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Simple Summary The atomic structure of a biological macromolecule determines its function. Discovering how one or more amino acid chains fold and interact to form a protein complex is critical, from understanding the most fundamental cellular processes to developing new therapies. However, this is far from a straightforward task, especially when studying a membrane protein. The functional link between the oligomeric state and complex composition of the proteins involved in the active mitochondrial transport of cytosolic pyruvate is a decades-old question but remains urgent. We present a brief historical review beginning with the identification of the so-called mitochondrial pyruvate carrier (MPC) proteins, followed by a rigorous conceptual analysis of technical approaches in more recent biochemical studies that seek to isolate and reconstitute the functional MPC complex(es) in vitro. We correlate these studies with early kinetic observations and current experimental and computational knowledge to assess their main contributions, identify gaps, resolve ambiguities, and better define the research goal. 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.

The active transport of glycolytic pyruvate across the inner mitochondrial membrane is thought to involve two mitochondrial pyruvate carrier subunits, MPC1 and MPC2, assembled as a 150 kDa heterotypic oligomer. Here, the recombinant production of human MPC through a co-expression strategy is first described; however, substantial complex formation was not observed, and predominantly individual subunits were purified. In contrast to MPC1, which co-purifies with a host chaperone, we demonstrated that MPC2 homo-oligomers promote efficient pyruvate transport into proteoliposomes. The derived functional requirements and kinetic features of MPC2 resemble those previously demonstrated for MPC in the literature. Distinctly, chemical inhibition of transport is observed only for a thiazolidinedione derivative. The autonomous transport role for MPC2 is validated in cells when the ectopic expression of human MPC2 in yeast lacking endogenous MPC stimulated growth and increased oxygen consumption. Multiple oligomeric species of MPC2 across mitochondrial isolates, purified protein and artificial lipid bilayers suggest functional high-order complexes. Significant changes in the secondary structure content of MPC2, as probed by synchrotron radiation circular dichroism, further supports the interaction between the protein and ligands. Our results provide the initial framework for the independent role of MPC2 in homeostasis and diseases related to dysregulated pyruvate metabolism.