Wild-type and mutant forms of rab7 are inducibly expressed in stable transfectants. Stable BHK fibroblasts were cultured in the absence of tetracycline (−Tet) for various times to induce the expression of recombinant rab7 proteins. Duplicate control samples were maintained continuously in media containing 3 μg/ml tetracycline (+Tet). (A) Cell lysates prepared 24 h after transfer to medium lacking tetracycline compared to control samples. (B) A representative time course of rab7 induction upon transfer of cells to medium lacking tetracycline for 0–18 h. Immunoblots were probed with antibodies against rab7 and actin (as a control for protein loading). In all cases, an affinity-purified anti-rab7 antibody was used. The antibody was typically diluted to detect primarily the overexpressed rab7 protein. In the case of the rab7N125I samples shown in B, slightly higher levels of antibody were used to allow simultaneous detection of the endogenous wild-type rab7 protein and the induction of the mutant protein. rab7wt, cells expressing wild-type rab7; rab7N125I, cells expressing a mutant form of rab7. An immunoblot for actin served as a control for protein loading.

CI-MPR and its ligands accumulate in light membranes in cells expressing the mutant protein rab7N125I. Stable BHK fibroblasts were cultured in the absence of tetracycline for 18 h to allow for overexpression of wild-type and mutant rab7 proteins. PNS were prepared and fractionated on Percoll gradients, as described in Materials and Methods. Percoll gradient fractions derived from cells (A) overexpressing wild-type rab7 and (B) overexpressing rab7N125I. The distribution of early endosomes was monitored using rab5 and EEA1 as markers. Late endosomes were detected using wild-type rab7 (wt) as a marker; N125I denotes the overexpressed mutant protein. Lysosomes were detected using lgp120 as a marker. The distributions of CI-MPR and hamster cathepsin D (P, procathepsin D; I, intermediate form) were followed using specific antibodies. All immunoblot analysis was conducted as detailed in Materials and Methods. (C) The distribution of β-hexosaminidase was measured enzymatically after Percoll gradient fractionation of cells expressing wild-type rab7 (closed squares, solid line) or rab7N125I (open squares, dashed line) proteins. All immunoblots are representative examples of data from four independent trials. The β-hexosaminidase activities represent average values of three separate experiments. SD values have been omitted for clarity. The average SD was ±0.94 for the wild-type rab7 samples and ±0.54 for the rab7N125I samples.

Newly synthesized CI-MPR and immature cathepsin D are present in early endocytic compartments in cells expressing rab7N125I. Stable BHK fibroblasts were cultured in the absence of tetracycline for 18 h to allow for overexpression of wild-type and mutant rab7 proteins. Cells were metabolically labeled and incubated for 30 or 120 min in medium containing excess unlabeled amino acids. During the final 10 min of the chase period, HRP was added to a final concentration of 5 mg/ml. Cells were subsequently cooled on ice, and individual dishes were subjected to DAB cross-linking (+) and controls were left untreated (−) (details in Materials and Methods). After cell lysis and removal of cross-linked material by centrifugation, immunoprecipitation of (A) hamster cathepsin D, (C) hamster CI-MPR, or (E) hamster lgp120 was carried out as detailed in Materials and Methods. Positions of procathepsin D (P), the single chain intermediate (I) form of the enzyme, the heavy and light chains of the two-chain mature form (M_H, M_L) of cathepsin D, and the immature (I) and mature (M) forms of CI-MPR and lgp120 are indicated. Quantitation of the amounts of (B) intermediate cathepsin D, (D) mature CI-MPR, and (F) mature lgp120 immunoprecipitated with (+) or without (−) DAB cross-linking. Samples were derived from cell lysates overexpressing wild-type (wt) rab7 or mutant rab7N125I proteins, as indicated. All values were normalized to total protein and are the average of triplicate determinations (+SD) from one of three representative and independent experiments.

Light membranes containing CI-MPR and its ligands are derived from the endocytic pathway and not from the Golgi complex. Stable BHK fibroblasts were induced to overexpress the mutant rab7N125I protein by culture in the absence of tetracycline for 18 h. Mannose 6–phosphate was included in the culture medium during this induction period to prevent the reinternalization of secreted ligands. HRP (5 mg/ ml) was internalized for 30 min, and the Golgi was labeled with NBD-ceramide, as described in Materials and Methods. PNS were prepared and fractionated on Percoll gradients. The top four fractions were divided into two aliquots; one aliquot was subjected to DAB cross-linking (+), and the other aliquot was left untreated (−) as a control. After the removal of cross-linked material by centrifugation, the supernatant fractions were analyzed. (A) Schematic outline of the experiment. (B) Immunoblots for hamster cathepsin D (cath) (P, procathepsin; I, intermediate form) and rab5. (C) The activity of α-mannosidase II (Golgi marker, units × 1) and β-hexosaminidase (units x 1.5) were determined enzymatically, while NBD-sphingolipids (a trans-Golgi marker) were monitored fluorometrically (relative fluorescence ×40). The activities measured in each of the top three gradient fractions with (+) or without (−) DAB treatment have been plotted as averaged values with SD shown.

CI-MPR distribution is altered in cells expressing a mutant form of rab7. Stable BHK fibroblasts were cultured in the absence of tetracycline for 18 h to allow for overexpression of wild-type (rab7wt) or mutant rab7N125I proteins. Cells were then fixed and stained with an affinity-purified polyclonal antibody directed against CI-MPR (left) or a mouse mAb against lgp120 (right). Antibody complexes were visualized with appropriate secondary antibodies conjugated to Texas red or FITC.

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 (M_H, M_L) 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.

Rab7N125I expression causes newly synthesized CI-MPR and immature cathepsin D to accumulate in light membrane fractions. Stable BHK fibroblasts were cultured in the absence of tetracycline for 18 h to allow for overexpression of wild-type and mutant rab7 proteins. Cells were metabolically labeled and, after a 2-h chase period, were subjected to Percoll gradient fractionation, as described in Materials and Methods. Before immunoprecipitation, fractions were pooled as follows: pool I, fractions 1–4; pool II, fractions 5–8; and pool III, fractions 9–12. (A) Cathepsin D or (B) CI-MPR were immunoprecipitated from pooled fractions 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 (M_H, M_L) of cathepsin D are indicated. The data shown are representative of two independent trials.