets in (A and B) show peak fractions collected and analysed by SDS–PAGE and visualised by Coomassie Blue stain. Download figure Download PowerPoint Next, we used thermostability analysis to evaluate folding and stability of the hetero-complex and its components. For this purpose, we monitored the unfolding of protein populations in a temperature ramp in the presence of 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM), which reacts with cysteine residues becoming exposed due to denaturation (Fig 1D). Mpc1 and Mpc3 both have a single cysteine residue (Fig EV1). The Mpc1/Mpc3 hetero-complex and the individual Mpc3 protein had very similar unfolding profiles and melting temperatures (52.0 and 51.7°C, respectively), clearly showing that they are folded and stable in detergent solution. The Mpc1 protein on its own, however, did not display a thermal denaturation profile, and high fluorescence was detected throughout the temperature ramp. This result means that either Mpc1 is unfolded or its single cysteine is already exposed. To discriminate between these two possibilities, we also performed thermostability analysis by nano differential scanning fluorimetry (nanoDSF; Fig 1E), which relies on changes in the environment of endogenous tyrosine and tryptophan residues. Again, the hetero-complex and Mpc3 had similar apparent melting temperatures, 48.5 and 48.0°C, respectively, similar to those obtained with CPM. However, Mpc1 alone showed a peak at 64.5°C, which might correspond to the SDS-resistant dimer, possibly being an aggregation artefact. The ability of Mpc proteins to form hetero-complexes was also demonstrated when we expressed and purified the Mpc1/Mpc2 (MPCFERM) hetero-complex (Bender et al, 2015) using a histidine tag on Mpc2 (Fig EV4A and B). We also compared the Mpc1/Mpc2 hetero-complex to the histidine-tagged Mpc2, expressed and purified alone (Fig EV4). Although Mpc2 can be expressed in mitochondria equally well on its own or together with Mpc1 (Fig EV4A), it could not be purified alone in sufficient quantities and could only be detected by peptide mass fingerprinting (Fig EV4B and Table EV1). Interestingly though, when they were expressed together, Mpc2 with Mpc1 successfully formed a hetero-complex, which was purified and had an apparent melting temperature of 42°C (Fig EV4C). Click here to expand this figure. Figure EV4. Purification and stability analysis of the hetero-complex Mpc1/Mpc2 The individual histidine-tagged Mpc1 (Mpc1his), histidine-tagged Mpc2 (Mpc2his) or the Mpc1/Mpc2 hetero-complex (Mpc1/Mpc2his) was expressed in the triple mpc knock-out strain SHY15 and detected with antibodies raised against Mpc1 (left) or against the histidine tag (right). SDS–PAGE analysis of purified Mpc1, Mpc2 and Mpc1/Mpc2 proteins, visualised with Coomassie Blue stain. As the yield of the purified Mpc2 was very low, the protein was not visible with Coomassie Blue stain, but was identified on the gel by peptide mass finger printing (Table EV1). The stability of the purified Mpc1/Mpc2 or Mpc2 was assessed via the CPM method. Thermal denaturation profiles (upper panel) were used to calculate the first derivative of the data (lower panel). The number is the apparent melting temperature for the Mpc1/Mpc2 hetero-complex. Time course of pyruvate homo-exchange by the Mpc1/Mpc2 hetero-complex in liposomes in comparison with empty liposomes at a ΔpH of 1.6 (n = 2). Download figure Download PowerPoint This is the first purification of MPC hetero-complexes, demonstrating that different Mpc protomers can form stable interactions. Of the two hetero-complexes, the principal complex Mpc1/Mpc3 was purified at higher yields, was 10°C more thermostable and was, therefore, selected for characterisation of its oligomeric state. The Mpc1/Mpc3 hetero-complex is a dimer We showed that the protomers are present in a 1:1 molar ratio in the hetero-complexes (Fig 1C), but this does not resolve the issue of the overall mass of the complex. Previous work on the yeast Mpc proposed that the protein is a multimeric complex of 150 kDa, based on its electrophoretic mobility by blue native gel electrophoresis (Bricker et al, 2012) and this notion has