biostudies-literatureUnknownBoopathy SMitochondrial 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.bioRxiv : the preprint server for biology2023.12.29.573615https://www.ebi.ac.uk/biostudies/studies/S-EPMC10793391biostudies-literatureIdentification of SLC25A46 interaction interfaces with mitochondrial membrane fusogens Mfn2 and Opa1.PMC10793391Ponce JBoopathy SMcDonald JLuce BEChao LHMakhlouta Lugo CHakim PUeberheide BMfalseIdentification 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.2023-01-01T00:00:00Z2023 Dec2025-04-25T23:24:43.599Z2025-04-06T09:24:24.818ZS-EPMC107933913823481310.1101/2023.12.29.573615