Figure 3.

Sulfated POMC products migrate through intracellular compartments with increasing densities. (A) Sulfated POMC was recovered in light-density membranes immediately after pulse labeling. To localize the compartment for sulfation of POMC, AtT-20 cells were labeled with [³⁵S]sulfate for 5 min and homogenized. Membranes from a PNS were fractionated on a D₂O-Ficoll density gradient and labeled POMC products in each fraction were analyzed by SDS-PAGE and PhosphorImager. Under this condition, only unprocessed POMC was recovered. □, Relative amount of sulfate-labeled POMC (in arbitrary scanning units). To localize a constitutive secretory marker on this gradient, AtT-20 cells were treated with xyloside for 30 min and then pulse-labeled with [³⁵S]sulfate for 5 min to label GAG chains. Cells were homogenized and membranes were fractionated as above. The amount of labeled GAG chains in each fraction was assayed by a CPC precipitation/filtration assay. •, Amount of GAG-associated radioactivity (in cpm) in each fraction. To localize rab 11-containing membranes on the same gradient, AtT-20 cells stably transfected with an influenza hemagglutinin epitope-tagged rab 11 were used, and rab11 immunoreactivity in each fraction was determined by immunoblotting using anti-hemagglutinin antibody. ○, Relative intensity obtained from scanning the Western blot. (B) Cells were continuously labeled with [³⁵S]sulfate for 3 h. Xyloside was added to the cells during the last 30 min to label newly synthesized [³⁵S]GAG chains. Cells were then homogenized and the membranes were fractionated as above. One half of each fraction was assayed for GAG, and the other half was subjected to immunoprecipitation with anti-ACTH. •, Amount of GAG-associated radioactivity (in cpm) in each fraction; □, distribution of labeled mature ACTH in arbitrary scanning units. Because no chase was performed, a small peak corresponding to newly labeled POMC was also present on the top of this gradient (not shown). POMC, GAG, and rab11 peak at a density of 1.12 g/ml, and mature ACTH peaks at a density of 1.20 g/ml.