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 interorganelle communication. We characterized RAB5C (RAB5C, member RAS oncogene family) as an endosomal modulator of mitochondrial homeostasis, and SLC27A2 (solute carrier family 27 (fatty acid transporter), member 2) as a novel partner of MFN2 relevant in autophagy. We conclude that MFN proteins participate in nutrient-modulated pathways involved in organelle communication and autophagy.Abbreviations: ACTB: actin, beta; ATG2: autophagy related 2; ATG5: autophagy related 5; ATG12: autophagy related 12; ATG14: autophagy related 14; ATG16L1: autophagy related 16 like 1; Baf A1: bafilomycin A(1); BECN1: beclin 1, autophagy related; BFDR: Bayesian false discovery rate; Cas9: CRISPR-associated endonuclease Cas9; CRISPR: clustered regularly interspaced short palindromic repeats; DNM1L/DRP1: dynamin 1-like; ER: endoplasmic reticulum; Faa1: fatty acid activation 1; FC: fold change; FDR: false discovery rate; FIS1: fission, mitochondrial 1; GABARAP: gamma-aminobutyric acid receptor associated protein; GABARAPL2: GABA type A receptor associated protein like 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HA: hemagglutinin; KO: knockout; LIR: LC3-interacting region; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MARCHF5: membrane associated ring-CH-type finger 5; MDVs: mitochondria-derived vesicles; MFN1: mitofusin 1; MFN2: mitofusin 2; NDFIP2: Nedd4 family interacting protein 2; OMM: outer mitochondrial membrane; OPA1: OPA1, mitochondrial dynamin like GTPase; OXPHOS: oxidative phosphorylation; PE: phosphatidylethanolamine; PINK1: PTEN induced putative kinase 1; PS: phosphatidylserine; RAB5C: RAB5C, member RAS oncogene family; S100A8: S100 calcium binding protein A8 (calgranulin A); S100A9: S100 calcium binding protein A9 (calgranulin B); SLC27A2: solute carrier family 27 (fatty acid transporter), member 2; TIMM44: translocase of inner mitochondrial membrane 44; TOMM20: translocase of outer mitochondrial membrane 20; ULK1: unc-51 like kinase 1; VCL: vinculin; VDAC1: voltage-dependent anion channel 1; WT: wild type.