The mitochondrial pyruvate carrier (MPC) occupies a central metabolic node by transporting cytosolic pyruvate into the mitochondrial matrix and linking glycolysis with mitochondrial metabolism. Two reported human MPC1 mutations cause developmental abnormalities, neurological problems, metabolic deficits, and for one patient, early death. We aimed to understand biochemical mechanisms by which the human patient C289T and T236A MPC1 alleles disrupt MPC function. MPC1 C289T encodes 2 protein variants, a misspliced, truncation mutant (A58G) and a full-length point mutant (R97W). MPC1 T236A encodes a full-length point mutant (L79H). Using human patient fibroblasts and complementation of CRISPR-deleted, MPC1-null mouse C2C12 cells, we investigated how MPC1 mutations cause MPC deficiency. Truncated MPC1 A58G protein was intrinsically unstable and failed to form MPC complexes. The MPC1 R97W protein was less stable but, when overexpressed, formed complexes with MPC2 that retained pyruvate transport activity. Conversely, MPC1 L79H protein formed stable complexes with MPC2, but these complexes failed to transport pyruvate. These findings inform MPC structure-function relationships and delineate 3 distinct biochemical pathologies resulting from 2 human patient MPC1 mutations. They also illustrate an efficient gene pass-through system for mechanistically investigating human inborn errors in pyruvate metabolism.
Lalita Oonthonpan, Adam J. Rauckhorst, Lawrence R. Gray, Audrey C. Boutron, Eric B. Taylor
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