Figure 4.

L- or S-OPA1 alone is sufficient to support mitochondrial respiratory function. A, OXPHOS assessment by cell growth in galactose media. All cell lines including OPA1-KO cells grew well in the glycolytic media. In galactose media, OPA1-KO did not grow, whereas WT and OPA1 variant cells grew with no discernible difference in their growth rates. Experiments were done in quadruplicate and repeated three times. Representative data are presented. B and C, oxygen consumption rates (OCR) of OPA1 variant cells show no difference from that of WT, whereas OPA1-KO cells are respiration-deficient. The respiration control ratios of uncoupled maximum respiration (state 3u) to leak respiration under oligomycin (state 4o) were indistinguishable in OPA1 variants and WT. n = 6. Error bars are S.E. ***, p < 0.0001; #, p = 0.0002; **, p = 0.0089 (one-way ANOVA with Tukey's post hoc test). D and E, immunoblotting of WT and OPA1 variant cells in galactose and SIMH conditions. Cells were incubated in 10 mm glucose (Glc) or galactose (Gal) for 24 h (D). For SIMH, cells were incubated in HBSS for 3 h (Strv) or in 10 μm cycloheximide (Chx) for 6 h (E). There are no changes in L- and S-OPA1 in galactose and HBSS incubations. Cycloheximide incubation appears to increase L-OPA1 cleavage in WT and OPA1-v1 cells. Olm, oligomycin; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; Con, control.