Fig. 5. Overexpression of SKD1(E235Q) delays the maturation of cathepsin D and induces the hypersecretion of pro-cathepsin D. GFP-SKD1(E235Q)-expressing and normal NRK cells were pulse-labeled with [³⁵S]-methionine/cysteine for 15 minutes and chased for the periods indicated. The cell lysates and medium were processed for immunoprecipitation with anti-rat cathepsin D antibody and the immunoprecipitates were subjected to SDS-PAGE followed by autoradiography. In the uninfected cells, more than 40% of newly synthesized pro-cathepsin D (45 kDa) was processed into the mature form (43 kDa) within 1 hour and over 95% within 3 hours. In contrast, in infected cells, more than 90 and 50% of the pro-form remained after 1 and 3 hour of chasing, respectively. Hypersecretion of pro-form by the infected cells was also observed.