Fig. 4: CISD1 is destabilized by Parkin-dependent ubiquitination.

a Ubiquitination levels of CISD1 in HEK293T cells expressing Parkin WT or C431S (CS). The cells were treated with 20 µM MG132, 1 µM antimycin, and 10 µM oligomycin for 4 h and lysed for anti-Myc immunoprecipitation to compare the level of CISD1 ubiquitination by immunoblot analyses. Three independent experiments were conducted and the immunoblot band intensity for ubiquitinated CISD1 were quantitated (bar graphs in bottom). b Ubiquitination levels of CISD1 WT and K55R/K68R (K55/68R) double mutant in HEK293T cells expressing Parkin WT. The cells were treated with 20 µM MG132, 1 µM antimycin, and 10 µM oligomycin for 4 h and lysed for anti-Myc immunoprecipitation to compare the level of CISD1 ubiquitination by immunoblot analyses. Three independent experiments were conducted and the immunoblot band intensity for ubiquitinated CISD1 were quantitated (bar graphs in bottom). c Aligned CISD1 protein sequences of human (Q9NZ45.1), mouse (NP_598768.1), rat (B0K020.1), and Drosophila (Q9VAM6.1) with indication of the conserved lysine residues in red boxes. Three-dimensional human CISD1 protein structures, RCSB PDB-2QD0¹¹, show K55 and K68 ubiquitination sites using PyMOL v2.4.0. d Immunoblot analyses of CISD1 protein showed its reduced stability by Parkin-dependent K55 and K68 ubiquitination in HEK293T cells. The cells were treated with 100 µg/µl cycloheximide (CHX) for 12 or 16 h and analyzed the level of CISD1 protein. Three independent experiments were conducted and the immunoblot band intensity for CISD1 were quantitated (bar graphs in right). One-way ANOVA with Tukey’s multiple comparisons test was used (a, b, d). ****p < 0.0001. ***p < 0.001. **p < 0.01. *p < 0.05. ns represents not significant. Source data and the exact p values are included within the Source Data file. All data are presented as mean ± SD.