biostudies-literatureBoopathy SNational Institutes of HealthNIGMS NIH HHSNIGMS107740https://www.ebi.ac.uk/biostudies/studies/S-EPMC11459905biostudies-literatureUnknown300(10)Mitochondrial fusion requires the sequential merger of four bilayers to two. The outer-membrane solute carrier family 25 member (SLC25A46) interacts with both the outer and inner membrane dynamin family GTPases mitofusin 1/2 and optic atrophy 1 (Opa1). While SLC25A46 levels are known to affect mitochondrial morphology, how SLC25A46 interacts with mitofusin 1/2 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 an SLC25A46 interaction with Opa1 and Mfn2. We reveal that the bundle signaling element of Opa1 interacts with SLC25A46, and present evidence of an Mfn2 interaction involving the SLC25A46 cytosolic face. We validate these newly identified interaction interfaces and showThe Journal of biological chemistryIdentification of SLC25A46 interaction interfaces with mitochondrial membrane fusogens Opa1 and Mfn2.PMC11459905R35 GM142553R35GM1425531S10OD010582-01A1Kim HLPonce JBoopathy SLugo CMMcDonald JLuce BEChao LHHakim PUeberheide BMfalseIdentification of SLC25A46 interaction interfaces with mitochondrial membrane fusogens Opa1 and Mfn2.Mitochondrial fusion requires the sequential merger of four bilayers to two. The outer-membrane solute carrier family 25 member (SLC25A46) interacts with both the outer and inner membrane dynamin family GTPases mitofusin 1/2 and optic atrophy 1 (Opa1). While SLC25A46 levels are known to affect mitochondrial morphology, how SLC25A46 interacts with mitofusin 1/2 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 an SLC25A46 interaction with Opa1 and Mfn2. We reveal that the bundle signaling element of Opa1 interacts with SLC25A46, and present evidence of an Mfn2 interaction involving the SLC25A46 cytosolic face. We validate these newly identified interaction interfaces and show2024-01-01T00:00:00Z2024 Aug2025-04-18T15:57:33.991Z2025-04-07T02:54:18.912ZS-EPMC114599053922268410.1016/j.jbc.2024.107740