been largely accepted in the literature. However, the electrophoretic mobility on blue native gels depends on the associated detergent and lipids present during protein extraction, as both the detergent-lipid micelle (DL) and the protein bind Coomassie stain, leading to anomalous migration (Crichton et al, 2013). Therefore, blue native gel electrophoresis is not an appropriate technique for sizing of small membrane proteins. Another approach that has been used involves chemical cross-linking with 7 and 15 Å long cross-linkers. The cross-linked MPC proteins appeared on SDS–PAGE in multiple bands corresponding to monomers, dimers and different higher oligomers (Bender et al, 2015; Nagampalli et al, 2018), but it cannot be excluded that the detected states are the result of non-specific cross-linking events. Here, we determined the molecular mass, oligomeric state and subunit stoichiometry of the affinity-purified Mpc1/Mpc3 hetero-complex by using size-exclusion chromatography linked to multi-angle laser light scattering (SEC-MALLS; Fig 2A and B). This technique can determine the mass of the protein–detergent–lipid complex (PDL) and of the protein itself (Slotboom et al, 2008; ter Beek et al, 2011). The analysis showed that on average the mass of the Mpc1/Mpc3 hetero-complex is contributing 30.8 ± 1.4 kDa to a 163.3 ± 5.1 kDa protein–detergent–lipid complex (Table EV2 and Fig 2B). The protein mass corresponded well to the sum of the theoretical masses of Mpc1 (15 kDa) and Mpc3 (17.1 kDa), which demonstrates that the complex is a hetero-dimer. Similar results were obtained whether the protein was purified by a single affinity chromatography step or by an additional size-exclusion chromatography step (Table EV2). Figure 2. The Mpc proteins form dimeric complexes Nickel-affinity-purified Mpc1/Mpc3 hetero-complex used for SEC-MALLS analysis, showing a 1:1 stoichiometry of the protomers. SEC-MALLS analysis of the hetero-complex. The light scattering trace for Mpc1/Mpc3 is shown as a black line. The masses of the protein–detergent–lipid complex (PDL, green), the detergent–lipid micelle (DL, blue) and the protein (P, red) are indicated. Protein fractions across the peak were assessed by SDS–PAGE and visualised by Coomassie Blue staining (B, inset). Nickel-affinity-purified Mpc3 protein used for SEC-MALLS analysis. SEC-MALLS analysis of Mpc3, colour designation as in (B). The protein fractions across the peak (D, inset) were assessed as in (B). Data information: Data in (B and D) represent a characteristic experiment repeated independently five times for Mpc1/Mpc3 and three times for Mpc3. All biological repeats are summarised in Table EV2. Download figure Download PowerPoint To exclude other possible stoichiometries, we performed an internal consistency analysis (Wen et al, 1996), where theoretical molecular masses for the complex, corresponding to different possible subunit stoichiometries, were calculated and compared with the experimentally determined molecular mass. The analysis was only consistent with a Mpc1/Mpc3 hetero-dimeric complex, showing the smallest difference between the experimentally determined and theoretical molecular masses (Table 1). Additionally, since Mpc1 and Mpc3 co-purify in equimolar amounts and remain associated throughout the SEC-MALLS step (Fig 2B, inset), the possibility that the calculated mass corresponds to separate homo-dimers is eliminated. Table 1. Stoichiometry analysis of the Mpc1/Mpc3 complex Mpc1 Mpc3 Absorbance 0.1% (=1 g/l) In silico calculated Mw (kDa) Mw from SEC-MALLS (kDa) Difference (kDa) Monomer 1 0 1.628 15 32.92 17.92 0 1 1.868 17.12 28.69 11.57 Homo-dimer 2 0 1.633 29.97 32.9 2.93 0 2 1.869 34.21 28.68 −5.53 Hetero-dimer 1 1 1.757 32.1 30.83 −1.27 Homo-trimer 3 0 1.629 44.95 32.9 −12.05 0 3 1.869 51.31 29.6 −21.71 Hetero-trimer 2 1 1.716 47.07 31.21 −15.86 1 2 1.796 49.19 30.12 −19.07 Since Mpc3 can be purified on its own (Figs 1C and EV3) and is stable in solution, it was also analysed by SEC-MALLS (Fig 2C and D). The average molecular mass for the protein–detergent–lipid complex was 182.9 ± 7.9 kDa with the protein contributing 34.8 ± 0.7 kDa (Table EV2 and Fig 2D), which is