Calnexin-Assisted Biogenesis of the Neuronal Glycine Transporter 2 (GlyT2)
Figure 7
CNX-binding and folding properties of the N-glycan-deficient GlyT2 mutant.
(A) COS7 cells expressing the N1234 N-glycan-deficient mutant were pulse-labeled for 15 min with [35S]methionine/cysteine and chased for the indicated time. The cell lysates were immunoprecipitated with GlyT2 antibody or subjected to sequential immunoprecipitations with CNX and GlyT2 antibodies as described in the Materials and Methods and resolved by SDS-PAGE. Lower graph: densitometry of the fluorograms (n = 2) showing the percentage of N1234 bound to CNX in each condition (± S.E.M). (B) The membrane enriched fraction from COS7 cells expressing GlyT2 or the N1234 N-glycan-deficient mutant (12.5 µg) was digested for 15 min at 22°C with the concentrations of papain indicated as described in the Materials and Methods and analyzed in Western blots probed with antibodies against the GlyT2 N-terminus. (C) COS7 cells expressing wild-type GlyT2 or the N1234 mutant were treated with the vehicle alone (-) or 10 µg/ml tunicamycin (+) for 3 h at 37°C, pulse-labeled for 15 min with [35S]methionine/cysteine and then chased for 90 min at the temperature indicated. The cell lysates were immunoprecipitated with GlyT2 antibody or subjected to sequential immunoprecipitation with CNX and GlyT2 antibodies as described in the Materials and Methods and resolved by SDS-PAGE. Right panels: densitometry of the fluorograms (n = 3) showing the percentage of total GlyT2 precursor bound to CNX in each condition (up) and the total synthesized transporter (down). Bars represent the S.E.M. (n = 3). *p<0.05, **p<0.01 with respect to wild-type control (Student’s t-test). (D) The experimental conditions applied were as described in Figure 3B except that cells expressed the N1234 mutant and CNX overexpression was achieved by co-expressing CNX and N1234 at ratios of 0∶1, 4∶1 and 8∶1. *p<0.05, **p<0.01, ***p<0.001 with respect to the wild-type control (Student’s t-test).