Galaz, A. | bioRxiv Science

bioRxiv2019

A highly responsive pyruvate sensor reveals pathway-regulatory role of the mitochondrial pyruvate carrier MPC

Mitochondria generate ATP and building blocks for cell growth and regeneration, using pyruvate as the main substrate. Here we introduce PyronicSF, a user-friendly GFP-based sensor of improved dynamic range that enables real-time subcellular quantitation of mitochondrial pyruvate transport, concentration and flux. We report that cultured mouse astrocytes maintain mitochondrial pyruvate in the low micromolar range, below cytosolic pyruvate, which means that the mitochondrial pyruvate carrier MPC controls the decision between respiration and anaplerosis/gluconeogenesis in an ultrasensitive fashion. The functionality of the sensor in living tissue is demonstrated in the brain of Drosophila melanogaster larvae. Mitochondrial subpopulations are known to coexist within a given cell, which differ in their morphology, mobility, membrane potential, and vicinity to other organelles. The present tool can be used to investigate how mitochondrial diversity relates to metabolism, to study the role of MPC in disease, and to screen for small-molecule MPC modulators.

cell biology↗

bioRxiv2019

MCT4 is a high affinity transporter capable of exporting lactate in high-lactate environments

MCT4 is an H+-coupled transporter expressed in metastatic cancer cells, macrophages, and other highly glycolytic cells, where it extrudes excess lactate generated by the Warburg phenomenon or by hypoxia. Intriguingly, its reported Km for lactate, obtained with pH-sensitive probes, is more than an order of magnitude higher than physiological lactate. Here we examined MCT4-rich MDA-MB-231 cells using the FRET sensor Laconic and found a median Km for lactate uptake of only 1.7 mM, while parallel estimation in the same cells with a pH probe gave a Km of 27 mM. The median Km of MCT4 for lactate was 0.7 mM in MCT4-expressing HEK293 cells and 1.2 mM in human macrophages, suggesting that high substrate affinity is a robust property of the transporter. Probed with the FRET sensor Pyronic, MCT4 showed a Km for pyruvate of only 4.2 mM in MDA-MB-231 cells, as opposed to > 150 mM reported previously. We conclude that prior estimates of MCT4 affinity based on pH probes were severely biased by the confounding action of pH regulatory mechanisms. Numerical simulation showed that MCT4, but not MCT1 or MCT2, endows cells with the capability of lactate extrusion in high lactate environments. The revised kinetic properties and novel transport assays may help in developing small-molecule MCT4 blockers for research and therapy.

physiology↗