N-linked glycosylation and its impact on the electrophoretic mobility and function of the human proton-coupled folate transporter (HsPCFT)

The human proton-coupled folate transporter (HsPCFT, SLC46A1) mediates intestinal absorption of folates and transport of folates into the liver, brain and other tissues. On Western blot, HsPCFT migrates as a broad band (similar to 55 kDa), higher than predicted (similar to 50 kDa) in cell lines. Western blot analysis required that membrane preparations not be incubated in the loading buffer above 50 degrees C to avoid aggregation of the protein. Treatment of membrane fractions from HsPCFT-transfected HeLa cells with peptidyl N-glycanase F, or cells with tunicamycin, resulted in conversion to a similar to 35 kDa species. Substitution of asparagine residues of two canonical glycosylation sites to glutamine, individually, yielded a similar to 47 kDa protein; substitution of both sites gave a smaller (similar to 35 kDa) protein. Single mutants retained full transport activity; the double mutant retained a majority of activity. Transport function and molecular size were unchanged when the double mutant was hemagglutinin (HA) tagged at either the NH2 or COOH terminus and probed with an anti-HA antibody excluding degradation of the deglycosylated protein. Wild-type or deglycosylated HsPCFT HA, tagged at amino or carboxyl termini, could only be visualized on the plasma membrane when HeLa cells were first permeabilized, consistent with the intracellular location of these domains. (C) 2008 Elsevier B.V. All rights reserved.