biostudies-literatureGordaliza-Alaguero IInstituto de Salud Carlos IIIFundación BBVACenters of Excellence Severo Ochoa Award“La Caixa” FoundationFundació Marató de TV3FEDER -A Way of making EuropeERDF Operational Program in CataloniaEuropean Union’s Horizon 2020 Research and Innovation ProgrammeEuropean Regional Development FundCERCA Programe of the Generalitat de Catalunya957-978https://www.ebi.ac.uk/biostudies/studies/S-EPMC12013434biostudies-literatureUnknown21(5)MFN1 (mitofusin 1) and MFN2 are key players in mitochondrial fusion, endoplasmic reticulum (ER)-mitochondria juxtaposition, and macroautophagy/autophagy. However, the mechanisms by which these proteins participate in these processes are poorly understood. Here, we studied the interactomes of these two proteins by using CRISPR-Cas9 technology to insert an HA-tag at the C terminus of MFN1 and MFN2, and thus generating HeLa cell lines that endogenously expressed MFN1-HA or MFN2-HA. HA-affinity isolation followed by mass spectrometry identified potential interactors of MFN1 and MFN2. A substantial proportion of interactors were common for MFN1 and MFN2 and were regulated by nutrient deprivation. We validated novel ER and endosomal partners of MFN1 and/or MFN2 with a potential role in interorgaAutophagyEndogenous interactomes of MFN1 and MFN2 provide novel insights into interorganelle communication and autophagy.PMC12013434PID2021-122909NB-I00IU16–01598320132330LCF/PR/HR21/52410007PID2022-137576OB-I00945352Sanchez-Fernandez-de-Landa PIvanova SMartinez-Vicente MMartin-Malpartida PSeco JPalacin MArauz-Garofalo GRadivojevikj DGordaliza-Alaguero IVilaseca MZorzano AMacias MJGay MVillarreal LfalseEndogenous interactomes of MFN1 and MFN2 provide novel insights into interorganelle communication and autophagy.MFN1 (mitofusin 1) and MFN2 are key players in mitochondrial fusion, endoplasmic reticulum (ER)-mitochondria juxtaposition, and macroautophagy/autophagy. However, the mechanisms by which these proteins participate in these processes are poorly understood. Here, we studied the interactomes of these two proteins by using CRISPR-Cas9 technology to insert an HA-tag at the C terminus of MFN1 and MFN2, and thus generating HeLa cell lines that endogenously expressed MFN1-HA or MFN2-HA. HA-affinity isolation followed by mass spectrometry identified potential interactors of MFN1 and MFN2. A substantial proportion of interactors were common for MFN1 and MFN2 and were regulated by nutrient deprivation. We validated novel ER and endosomal partners of MFN1 and/or MFN2 with a potential role in interorga2025-01-01T00:00:00Z2025 May2026-06-12T04:58:59.078Z2026-06-12T03:08:19.802ZS-EPMC120134343967505410.1080/15548627.2024.2440843