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First published online 20 November 2002
doi: 10.1242/jcs.00214

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Secretory ribonucleases are internalized by a dynamin-independent endocytic pathway

Marcia C. Haigis^1,* and Ronald T. Raines^1,2,

¹ Department of Biochemistry, University of Wisconsin—Madison, Madison, Wisconsin 53706, USA
² Department of Chemistry, University of Wisconsin—Madison, Madison, Wisconsin 53706, USA
^* Current address: Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA

View larger version (37K): [in a new window]   Fig. 1. Three-dimensional structure of ribonuclease A and Onconase®. Ribbon diagrams of (A) RNase A and (B) ONC were created using the atomic coordinates derived by x-ray diffraction analysis (Wlodawer et al., 1988; Mosimann et al., 1994) and the program MOLSCRIPT (Kraulis, 1991). The location of the active site clefts and residues 19 (RNase A) and 16 (ONC), which were replaced with cysteine for labeling experiments, are indicated.

View larger version (69K): [in a new window]   Fig. 2. Binding of unlabeled RNase A, G88R RNase A and ONC to the surface of K-562 cells. Cells were incubated with a ribonuclease (1 µM) for 30 minutes at 4°C. Cells were then washed, fixed and processed for indirect immunofluorescence with antibodies generated against either RNase A or ONC. The appropriate FITC or TRITC-conjugated secondary antibody was used to visualize RNase A (green), G88R RNase A (green) and ONC (red) binding. Negative control samples were incubated in PBS in the absence of protein and processed with primary and secondary antibodies as described above.

View larger version (22K): [in a new window]   Fig. 3. Effect of metabolic inhibitors on ribonuclease-mediated cytotoxicity. (A) Cells were incubated with NaN3 (10 mM) and 2-deoxyglucose (50 mM) or PBS control for 1 hour at 37°C. ONC (10 µM) or K7A/G88R RNase A (25 µM) was then added, and cells were incubated for an additional 6 hours. DNA synthesis was measured as described in Materials and Methods. (B) Cells were incubated with NaN3 (10 mM) and 2-deoxyglucose (50 mM). BODIPYRNase A (1 µM) was then added, and cells were incubated for an additional 30 minutes at 4°C. Cells were pulsed at 37°C for 30 minutes, and the fluorescence of BODIPYRNase A was visualized directly.

View larger version (30K): [in a new window]   Fig. 4. Binding and internalization of RNase A. (A) JAR cells were incubated with BODIPYRNase A (1 µM) for 30 minutes at 4°C, and their fluorescence was visualized directly or after a 5 minute incubation at 37°C. (B) K-562 cells were treated as in A. (C) K-562 cells were incubated with unlabeled RNase A as described in A. After a 5 minute incubation at 37°C, cells were visualized directly or fixed, and internalized RNase A was detected using an appropriate primary and secondary antibody. (D) K-562 cells were incubated with fluoresceinRNase A (open; 1 µM) or OGRNase A (filled; 1 µM) for 20 minutes at 4°C. Fluorescence intensity was measured after a 0-6-minute incubation at 37°C with a FACScan flow cytometer. Each data point represents the mean(±s.e.) of the fluorescence from 10,000 cells in two separate experiments.

View larger version (145K): [in a new window]   Fig. 5. Co-internalization of BODIPYRNase A and OGONC in K-562, JAR, and HeLa cells. Cells were incubated with OGONC (green) or BODIPYRNase A (red; 1 µM, 10% labeled) for 20 minutes at 4°C. After a 5 minute incubation at 37°C, cells were washed three times with ice-cold PBS and fixed and their fluorescence was visualized directly.

