Figure 2.

OPA1 cleavage by OMA1 occurs in the IMS. Mitochondria were isolated from Oma1^−/− MEFs expressing OMA1-myc.

1. Submitochondrial localisation of OMA1. Mitochondria and mitoplasts, which were generated by hypotonic disruption of the OM (swelling), were incubated with or without proteinase K (PK; 50 μg/ml) and analysed by SDS–PAGE and immunoblotting. The IMS protein Smac and the matrix protein Hsp60 served as controls. Mitochondrial membranes were solubilised with Triton X-100 where indicated. The slightly reduced level of Smac upon PK treatment of mitochondria indicates partial disruption of the OM upon purification of mitochondria.
2. OMA1 is an integral membrane protein. Alkaline extracts of mitochondria in Na₂CO₃ (pH 11.5) were separated into soluble (S) and membrane (P) fractions by centrifugation and analysed by SDS–PAGE and immunoblotting. The integral inner membrane protein PHB2 and the soluble intermembrane space protein Smac served as controls. T, total.
3. Topological model of mature OMA1 in the IM. The C-terminal M48 metallopeptidase domain is exposed to the IMS, whereas the N-terminal domain is present at the matrix side of the IM.
4. FLAG-tagged OPA1 Sp1 or OPA1 Sp1-TCS, which contains a TEV-cleavage site (TCS) instead of S1, were transfected into cell lines allowing tetracycline-inducible expression of HA-tagged Su9-TEV (targeted to the matrix) or Smac-TEV (targeted to the IMS). Cells were treated with tetracycline when indicated and analysed by SDS–PAGE and immunoblotting.

Source data are available online for this figure.