Fig. 3. Membrane topology of mitofusin 2. (A) Structure of myc-tagged (M) Mfn2 (MF2) and Mfn2-mutants (MF2-IYFFT, MF2-NT, MF2-TMCT). (B) Antibody specificity. Cells were transfected with myc-tagged mitofusins (MF1, MF2) and Mfn2-fragments (MF2-IYFFT, MF2-NT, MF2-TMCT) and analyzed by western-blot with the indicated antibodies (myc, NG, CT). The images visualize overexpressed proteins at exposures where endogenous Mfn2 is hardly visible (NG, CT). Myc-specific antibodies decorate both mitofusins and the Mfn2-constructs (arrows). NG and CT do not decorate Mfn1 and are specific for their respective Mfn2-domains (arrows). (C,D) Western blot analysis of isolated mitochondria treated with Proteinase K for the indicated times (minutes) in the absence (C) or presence (D) of Triton X-100. Proteins of the intermembrane space (Cyt.c) and of the mitochondrial matrix (Hsp60) are protected in intact mitochondria (C) and are degraded after membrane solubilization (D). In the absence of detergent (C), Mfn2 is gradually degraded into fragments (asterisks) that are differentially recognized by domain-specific antibodies (NG, CT). These fragments are smaller than MF2-NT/MF2IYFFT (upper arrow) and MF2-TMCT (lower arrow), respectively. This reveals the cleavage of N-terminal and C-terminal domains and their exposure towards the protease-containing solution. The position and size (in kDa) of molecular mass markers is indicated by arrowheads. (E) Deduced membrane topology of Mfn2. OM and IM, outer and inner mitochondrial membranes.

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