Fig. 1
Identification and characterization of binding regions in MIEFs required for Mfn1, Mfn2, hFis1, and Drp1. a Exogenous MIEF1-V5 and MIEF2-V5 interact with both pro-fission (Drp1, hFis1, Mff) and pro-fusion (Mfn1, Mfn2) proteins. 293T cells were transfected with empty vector (as control), MIEF1-V5, or MIEF2-V5 and were in vivo crosslinked with 1% formaldehyde (FA). Cell lysates were used for co-IP with anti-V5 beads, followed by Western blotting with indicated antibodies. b, c Mfn1 and Mfn2 interact with MIEF1/2 at endogenous levels. 293T cells were in vivo crosslinked with 1% FA. Cell lysates were subjected to co-IP with Protein G beads pre-bound to mouse/rabbit normal IgG (negative control), anti-Mfn1 (b), or Mfn2 (c) antibodies, followed by immunoblotting with indicated antibodies. d Summary of MIEF1-mutated constructs used in the co-IP experiments. e Interaction of different MIEF1 mutants with Mfn1, Mfn2, Drp1, and hFis1. 293T cells were transfected with full-length MIEF1 and deletion mutants as indicated, and in vivo crosslinked with 1% FA. Cell lysates were used for co-IP with anti-V5 beads, followed by Western blotting with indicated antibodies. f Summary of MIEF2-mutated constructs used in the co-IP experiments. g Interaction of different MIEF2 mutants with Mfn1, Mfn2, Drp1, and hFis1. 293T cells were transfected with the full-length and the truncated MIEF2 mutants as indicated, and in vivo crosslinked with 1% FA. Cell lysates were used for co-IP with anti-V5 beads, followed by Western blotting with indicated antibodies. h Protein sequence alignments around the transmembrane domains (TM, in blue color) and the disordered regions (DR, gray color) comparing human MIEF1 (NP_061881) and MIEF2 (NP_631901). The amino acid sequence alignments of human MIEF1 and MIEF2 were generated by the CLUSTALW program (https://npsa-prabi.ibcp.fr). The regions that were deleted in MIEF1Δ50-108 and MIEF2Δ50-104 are indicated in the alignment. Red: identical; Green: strongly similar; Blue: weakly similar; Black: different amino acids