Figure 4.

MiD49/51 recruit GFP–Drp1 to the mitochondrial surface. (A) COS7 cells expressing GFP–Drp1 alone or with MiD49, MiD51 or huMilt1 were blind-counted for GFP–Drp1 localization (mean±s.e.m., n=3, 100 cells counted per experiment). (B) Mouse embryonic fibroblasts (MEFs) before (−4HT) and 24 h after (+4HT) induction of MiD51 were stained with MitoTracker Red (red) and visualized by epifluorescence. Western blot analysis confirmed MiD51 induction, with mitochondrial matrix B17.2L as a control. Scale bars, 20 μm. (C) MEFs transfected with GFP constructs were visualized by epifluorescence before (−4HT) and 24 h after (+4HT) MiD51 induction. (D) Western blot analysis of whole-cell (W), mitochondria (M) and cytosolic (C) fractions from MEFs before (−4HT) and after (+4HT) MiD51 expression. Cytosolic Hsc70 and mitochondrial matrix B17.2L acted as controls. (E) By using a yeast two-hybrid assay, a direct interaction between truncated MiD49^ΔN120 and Drp1 was observed, similar to the positive control p53 and large T-antigen. MiD49^ΔN120 did not interact with the soluble domain of hFis1 or the large T-antigen, similar to the negative control Lamin and T-antigen. (F) Coimmunoprecipitation of endogenous Drp1 with MiD49–GFP following 20-μM bis(maleimido)hexane crosslinking of HeLa cells. Western blot analysis was conducted with antibodies as indicated. Arrow indicates crosslinked species. Drp1, dynamin-related protein 1; GFP, green fluorescent protein; MiD49/51, mitochondrial dynamics proteins of 49 and 51 kDa; Miro1, Mitochondrial Rho GTPase 1; WB, western blot; 4HT, 4-hydroxytamoxifen.