Effects of the loss of capacity for N-glycosylation on the transport activity and cellular localization of the human reduced folate carrier

Abstract

The role of N-glycosylation in reduced folate carrier (RFC) transport and membrane targeting was examined in transport-deficient K562 (K500E) cells transfected with human RFC cDNAs. Treatment of cells expressing wild-type RFC with tunicamycin (0–3 μg) resulted in a progressive shift of the ∼85 kDa RFC on western blots to 65 kDa. At 3 μg/ml tunicamycin, the nearly complete loss of glycosylated RFC was accompanied by a ∼25% decreased rate of methotrexate uptake. A deglycosylated RFC cDNA construct in which asparagine-58 was replaced by glutamine (Gln 58-RFC) was expressed in K500E cells as a 65 kDa protein and restored transport capacity for methotrexate and (6 S)5-formyl tetrahydrofolate. With both wild-type and Gln 58-RFC constructs, expression of cDNA-encoded RFC protein far exceeded relative levels of RFC uptake. Wild-type and Gln 58-RFCs containing a hemagglutinin (HA) epitope at the carboxyl terminus were similarly functional and, by immunofluorescence staining with rhodamine-conjugated anti-HA antibody, were localized to plasma membranes. Collectively, our results demonstrate that N-glycosylation of human RFC plays no significant role in either transport function or membrane targeting. The discrepancy between the stoichiometries of RFC expression and transport activity for both wild-type RFC and Gln 58-RFC implies that identical regulatory controls and/or non-RFC transport components are necessary to completely restore transport function in the transfected cells.