Insights on the Quest for the Structure-Function Relationship of the Mitochondrial Pyruvate Carrier

Abstract

The molecular identity of the mitochondrial pyruvate carrier (MPC) was presented in 2012, forty years after the active transport of cytosolic pyruvate into the mitochondrial matrix was first demonstrated. An impressive amount of in vivo and in vitro studies has since revealed an unexpected interplay between one, two, or even three protein subunits defining different functional MPC assemblies in a metabolic-specific context. These have clear implications in cell homeostasis and disease, and on the development of future therapies. Despite intensive efforts by different research groups using state-of-the-art computational tools and experimental techniques, MPCs' structure-based mechanism remains elusive. Here, we review the current state of knowledge concerning MPCs' molecular structures by examining both earlier and recent studies and presenting novel data to identify the regulatory, structural, and core transport activities to each of the known MPC subunits. We also discuss the potential application of cryogenic electron microscopy (cryo-EM) studies of MPC reconstituted into nanodiscs of synthetic copolymers for solving human MPC2.

Keywords: function; membrane proteins; mitochondrial pyruvate carrier; structure.

Figure 1

Predicted features of human mitochondrial pyruvate carrier (MPC). A collection of elements, from either direct or correlational pieces of evidence and in silico predictions, can be projected for the two human MPC subunits. ^A Correlational evidence: the topology and the orientation of each subunit within the IMM bilayer can be inferred from the experimental study on yeast MPC [25], based on significant primary sequence identity between homologs, particularly within the transmembrane helices; ^B computational prediction: the presence and positional boundaries of the TMH (orange circles) were predicted by the Constrained Consensus Topology server (CCTOP, [39]), and the non-TM helices (light red circles) by PSI-PRED workbench [40]. Of all five collective transmembrane helices in MPC1 and MPC2, only TMH 3 in MPC2 was predicted to contribute to cavity formation (yellow circles framed in red), according to MEMSAT-SVM [41,42]; ^C Experimental evidence: pathogenic variants (red diamonds) that severely compromise pyruvate transport activity have been described in patients [43], while still allowing for heterocomplex formation. For instance, mutations such as Leu79His and Arg97, have been described exclusively in MPC1, although at non-transmembrane regions [43]; ^D Experimental evidence: post-translational modifications, such as lysine acetylation (green squares), and phosphorylation (blue squares), have been identified exclusively in human MPC1, in a high-throughput proteomics study [44,45,46]. No signal peptides could be identified by primary sequence analysis.

Figure 2

MPC2–EGFP reconstitution into styrene maleic acid (SMA) lipid particles and cryogenic electron microscopy (cryo-EM) analysis. (a) SMA encapsulation of MPC2–EGFP showing green fluorescence during initial purification steps. (b) Size exclusion chromatography of MPC2–EGFP encapsulated in SMA. Peaks correspond to fluorescence emission at 488 nm. According to column calibration, the dominant peak was observed at an elution volume that corresponded to a molecular weight of around 300–400 KDa. Corresponding eluted peaks showing bands of 30 and 40 kDa for the MPC2–EGFP fusion in both Coomassie and fluorescence SDS-PAGE. (c) A two-dimensional classification was applied to the extracted MPC2–EGFP–SMA–lipid–protein (SMALP) particles. (d) Schematic representation of the predicted SMA-encapsulated human MPC2–GFP complex.