Identification of SLC25A46 interaction interfaces with mitochondrial membrane fusogens Mfn2 and Opa1

Mitochondrial fusion requires the sequential merger of four bilayers to two. The outer-membrane solute carrier protein SLC25A46 interacts with both the outer and inner-membrane dynamin family GTPases Mfn1/2 and Opa1. While SLC25A46 levels are known affect mitochondrial morphology, how SLC25A46 interacts with Mfn1/2 and Opa1 to regulate membrane fusion is not understood. In this study, we use crosslinking mass-spectrometry and AlphaFold 2 modeling to identify interfaces mediating a SLC25A46-Opa1-Mfn1/2 complex. We reveal that the bundle signaling element of Opa1 interacts with SLC25A46, and the helical repeat 1 region of Mfn2 interacts with the SLC25A46 N-terminus. We validate these newly identified interaction interfaces and show that they play a role in mitochondrial network maintenance. ### Competing Interest Statement L.H.C. is an advisor for Stealth Biotherapeutics. The remaining authors declare that there are no competing financial interests. * Abbreviations and nomenclature : OMM : outer mitochondrial membrane IMM : inner mitochondrial membrane SLC : solute carrier family SLC25A46 : solute carrier family 25 member A46 Opa1 : optic atrophy 1 Mfn1/2 : mitofusin 1/2 iPSC : induced pluoripotent stem cells BSE : bundle signaling element HR1/2 : heptad repeat 1/2 ER : endoplasmic reticulum MGD : minimal GTPase domain DDM : n-Dodecyl-β-D-Maltopyranoside GDN : glyco-diosgenin PA : phosphatidic acid TM : transmembrane segment MTS : mitochondrial targeting sequence IMS : inter-membrane space