Figure 6. Mitofusin degradation by the proteasome is dependent on VCP.

A. Western blots in YFP-Parkin stable HeLa cells against MFN1 and 2, VDAC and tubulin at different time points after CCCP treatment. Ubiquitinated forms of MFNs 1/2 can be observed migrating more slowly. B. Western blots in YFP-Parkin stable HeLa cells against MFN1/2, VDAC, and tubulin. Cells were treated for 12 h with CCCP and either proteasome inhibitors (MG132 or epoxomicin) or the autophagy inhibitor bafilomycin. Ubiquitinated forms of MFN1/2 can be again observed migrating more slowly, particularly with proteasome inhibition. C. Quantification of total MFN expression levels normalized against tubulin in YFP-Parkin stable HeLa cells treated for 12 h with CCCP and either proteasome inhibitors (MG132 or epoxomicin), or the autophagy inhibitor bafilomycin. Error bars indicate standard deviation from triplicates. D. Knockdown of VCP stabilizes MFNs 1 and 2. Western blots in YFP-Parkin stable HeLa cells against MFN1/2, VCP and tubulin. Cells were transfected with non-targeting or VCP-targeting siRNA and treated for 12 h with DMSO or CCCP. E. FLAG IP in HeLa cells cotransfected with YFP-Parkin and VCP-FLAG and treated with CCCP for the indicated times. Immunoprecipitation samples were immunoblotted against MFN1/2 and VCP. Following mitochondrial depolarization VCP interacts with MFN2. F. Total dMfn-HA accumulates in PINK1^B9 (lane 2) and Park²⁵ (lane 3) null mutants. Notably, ubiquitinated dMfn is decreased in PINK1^B9 null mutants (lane 2) and absent in Park²⁵ null mutants (lane 3). G. Overexpression of dVCP in the compound eye destabilizes dMfn-HA (lane 1), whereas knockdown of endogenous dVCP in the compound eye leads to accumulation of ubiquitinated dMfn-HA (lane 3). H. Immunoprecipitation of HA-dMfn and endogenous (protein-trap) GFP-dVCP from Drosophila brain extract.