Figure 4. UPS mediated degradation of ARTS by Parkin protects cells from apoptosis.

A. and B. Parkin inhibits apoptosis induced by ARTS. COS-7 cells were co-transfected with 1myc-Parkin and 6myc-ARTS. Over-expression of ARTS alone was sufficient to induce apoptosis in these cells. Apoptosis is exhibited by the presence of the apoptotic markers cleaved caspase-3 (A) and H2AX (B). Thus, overexpression of Parkin can strongly inhibit ARTS-induced apoptosis. C. Co-transfection of 1myc-Parkin and AU5-ARTS confers a protective effect against cell death. Cell viability was quantified using XTT-based assay (see Materials and Methods). The results are represented as a ratio between each transfection and the negative control (mean± SE, n = 6). The viability of cells transfected with empty vector was defined as the baseline viability of the assay. Cell viabilities were recorded in relation to this baseline. D. ARTS binds to Parkin both at the mitochondria and in the cytosol. Sub cellular fractionation was performed using 140 µg/ml digitonin. Apoptosis was induced using 1.5 µM STS for 2 h. Immunoprecipitation (IP) of ARTS from both cytosolic and mitochondrial fractions revealed basal binding of ARTS to Parkin under non apoptotic as well as under apoptotic conditions.