Fig. 5. The coiled-coil domains of Mfn2 interact with each other and mediate mitochondrial localization of mistargeted Mfn2-fragments. (A-D) HeLa cells were co-transfected with plasmids encoding Myc-tagged Mfn2-fragments (MF2-NT, MF2-IYFFT) and with a plasmid encoding the transmembrane and C-terminal domain of Mfn2 (F2-TMCT). Molecules were visualized with antibodies against myc and CT, respectively. (A) Soluble Mfn2-fragments truncated before the transmembrane domain (MF2-NT) localize to mitochondria upon co-expression of the transmembrane and C-terminal domains of Mfn2 (F2-TMCT). (B) MF2-NT molecules devoid of their coiled-coil domain (MF2-NTC1) remain cytosolic despite the co-expression of F2-TMCT. (C,D) Mfn2-mutants normally targeted to the endoplasmic reticulum (MF2-IYFFT) localize to mitochondria upon co-expression of F2-TMCT. (D) Co-expressed Mfn2-fragments can redistribute and cluster mitochondria in the perinuclear region. (E) Cells transfected with the indicated plasmids were processed for immunofluorescence and the localization of MF2-NT or MF2-IYFFT was determined. Where indicated (C1, C2), molecules were devoid of their coiled-coil domains. The results are expressed as percentage of transfected cells in which MF2-NT or MF2-IYFFT localize to mitochondria (n200 cells for each category). The mitochondrial relocalization of MF2-NT and MF2-IYFFT by F2-TMCT depends on the presence of coiled-coil domains on both Mfn2-fragments. Bars, 15 µm.