Fig. 1.

Effect of IFG on N370S GlcCerase biosynthesis and trafficking. (A) Molecular structure of IFG. (B) Cells were cultured for 5 days with or without 30 μM IFG before labeling with 750 μCi of [³⁵S]methionine/[³⁵S]cysteine for 1 h. After labeling medium was replaced with normal growth medium, cells were harvested at the indicated times, and the GlcCerase was immunoprecipitated and analyzed by SDS/PAGE and autoradiography, as described in Methods. (C) Cells cultured in the absence of IFG were labeled with 750 μCi of [³⁵S]methionine/[³⁵S]cysteine for 1 h. After the labeling medium was replaced with normal growth medium, the cells were chased for various times. In lanes 5–7, IFG was added to a final concentration of 30 μM at the chase times indicated. Newly synthesized GlcCerase molecules were immunoprecipitated and treated with (lanes 2 and 8) or without peptide:N-glycosidase F (PNGase F) (lanes 1, 3–7) to remove all N-glycans before SDS/PAGE and autoradiography. The asterisks in B and C denote nonspecific bands.