Logo of jcellbiolHomeThe Rockefeller University PressEditorsContactInstructions for AuthorsThis issue

Figure 5

An external file that holds a picture, illustration, etc.
Object name is JCB14530.f5a.jpg
An external file that holds a picture, illustration, etc.
Object name is JCB14530.f5b.jpg

Rab7N125I expression leads to diminished cathepsin D processing in endosomes, but Golgi processing remains normal. Stable BHK fibroblasts were cultured in the absence of tetracycline for 18 h to allow for overexpression of wild-type and mutant rab7 proteins. (A) Processing of cathepsin D to the intermediate species is kinetically delayed, and formation of the mature protein is inhibited in cells overexpressing rab7N125I. Cells were metabolically labeled and then transferred to medium containing excess unlabeled amino acids and mannose 6–phosphate to prevent reinternalization of secreted ligand. After the indicated times, cells were collected and hamster cathepsin D was immunoprecipitated as described in Materials and Methods. Immunoprecipitates were resolved by SDS-PAGE, and positions of procathepsin D (P), the single chain intermediate (I) form of the enzyme, and the heavy and light chains of the two-chain mature form (MH, ML) of cathepsin D are indicated. (B) Acquisition of complex carbohydrates by CI-MPR in the Golgi follows similar kinetics in cells expressing wild-type or mutant rab7N125I proteins. Cells were metabolically labeled and harvested as described above. Hamster CI-MPR was immunoprecipitated as detailed in Materials and Methods, and immunoprecipitates were resolved by SDS-PAGE. Positions of the immature (I) and mature (M) forms of the receptor are indicated. Data shown are representative of three independent trials.

Images in this article

  • Figure 4
  • Figure 1
  • Figure 2
  • Figure 3
  • Figure 5
  • Figure 7
  • Figure 6
Click on the image to see a larger version.