Monitoring Mitochondrial Pyruvate Carrier Activity in Real Time Using a BRET-Based Biosensor: Investigation of the Warburg Effect

Abstract

The transport of pyruvate into mitochondria requires a specific carrier, the mitochondrial pyruvate carrier (MPC). The MPC represents a central node of carbon metabolism, and its activity is likely to play a key role in bioenergetics. Until now, investigation of the MPC activity has been limited. However, the recent molecular identification of the components of the carrier has allowed us to engineer a genetically encoded biosensor and to monitor the activity of the MPC in real time in a cell population or in a single cell. We report that the MPC activity is low in cancer cells, which mainly rely on glycolysis to generate ATP, a characteristic known as the Warburg effect. We show that this low activity can be reversed by increasing the concentration of cytosolic pyruvate, thus increasing oxidative phosphorylation. This biosensor represents a unique tool to investigate carbon metabolism and bioenergetics in various cell types.

MPC1-like Is a Placental Mammal-specific Mitochondrial Pyruvate Carrier Subunit Expressed in Postmeiotic Male Germ Cells

“…Inserts containing the mMPC1-FLAG, mMPC1L-FLAG and mMPCLP-FLAG coding sequences were then introduced into the BamHI/ SalI sites of pWPT using Gibson assembly. Generation of the MPC1-Venus, MPC2-Venus, MPC2-RLuc8, and matrix or intermembrane space localized Venus (Mx-Venus and IMSVenus, respectively) encoding constructs were described previously (17). Phusion polymerase (New England Biolabs) was used for all PCRs, and all constructs were sequence-validated (Fasteris).…”

“…We concluded previously that binding of pyruvate to the MPC followed by pyruvate transport across the IMM leads to conformational changes in the MPC1/MPC2 heteromers, which result in a change in BRET intensity and which allow us to monitor the pyruvate import activity of the MPC in real time (17). To determine the activity of the MPC1L/MPC2 heteromers in this assay, we performed time course experiments using cells co-transfected with MPC1L-RLuc8/MPC2-Venus, MPC2-RLuc8/MPC1L-Venus, or the positive control plasmids MPC2-RLuc8/MPC1-Venus (Fig.…”

“…To confirm that MPC1L associates physically with MPC2, we took advantage of the bioluminescence resonance energy transfer (BRET) assay that we have recently developed to monitor the activity of the MPC in real time (17). In the earlier study, either MPC1 or MPC2 (from human origin) was fused to a BRET donor (Renilla luciferase, RLuc8), and co-expressed with the corresponding complementary subunit fused to Venus, the BRET acceptor.…”

“…To confirm that MPC1L can sustain pyruvate import into mammalian mitochondria, we took advantage of previously described MPC-deficient mouse embryonic fibroblasts (MEFs) in which MPC1 expression had been ablated using a gene trap strategy (17,25). These cells display no detectable MPC1 by Western blotting analysis (Fig.…”

“…A decrease in MPC activity has been shown to perturb whole body glucose homeostasis through effects on glucose-stimulated insulin secretion (11,12) and gluconeogenesis (13,14). In addition, reduced MPC expression (15,16) and activity (17)(18)(19) has been observed in cancer cells, contributing to the Warburg effect. This favors cell growth and metastasis and promotes the establishment and maintenance of the cancer stem cell compartment (15,16,20).…”