View larger version (33K): [in a new window]   Fig. 6. Dose-dependent internalization of OGRNase A and OGONC. (A) HeLa cells were grown on coverslips in the wells of a six-well plate, incubated with 30% labeled OGRNase A or OGONC (0.01, 0.1, 1 and 10 µM), washed, and fixed as described for Fig. 5. The arrows correspond to the IC50 values for G88R RNase A and ONC (Table 2). (B) HeLa cells were pulsed with OGRNase A (circles) or OGONC (squares) as described for A, except that cells were treated with trypsin before fixation. Fluorescence was quantified using a FACScan flow cytometer. Each data point represents the mean (±s.e.) of the fluorescence from 10,000 cells in two separate experiments.

View larger version (104K): [in a new window]   Fig. 7. Endocytosis in K-562 cells. K-562 cells were co-incubated with BODIPYRNase A (1 µM) and either FM1-43 (1 µg/ml) or BODIPY-FL-labeled transferrin (50 µg/ml) for 20 minutes at 4°C. Cells were pulsed at 22°C for 15 minutes, washed and fixed. The fluorescence of BODIPYRNase A (red), FM 1-43 (green) and BODIPY-FL-labeled transferrin (green) was visualized directly.

View larger version (23K): [in a new window]   Fig. 8. Effect of dynamin on ribonuclease-mediated cytotoxicity. (A) K44A Dyn cells grown for 2 days in the presence (induced) or absence (uninduced) of tetracycline were fixed and stained with TRITC-conjugated phalloidin to detect actin filaments (red). The production of wild-type dynamin and its K44A variant was monitored by immunoblotting using anti-HA antibodies. (B) The toxicity of G88R RNase A and ONC was measured for cells overproducing wild-type dynamin or K44A dynamin. Cells were grown in the absence of tetracycline for 2 days to induce overproduction. Then, increasing concentrations of G88R RNase A and ONC were added. After a 48 hour incubation at 37°C, DNA synthesis was measured as described in Materials and Methods. As a control, the effects of G88R RNase A and ONC were measured on cells not overproducing either form of dynamin. Each point represents the percent of [methyl-3H]thymidine incorporated compared to a PBS control and is the mean (±s.e.) of at least three separate experiments with triplicate samples.

View larger version (38K): [in a new window]   Fig. 9. Dynamin-independent internalization of RNase A. (A) Induced and uninduced K44A Dyn cells were co-incubated with OGRNase A (1 µM) or TAMRAtransferrin (50 µg/ml) for 20 minutes at 4°C. After a 15 minute incubation at 22°C, cells were washed and fixed; and the fluorescence of OGRNase A (green) and TAMRAtransferrin (red) was visualized directly. (B) Fractions of total uninduced (white) or induced (hatched) cells that contained internalized OGRNase A or TAMRAtransferrin. Data are the mean (±s.e.) of two separate experiments with n=65 and 105 cells for uninduced and induced samples, respectively.

View larger version (20K): [in a new window]   Fig. 10. Effect of drugs on ribonuclease-mediated cytotoxicity. The effect of G88R RNase A and ONC inhibition of cellular DNA synthesis was measured in the presence of NH4Cl (20 mM), monensin (10 µM) and brefeldin A (10 µM). K-562 cells were pre-incubated with each drug for 2 hours at 37°C and then with G88R RNase A (circles) and ONC (squares). Open symbols represent control samples without drug treatment; filled symbols represent samples pre-incubated with drugs. Each point represents the percentage of [methyl-3H]thymidine incorporated compared with a PBS control (with or without drug) and is the mean (±s.e.) of at least three separate experiments with triplicate samples.

View larger version (29K): [in a new window]   Fig. 11. Model of the cytotoxic internalization of ribonucleases. RNase A and ONC bind to the cell surface and are internalized by dynamin-independent endocytosis. Like diptheria toxin, both ribonucleases probably reach the cytosol by translocation from an endosome. Unlike diphtheria toxin, the low pH of acidic endosomes is not a prerequisite for the translocation of RNase A and ONC to the cytosol. The entry of ONC to the Golgi is inefficient for cytotoxicity. Unlike ricin and Shiga toxin, both ribonucleases reach the cytosol by translocation from a pre-ER compartment.