Characteristic Analysis of Homo- and Heterodimeric Complexes of Human Mitochondrial Pyruvate Carrier Related to Metabolic Diseases

Abstract

Human mitochondrial pyruvate carriers (hMPCs), which are required for the uptake of pyruvate into mitochondria, are associated with several metabolic diseases, including type 2 diabetes and various cancers. Yeast MPC was recently demonstrated to form a functional unit of heterodimers. However, human MPC-1 (hMPC-1) and MPC-2 (hMPC-2) have not yet been individually isolated for their detailed characterization, in particular in terms of their structural and functional properties, namely, whether they exist as homo- or heterodimers. In this study, hMPC-1 and hMPC-2 were successfully isolated in micelles and they formed stable homodimers. However, the heterodimer state was found to be dominant when both hMPC-1 and hMPC-2 were present. In addition, as heterodimers, the molecules exhibited a higher binding capacity to both substrates and inhibitors, together with a larger structural stability than when they existed as homodimers. Taken together, our results demonstrated that the hetero-dimerization of hMPCs is the main functional unit of the pyruvate metabolism, providing a structural insight into the transport mechanisms of hMPCs.

The Multifaceted Pyruvate Metabolism: Role of the Mitochondrial Pyruvate Carrier

“…Functional tests with yeast MPC1 and MPC3 following reconstitution of the carrier in liposomes showed that only heterodimers were able to transport pyruvate [ 10 ]. In another report, MPC2 homodimers were also reported to be functional [ 11 ], although this was not supported by the results of Tavoulari et al [ 10 ] or by other data reporting that mitochondria from ΔMPC1 mutants were unable to import pyruvate [ 5 , 6 , 12 ]. The reason for this discrepancy remains unclear.…”

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

“…Lee and colleagues recently reported the extraction and purification of human MPC1 and MPC2, likewise in the detergent DDM [36]. Based on previous successes, including many high-resolution structures, DDM is historically considered the universal choice of detergent for α-helical membrane proteins [55].…”

“…By contrast, Lee and co-workers employed conventional baculovirus plasmids co-infected in insect cells to express the individual human MPC proteins. In this case, MPC1 was fused at the C-terminal to a cleavable His-tag followed by a FLAG sequence, while MPC2 was tagged at its N-terminal either to a cleavable standard His-tag or to a longer poly-His + T4 lysozyme tag [ 36 ]. In this study, MPC1 tagged at its N-terminal and showed low expression.…”

“…Earlier in 2020, Lee and colleagues separately over-expressed human MPC1 and MPC2 in insect cells to purify not only heterodimers but also homodimers of MPC [ 36 ]. Based on SDS-PAGE, they claimed to have isolated both configurations of MPC in a complex with detergent, which allowed for stability studies by circular dichroism analysis.…”

“…The authors also presented computational models for MPC homo- and heterodimers based on SemiSWEET to conclude that MPC1:MPC2 is more stable and has an improved affinity to the substrate and inhibitors when compared to MPC2:MPC2. Transport studies for the isolated complexes in a lipidic vesicle system were not reported [ 36 ].…”