Edited by Mike Shipston Monocarboxylate transporter 4 (MCT4) is an H ؉-coupled symporter highly expressed in metastatic tumors and at inflammatory sites undergoing hypoxia or the Warburg effect. At these sites, extracellular lactate contributes to malignancy and immune response evasion. Intriguingly, at 30-40 mM, the reported K m of MCT4 for lactate is more than 1 order of magnitude higher than physiological or even pathological lactate levels. MCT4 is not thought to transport pyruvate. Here we have characterized cell lactate and pyruvate dynamics using the FRET sensors Laconic and Pyronic. Dominant MCT4 permeability was demonstrated in various cell types by pharmacological means and by CRISPR/Cas9-mediated deletion. Respective K m values for lactate uptake were 1.7, 1.2, and 0.7 mM in MDA-MB-231 cells, macrophages, and HEK293 cells expressing recombinant MCT4. In MDA-MB-231 cells MCT4 exhibited a K m for pyruvate of 4.2 mM, as opposed to >150 mM reported previously. Parallel assays with the pH-sensitive dye 2,7-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) indicated that previous K m estimates based on substrate-induced acidification were severely biased by confounding pH-regulatory mechanisms. Numerical simulation using revised kinetic parameters revealed that MCT4, but not the related transporters MCT1 and MCT2, endows cells with the ability to export lactate in highlactate microenvironments. In conclusion, MCT4 is a high-affinity lactate transporter with physiologically relevant affinity for pyruvate. Cancer cells ferment glucose to lactate in the presence of oxygen, a phenomenon originally described by Otto Warburg and colleagues in the 1920s and later found to promote tumor growth and malignancy (1-4). In addition to fostering glycolysis by end product removal, cytosolic alkalinization, and NADH recycling, the co-extrusion of lactate and protons causes inter

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