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 proteins that reside in the plasma membrane of almost all cells and which mediate the 1:1 electroneutral transport of a proton and a lactate ion. The isoform MCT3 is restricted to the basal membrane of the retinal pigment epithelium where it regulates lactate levels in the neural retina. Kinetic analysis 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 gene and a green fluorescent protein-tagged MCT3 construct in Saccharomyces cerevisiae. Since L-lactate metabolism in yeast depends on the CYB2 gene, we disrupted CYB2 to study the MCT3 transporter activity free from the complications of metabolism. Under these conditions L-lactate uptake varied inversely 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" inhibitors of lactate transport. Last, studies with diethyl pyrocarbonate and p-chloromercuribenzenesulfonate set limitations on the locus of potential residues involved in the critical site of lactate translocation.
Laboratory of Cell Biochemistry and Biology, Laboratory of Biochemical Pharmacology, NIDDK, and Laboratory of Neurobiology, NINDS, National Institutes of Health, Bethesda, Maryland 20892, USA.
E F
EF
Grollman
N J
NJ
Philp
P
P
McPhie
R D
RD
Ward
B
B
Sauer
D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects / Q000235:genetics; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D002645:Chickens; D003373:Coumaric Acids / Q000494:pharmacology; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects; D005822:Genetic Vectors / Q000138:chemical synthesis / Q000378:metabolism; D049452:Green Fluorescent Proteins; D019344:Lactic Acid / Q000037:antagonists & inhibitors / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D010693:Phloretin / Q000494:pharmacology; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D014170:Transformation, Genetic
D002352:Carrier Proteins; D003373:Coumaric Acids; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; C007175:alpha-cyano-4-hydroxycinnamate; D019344:Lactic Acid; D002731:4-Chloromercuribenzenesulfonate; D004047:Diethyl Pyrocarbonate; D010693:Phloretin