Figure 8. Cdc48 regulates mitochondrial fusion via Ubp12 and Ubp2.

(A) Steady state levels of Fzo1 in Δubp2 Δubp12 upon mutation of CDC48. Total cellular extracts of wt, cdc48-2, Δubp2 Δubp12 and Δubp2 Δubp12 cdc48-2 cells were analyzed by SDS-PAGE and immunoblotting using Fzo1- and Tom40-specific antibodies. Bottom panel, quantification of five independent experiments, including SD. (B) Steady state levels of Fzo1 in Δubp2 cells upon deletion of CDC48. Total cellular extracts of wt, cdc48-2, Δubp2 and Δubp2 cdc48-2 cells were analyzed by SDS-PAGE and immunoblotting using Fzo1- and Tom40-specific antibodies. Bottom panel, quantification of five independent experiments, including SD. (C) Mitochondrial morphology of cdc48-2 cells upon overexpression of Ubp2. Wt or cdc48-2 mutant cells expressing Ubp2 or the corresponding empty vector were analyzed for mitochondrial tubulation after expressing a mitochondrial-targeted GFP plasmid, as in Figure 1A. Quantification from three different experiments (with more than 200 cells each), including SE, as described (Cumming et al., 2007). ns, p>0.05. **p≤0.01, ***p≤0.001 (One-way ANOVA, Tukey’s multiple comparison test). (D) Role of Ubp2 overexpression on the respiratory capacity of CDC48-deficient cells. A spot assay was performed as described in Figure 4B with the indicated cells but using synthetic media supplemented with lactate (SCLac) and incubated for 4 days. PoS, Ponceau S staining.

Figure 8—figure supplement 1. Cdc48 regulates mitochondrial fusion via Ubp12 and Ubp2.

Physical interaction between Ubp2 and Cdc48. The catalytically inactive variant Ubp2^C745S-Flag or the corresponding empty vector were expressed in Δubp12 cells and analyzed for Cdc48 interaction, as in 2A. Crude mitochondrial extracts were lysed and Flag-tagged Ubp2^C745S was precipitated using Flag-coupled beads. The eluate was analyzed by SDS-PAGE and immunoblotting using Flag- and Cdc48-specific antibodies. PoS, Ponceau S staining; IP, immunoprecipitation; WB, western blot.