Gordaliza-Alaguero Isabel, Sànchez-Fernàndez-de-Landa Paula, Radivojevikj Dragana, Villarreal Laura, Arauz-Garofalo Gianluca, Gay Marina, Martinez-Vicente Marta, Seco Jorge, Martín-Malpartida Pau, Vilaseca Marta, Macías María J, Palacin Manuel, Ivanova Saška, Zorzano Antonio
Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Baldiri Reixac, Barcelona, Spain.
Departament de Bioquímica i Biomedicina Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain.
Autophagy. 2025 May;21(5):957-978. doi: 10.1080/15548627.2024.2440843. Epub 2024 Dec 24.
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.: 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; 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.
MFN1（线粒体融合蛋白1）和MFN2是线粒体融合、内质网（ER）-线粒体并置以及巨自噬/自噬过程中的关键蛋白。然而，这些蛋白参与这些过程的机制尚不清楚。在这里，我们通过使用CRISPR-Cas9技术在MFN1和MFN2的C末端插入一个HA标签，从而构建出内源性表达MFN1-HA或MFN2-HA的HeLa细胞系，研究了这两种蛋白的相互作用组。通过HA亲和分离和质谱鉴定出MFN1和MFN2的潜在相互作用蛋白。很大一部分相互作用蛋白是MFN1和MFN2共有的，并且受营养剥夺的调节。我们验证了MFN1和/或MFN2的新型内质网和内体伙伴在细胞器间通讯中可能发挥的作用。我们将RAB5C（RAB5C，RAS癌基因家族成员）鉴定为线粒体稳态的内体调节因子，将SLC27A2（溶质载体家族27（脂肪酸转运蛋白）成员2）鉴定为与自噬相关的MFN2的新型伙伴。我们得出结论，MFN蛋白参与了与细胞器通讯和自噬相关的营养调节途径。：ACTB：肌动蛋白，β；ATG2：自噬相关蛋白2；ATG5：自噬相关蛋白5；ATG12：自噬相关蛋白12；ATG14：自噬相关蛋白14；ATG16L1：自噬相关蛋白16样蛋白1；巴弗洛霉素A1；BECN1：贝克林1，自噬相关；BFDR：贝叶斯错误发现率；Cas9：CRISPR相关内切酶Cas9；CRISPR：成簇规律间隔短回文重复序列；DNM1L/DRP1：动力蛋白1样蛋白；内质网；Faa1：脂肪酸激活蛋白1；FC：倍数变化；错误发现率；FIS1：线粒体分裂蛋白1；GABARAP：γ-氨基丁酸受体相关蛋白；GABARAPL2：GABA A型受体相关蛋白样蛋白2；GAPDH：甘油醛-3-磷酸脱氢酶；HA：血凝素；敲除；LIR：LC3相互作用区域；MAP1LC3B/LC3B：微管相关蛋白1轻链3β；MARCHF5：膜相关环-CH型指蛋白5；MDVs：线粒体衍生囊泡；MFN1：线粒体融合蛋白1；MFN2：线粒体融合蛋白2；NDFIP2：Nedd4家族相互作用蛋白2；线粒体外膜；OPA1：OPA1，线粒体动力蛋白样GTP酶；氧化磷酸化；PE：磷脂酰乙醇胺；PINK1：PTEN诱导的假定激酶1；PS：磷脂酰丝氨酸；RAB5C：RAB5C，RAS癌基因家族成员；S100A8：S100钙结合蛋白A8（钙粒蛋白A）；S100A9：S100钙结合蛋白A9（钙粒蛋白B）；SLC27A2：溶质载体家族27（脂肪酸转运蛋白）成员2；TIMM44：线粒体内膜转位酶44；TOMM20：线粒体外膜转位酶20；ULK1：unc-51样激酶1；VCL：纽蛋白；VDAC1：电压依赖性阴离子通道1；野生型。
