Determination of transport kinetics of chick MCT3 monocarboxylate transporter from retinal pigment epithelium by expression in genetically modified yeast

Monocarboxylate transporters (MCTs) comprise a group of highly homologous p roteins that reside in the plasma membrane of almost all cells and which me diate the 1:I electroneutral transport of a proton and a lactate ion. The i soform MCT3 is restricted to the basal membrane of the retinal pigment epit helium where it regulates lactate levels in the neural retina. Kinetic anal ysis of this transporter poses formidable difficulties due to the presence of multiple lactate transporters and their complex interaction with MCTs in adjacent cells. To circumvent these problems, we expressed both the MCT3 g ene and a green fluorescent protein-tagged MCT3 construct in Saccharomyces cerevisiae. Since L-lactate metabolism in yeast depends on the CYB2 gene, w e disrupted CYB2 to study the MCT3 transporter activity free from the compl ications of metabolism. Under these conditions L-lactate uptake varied inve rsely with pH, greater uptake being associated with lower pH. Whereas the V -max was invariant, the K-m increased severalfold as the pH rose from 6 to 8. In addition, MCT3 was highly resistant to a number of "classical" inhibi tors of lactate transport. Last, studies with diethyl pyrocarbonate and p-c hforomercuribenzenesulfonate set limitations on the locus of potential resi dues involved in the critical site of lactate translocation.