Human mitochondrial pyruvate carrier 2 as an autonomous membrane transporter

Raghavendra Sashi Krishna Nagampalli,Carolline Fernanda Rodrigues Ascenção,Sílvio Roberto Consonni,Amanda Cristina Teixeira Silva,José Edwin Neciosup Quesñay,Adriana Franco Paes Leme,Àngela María ,Bianca Alves Pauletti,Isabel Moraes,J.r Birch ,Richard M B M Girard ,Heitor Gobbi Sebinelli,Ariel Mariano Silber,Kleber G Franchini ,Pietro Ciancaglini,Zeyaul Islam,José Farias De Oliveira ,Douglas Adamóski ,Sandra Martha Gomes Dias,André Luís Berteli Ambrósio

doi:10.1038/s41598-018-21740-z

Abstract

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.

Highlights

Four decades after the demonstration of the protein-mediated transport of pyruvate across the inner mitochondrial membrane (IMM)[1], two concurrent studies identified the oligomeric complex formed by MPC1 and MPC2 as necessary and sufficient for this task[2,3]; MPC1 and MPC2 were proposed to function together via the formation of an oligomeric structure of approximately 150 kDa2
Human MPC1 and MPC2 proteins were co-expressed from codon-optimized genes in a heterologous yeast system (Saccharomyces cerevisiae JRY472) that was modified to lack endogenous MPC (Δmpc1/2/3)
A bi-directional expression plasmid was initially engineered to contain MPC1 fused to an 8xHis tag and MPC2 linked to a cleavable monomeric GFP (Fig. 1A)

Summary

Introduction

Four decades after the demonstration of the protein-mediated transport of pyruvate across the inner mitochondrial membrane (IMM)[1], two concurrent studies identified the oligomeric complex formed by MPC1 and MPC2 as necessary and sufficient for this task[2,3]; MPC1 and MPC2 were proposed to function together via the formation of an oligomeric structure of approximately 150 kDa2. The ectopic expression of human MPC2 improved oxygen consumption and stimulated growth under nutrient-depleted conditions compared to yeast cells lacking endogenous MPC Most importantly, these observations are consistent with those of early and recent publications, suggesting that mitochondrial pyruvate transport by MPC is a rapid and specific process that depends on co-proton import and redox balance and is sensitive to inhibition by a small molecule[14,15,16,17]. Our work has immediate implications for the development of small-molecule-oriented therapeutics that target MPC2 in pyruvate-related diseases such as cancer, Alzheimer’s disease, and diabetes[9,13,18,19,20,21]

Methods

Results

Conclusion