Figure 1.

MiD49/51 are mitochondrial outer-membrane proteins that affect mitochondrial morphology. (A) Sequence alignment with conserved residues shown in bold and predicted transmembrane domains underlined. (B) COS7 cells expressing GFP fusion constructs and stained with MitoTracker Red (red) and Hoechst (blue; upper panels). HeLa cells expressing MiD–GFP fusion constructs and immunostained for cytochrome c or Tom20 (lower panels). (C) COS7 cells expressing different constructs were blind-counted for mitochondrial morphology (mean±s.e.m., n=3, 100 cells counted per experiment). (D) Fractionated COS7 cells were analysed by immunoblotting using antibodies as indicated. (E) COS7 mitochondria were subjected to proteinase K (PK) or sodium carbonate (Na Carb) extraction, followed by western blot analysis using antibodies as indicated. (F) MiD49¹⁻⁵⁰–GFP and MiD49⁵⁰⁻⁴⁵⁴–GFP were expressed in COS7 cells and stained with MitoTracker Red and Hoechst. Scale bars, 20 μm. GFP, green fluorescent protein; IMS, intermembrane space; MiD49/51, mitochondrial dynamics proteins of 49 and 51 kDa; Mito, mitochondria; P, pellet; S, supernatant; TMD, transmembrane domain.