FIGURE 2:

MARCH5 controls ubiquitination and proteasomal degradation of MiD49. (A) Total cell lysates obtained from HeLa cells transfected with five different MARCH5 shRNA constructs (1–5; lanes 4–8), two different p97 shRNA constructs (lanes 2 and 3), or a GFP shRNA construct (Control; lane 1) were analyzed by Western blot as indicated. (B) Wild-type and MARCH5^−/− cells were subjected to cell fractionation to obtain total-cell lysates (T), mitochondria-enriched heavy membrane fractions (M), and postmitochondrial cytosolic fractions (C), followed by Western blot as indicated. *, an x-reactive band detectable with anti-MARCH5 antibody. (C) Western blot analysis of vector (Control)- and MYC-MARCH5 (MARCH5)-transfected MARCH5^−/− cells. Cells were analyzed for the levels of MiD49 and MYC-MARCH5. Tom20 was used as a loading control. (D) Wild-type and MARCH^−/− cells were treated with CHX as indicated, followed by Western blot to detect MiD49 (two exposures of the same MiD49 blot are shown), Drp1 and Mff. Tom20 served as a loading control. (E and F) Relative MiD49 (E) and Mff (F) protein levels in wild-type and MARCH^−/− cells were quantified and plotted as a function of time of CHX treatment. Protein levels detected in untreated samples (0 min) were set at 1. (G and H) Control or MG132-treated (8 h) wild-type and MARCH5^−/− cells transfected with MYC-MID49 were subjected to MYC immunoprecipitation under denatured conditions. Samples were analyzed by Western blot for MYC-tag (to detect MYC-MiD49; G) and Ub (H) as indicated (top panels in G and H). Inputs (1% of lysates used for immunoprecipitation) are shown in the bottom panels in G and H. (I) MARCH5^−/− cells transfected with MYC-MiD49 alone, cotransfected with MYC-MiD49 and YFP-MARCH5 or MYC-MiD49 and YFP-MARCH5^H43W, or MYC-MiD49 and YFP-vector were treated with MG132 for 8 h, followed by MYC immunoprecipitation under denatured conditions and then Western blot as indicated. *, antibody heavy chain.