approximately twice the theoretical mass of Mpc3 (17.1 kDa). Thus, Mpc3 forms homo-dimers in the absence of Mpc1. The Mpc1/Mpc3 hetero-dimer is the active mitochondrial pyruvate carrier To investigate whether the Mpc1/Mpc3 hetero-dimer is capable of pyruvate transport, we established a reconstitution and transport protocol. We prepared proteoliposomes of the purified hetero-dimer loaded with 5 mM unlabelled pyruvate in buffer at pH 8.0 (Fig 3) or 7.4 (Fig EV5), and we initiated pyruvate homo-exchange by addition of radiolabelled pyruvate (50 μM) on the outside. To evaluate the pH dependence of transport, we performed our assays with external buffer at different pH units (Figs 3 and EV5). Figure 3. The Mpc1/Mpc3 hetero-complex transports pyruvate Time course of pyruvate homo-exchange by the Mpc1/Mpc3 hetero-complex in liposomes (n = 8) in comparison with empty liposomes (n = 6) at a ΔpH of 1.6. Kinetic analysis of pyruvate homo-exchange at ΔpH of 1.6 (n = 3). The hetero-complex was assayed for initial rates of uptake in the concentration range of 25–600 μM. The calculated KM was 299 μM in this experiment and 318 and 409 μM in two additional biological repeats. Time course of pyruvate homo-exchange by the Mpc1/Mpc3 hetero-complex in physiological pH (n = 4) compared to empty liposomes (n = 4). In the absence of a ΔpH, the time course of pyruvate homo-exchange was similar for the Mpc1/Mpc3 proteoliposomes and the empty liposomes (n = 4). Inhibition of [14C]-pyruvate homo-exchange by UK5099 (1–100 μM), Zaprinast (1–1,000 μM), lonidamine (10–10,000 μM) and 7ACC2 (5–500 μM). Data points represent the mean of three technical replicates of a typical experiment. The IC50 measurements have also been independently replicated, three times for UK5099, Zaprinast and 7ACC2 (average IC50: 9 ± 7, 18 ± 8 and 27 ± 13 μM, respectively) and two times for lonidamine (average IC50: 118 ± 24 μM). [14C]-pyruvate homo-exchange inhibition by the TZDs, pioglitazone and rosiglitazone (n = 6). Data information: Data have been independently replicated: (A) four biological repeats, (B) three biological repeats, (C, D) two biological repeats, (E, F) three biological repeats for UK5099, Zaprinast, 7ACC2 and two biological repeats for lonidamine and the TZDs. The error bars represent the standard error of the mean in (A) and the standard deviation in (C, D and F). Download figure Download PowerPoint Click here to expand this figure. Figure EV5. Effect of pH on pyruvate homo-exchange in Mpc1/Mpc3-containing proteoliposomes compared to diffusion of pyruvate into empty liposomesPyruvate homo-exchange of Mpc1/Mpc3 proteoliposomes or empty liposomes was tested in three conditions. In the absence of a ΔpH, using an internal buffer pH of 7.4 and external buffer pH of 7.4 (n = 2). At a ΔpH of 1.0, using an internal buffer pH of 7.4 and external buffer pH of 6.4 (n = 4). At a ΔpH of 2.0, using an internal buffer pH of 7.4 and external buffer pH of 5.4 (n = 2). Download figure Download PowerPoint In proteoliposomes loaded with internal buffer at pH of 7.4 (Fig EV5), we observed that diffusion, as determined by [14C]-pyruvate accumulation in empty liposomes, was pH-dependent and highest in acidic pH of 5.4, when pyruvate accumulation into empty liposomes reached levels even higher than into proteoliposomes. This result is consistent with previous studies suggesting that pyruvate can cross the mitochondrial membrane via “absorption”, depending on its protonation state (Klingenberg, 1970; Zahlten et al, 1972; Bakker & van Dam, 1974). Therefore, the selection of internal and external pH units is critical. Our finalised pyruvate transport protocol (Fig 3) was based on achieving the maximal ΔpH under conditions where the diffusion is minimal. When we selected an internal pH of 8.0 and external pH of 6.4, yieldi" @default.
• W2939652298 created "2019-04-25" @default.
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• W2939652298 date "2019-04-12" @default.
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• W2939652298 title "The yeast mitochondrial pyruvate carrier is a hetero‐dimer in its functional state" @default.
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