Functional coupling between a bafilomycin A1-sensitive proton pump and a probenecid-sensitive folate transporter in...

The mechanism of transport of 5-methyltetrahydrofolate, the predominant form of folate in human blood, was investigated in JAR human placental choriocarcinoma cells. Northern analysis revealed the presence of the folate receptor mRNA in these cells. Accumulation of 5-methyltetrahydrofolate in these cells was saturable and inhibited by other folate analogs. The transport of 5-methyltetrahydrofolate into the cytoplasm was decreased by the anion transport inhibitors probenecid and 4,4'-diisothiocyanostilbene 2,2'-disulfonic acid. The cytosolic transport was dependent on a transmembrane H+ gradient because NH4Cl and chloroquine which alkalinize acidic compartments in the cell and protonophores which dissipate transmembrane H+ gradients inhibited the transport. The inhibitors of receptor-mediated endocytosis, monodansylcadaverine and bacitracin, had no effect on the cytosolic transport nor on the accumulation. Bafilomycin A1, a specific inhibitor of the vacuolar type (V-type) H(+)- pump, caused a significant reduction in the cytosolic transport of 5-methyltetrahydrofolate, without affecting the binding of the vitamin to its membrane bound receptor. It is concluded that (a) the JAR cells transport 5-methyltetrahydrofolate via a specific probenecid-sensitive folate transporter, (b) the transporter is driven by a transmembrane H+ gradient, and (c) the H+ gradient involved in this process is generated by a bafilomycin A1-sensitive V-type H(+)-pump present in the plasma membrane. The results strongly suggest that the transport of folate in these cells occurs by potocytosis involving a functional coupling between the folate receptor, the folate transporter and the H+ pump.