FIGURE 1:

OMA1 and YME1L differentially regulate OPA1 fusion activity. (A) Schematic of mouse OPA1 protein isoforms, showing the origin of protein bands a–e. MEFs express isoforms 1, 5, 7, and 8. Isoforms 1 and 7 produce long forms that constitute bands a and b and short forms that constitute bands c–e. Isoforms 5 and 8 produce exclusively short forms expected to comigrate with bands c–e, but for simplicity, bands c–e are labeled according to their origin from isoforms 1 and 7. The MPP (mitochondrial processing peptidase), S1 (by OMA1), and S2 (by YME1L) cleavage sites are indicated with arrows. The orange line and arrows show that bands c and d are derived from long isoform 7/a, and the pink line and arrow show that band e is derived from the long isoform 1/b. (B) Western blot analysis of OPA1 bands in Oma1 and Yme1l mutant MEFs. Five OPA1 bands (a–e) are apparent in wild-type MEFs. Oma1-null MEFs lack c and e; Yme1l-null MEFs lack d; and Oma1/Yme1l-null MEFs lack all the short forms, c–e. Tubulin was used as loading control. (C) Representative images of mitochondrial morphology (mito-DsRed) in WT and protease-null MEFs in different media. GLY: high glucose medium; OXI: OXPHOS-inducing medium; CHX: high glucose medium with 10 μM cycloheximide. Insets show magnified view. Scale bar, 5 μm. (D) Quantification of mitochondrial morphology of cells in C. In each experiment, 100 cells were scored. Error bars show SD from three independent experiments. (E) Comparison of mitochondrial fusion rates in vivo in different media between WT and Oma1/Yme1l-null MEFs. Fusion activity was measured by the intensity reduction of PA-GFP as a function of time. Error bars represent SDs from at least six independent measurements.