Structural and Functional Characterisation of the Human Mitochondrial Pyruvate Carrier

Abstract: The metabolism of pyruvate in the cell is abnormally regulated in several human diseases, such as type-II diabetes, cancer and neurodegenerative disorders, in which the human mitochondrial pyruvate carrier (HuMPC) sits at a pivotal point. It transports pyruvate across the inner mitochondrial membrane and consists of two proteins, MPC1 and MPC2, and is a potential drug target against these diseases. There is currently no three-dimensional structure at atomic resolution nor unambiguity of the biological oligomeric composition of this pyruvate transporting complex, although many studies advocate for a heterodimer of MPC1/2 being the main functional unit. In this study, the pyruvate transport activity of HuMPC complexes was investigated as well as the presence of a MPC1/2 heterocomplex. The pyruvate transport activity of human MPC1 alone, MPC2 alone and MPC1/2 mixed together was investigated with a proteoliposome assay based on the enzymatic activity of lactate dehydrogenase (LDH). In addition, the presence of the MPC1/2 complex in proteoliposomes was examined with nano-differential scanning fluorimetry, as well as with cross-linking trials for further analyses with mass spectrometry. Pure HuMPC protein was obtained by affinity chromatography from solubilised Pichia pastoris membranes. Here it was demonstrated that MPC2 and MPC1/2 displayed higher pyruvate transport activity compared to MPC1. Moreover, the nano-DSF measurement did not give any concrete evidence of the presence of the MPC1/2 heterocomplex. The mass spectrometric analyses did not identify any cross-links between MPC1 and MPC2 in the MPC1/2 proteoliposomes but more peptides of MPC2 than MPC1 could be identified. In conclusion, the true pyruvate transporting complex of HuMPC still remains unclear based on these results, as well as the existence of the MPC1/2 heterocomplex, which requires further investigation.