The role of N-glycosylation in transport function and surface targeting of the human solute carrier PAT1

Abstract 

In the present study we show in the Xenopus laevis expression system that the proton-coupled amino acid transporter 1 (PAT1, SLC36A1) is glycosylated at asparagine residues N174, N183 and N470. To determine the functional role of N-glycosylation, glycosylation-deficient mutants were analyzed by two-electrode voltage-clamp measurements after expression in X. laevis oocytes. Single replacements of asparagine residues had no effect on transport activity. However, multiple substitutions resulted in a decreased transport rate, leaving K_t unchanged. Immunofluorescence localisation revealed a diminished plasma membrane expression of glycosylation-defective mutants. This indicates that N-glycans are not required for transport function, but are important for membrane targeting.

Abbreviations: HA, hemagglutinin, LYAAT1, lysosomal amino acid transporter, PAT1, proton-coupled amino acid transporter 1, PAGE, polyacrylamide gel electrophoresis, PNGase F, Peptide: N-glycosidase F, SLC, solute carrier, X. laevis, Xenopus laevis

Keywords: hPAT1, Solute carrier, Glycosylation, Peptide: N-glycosidase F, Xenopus laevis oocyte