Membrane Transport of Folate Compounds

Abstract

All eukaryotic cells and some prokaryotes that are unable to synthesize folic acid utilize membrane-associated transport systems for acquisition of the pre-formed vitamin or its coenzyme forms from external sources. These transport systems, in addition to providing folates essential for cell replication, are also important because of their role in the internalization of antifolates such as Methotrexate (MTX) that are used extensively in cancer chemotherapy. Information about the components and mechanism of folate transport systems has been derived, in large part, from studies with Lactobacillus casei and L1210 mouse leukemia cells, which serve as convenient models for prokaryotes and eukaryotes, respectively. L. casei contain a single folate transport system whose K_t value (i. e., concentration for half-maximum rate of uptake) for the preferred substrate folate is in the nanomolar range. The hydrophobic membrane-associated folate transport protein (18 kDa) has been purified to homogeneity and characterized. Expression of this transporter is repressed in cells grown on high concentrations (μM) of folate. L1210 cells contain two separate transport systems for folate compounds: (1) the low affinity system (K_t values for the preferred substrates 5-methyl- and 5-formyltetrahydrofolate and MTX in the μM range); and (2) the high affinity system (Kt for folate in the nM range). Fluorescein and biotin derivatives of MTX and folate, after conversion to N-hydroxysuccinimide esters, can be attached covalently to the transporters. These probes have been used for visualizing the transporters by fluorescence and electron microscopy and for their purification to homogeneity. The μM transporter (43 kDa) is a non-glycosylated, integral membrane protein, while the nM counterpart (39 kDa) is heavily glycosylated and anchored exofacially to the membrane by a glycosylphosphatidylinositol component.