Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2022/02/PXD030730/peptides.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2022/02/PXD030730/peptides__1_.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2022/02/PXD030730/20210707-02-blank.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2022/02/PXD030730/20210707-06-MiNT.rawprimary200OKrachel@mail.huji.ac.ilHenri-Baptiste MarjaultMass SpectrometryTop-down proteomicsBreast CancerMitochondriaPull-downCancerhttps://www.ebi.ac.uk/pride/archive/projects/PXD030730Epithelial CellCell CultureAfter the last step of immunoprecipitation, beads were washed twice with 150 mM NaCl, 50 mM Tris-HCl pH 7.4. Packed beads were then resuspended in 100 μl 8M urea, 10 mM DTT, 25 mM Tris-HCl pH 8.0 and incubated for 30 min at 22°C. Next, Iodoacetamide (55 mM) was added and beads were incubated for 30 min (22°C, in the dark), followed by addition of DTT (20 mM). The Urea was diluted by the addition of 6 volumes of 25 mM Tris-HCl pH 8.0. Trypsin was added (0.3 μg/ sample) and the beads were incubated overnight at 37°C with gentle agitation. The beads were spun down and the peptides in the supernatant were desalted on C18 home-made Stage tips.PridenanoLC-MS/MS analysis MS analysis was performed using a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific, Waltham, MA USA) coupled on-line to a nanoflow UHPLC instrument, Ultimate 3000 Dionex (Thermo Fisher Scientific, Waltham, MA USA). Peptides (0.35 μg, as estimated by O.D.280 nm) were separated over a non-linear 60 min gradient (0 - 80% acetonitrile) run at a flow rate of 0.3 μl/min on a reverse phase 25-cm-long C18 column (75 μm ID, 2 μm, 100Å, Thermo PepMapRSLC). The survey scans (380–2,000 m/z, target value 3E6 charges, maximum ion injection times 50 ms) were acquired and followed by higher energy collisional dissociation (HCD) based fragmentation (normalized collision energy 25). A resolution of 70,000 was used for survey scans and up to 15 dynamically chosen most abundant precursor ions, with “peptide preferable” profile were fragmented (isolation window 1.6 m/z). The MS/MS scans were acquired at a resolution of 17,500 (target value 1E5 charges, maximum ion injection times 120 ms). Dynamic exclusion was 60 sec. Data were acquired using Xcalibur software (Thermo Scientific). To avoid a carryover, the column was washed with 80% acetonitrile, 0.1% formic acid for 25 min between samples. MS data analysis Mass spectra data were processed using the MaxQuant computational platform, version 1.5.3.1254. Peak lists were searched against translated coding sequences of the human proteome obtained from Uniprot. The search included cysteine carbamidomethylation as a fixed modification and oxidation of methionine as variable modifications and allowed up to two miscleavages. The match-between-runs option was used. Peptides with a length of at least seven amino-acids were considered and the required FDR was set to 1% at the peptide and protein level. Protein identification required at least 2 unique or razor peptides per protein. Relative protein quantification in MaxQuant was performed using the label-free quantification (LFQ) algorithm. Protein contaminants and proteins identified by less than 2 peptides were excluded from the analysis.ProteomicsRachel NechushtaiPARTIALThe Alexander Silberman Institute of Life Science and The Wolfson Centre for Applied Structural Biology, Faculty of Science and Mathematics, The Edmond J. Safra Campus at Givat Ram,TheHebrew University of Jerusalem, Jerusalem, 91904, Israel.Homo Sapiens (human)35135884 Karmi O, Marjault HB, Bai F, Roy S, Sohn YS, Darash Yahana M, Morcos F, Ioannidis K, Nahmias Y, Jennings PA, Mittler R, Onuchic JN, Nechushtai R. A VDAC1-mediated NEET protein chain transfers [2Fe-2S] clusters between the mitochondria and the cytosol and impacts mitochondrial dynamics. Proc Natl Acad Sci U S A. 2022 119(7) 10.1073/pnas.2121491119henri.margault@mail.huji.ac.ilHebrew University of JerusalemIsraelMitochondrial inner NEET (MiNT) and the outer mitochondrial membrane (OMM) mitoNEET (mNT) proteins belong to the NEET protein family. This family plays a key role in mitochondrial labile iron and reactive oxygen species (ROS) homeostasis. NEET proteins contain labile [2Fe-2S] clusters which can be transferred to apo-acceptor proteins. In eukaryotes, the biogenesis of [2Fe-2S] clusters occurs within the mitochondria by the iron-sulfur cluster (ISC) system; the clusters are then transferred to [2Fe-2S] proteins within the mitochondria or exported to cytosolic proteins and the cytosolic iron-sulfur cluster assembly (CIA) system. The last step of export of the [2Fe-2S] is not yet fully characterized. Here we show that MiNT interacts with voltage-dependent anion channel 1 (VDAC1), a major OMM protein that connects the intermembrane space with the cytosol and participates in regulating the levels of different ions including mitochondrial labile iron (mLI). We further show that VDAC1 is mediating the interaction between MiNT and mNT, in which MiNT transfers its [2Fe-2S] clusters from inside the mitochondria to mNT that is facing the cytosol. This MiNT-VDAC1-mNT interaction is shown both experimentally and by computational calculations. Additionally, we show that modifying MiNT expression in breast cancer cells affects the dynamics of mitochondrial structure and morphology, mitochondrial function, and breast cancer tumor growth. Our findings reveal a pathway for the transfer of [2Fe-2S] clusters, which are assembled inside the mitochondria, to the cytosol.A VDAC1-mediated NEET protein chain transfers [2Fe-2S] clusters between the mitochondria and the cytosol and impacts mitochondrial dynamics.Karmi Ola O, Marjault Henri-Baptiste HB, Bai Fang F, Roy Susmita S, Sohn Yang-Sung YS, Darash Yahana Merav M, Morcos Faruck F, Ioannidis Konstantinos K, Nahmias Yaakov Y, Jennings Patricia A PA, Mittler Ron R, Onuchic José N JN, Nechushtai Rachel RHac-1/Dark, Co-Immunoprecipitations, GRP1/cytohesin 1, Dapaf-1/HAC-1, chlorure d'hydrogene, DmelCG6829, Psychomotor agitation, classic hairy cell leukaemia, Carbamide, Hydrogenchlorid, Basodexan, Apaf-1, Co Immunoprecipitation, CG11633, Dark/Hac-1/dApaf1, cytohesin/GRP1, Karbamid, Polypeptides, Hac1, halite, hydrogen chloride, Dark/Hac-1/dApaf-1, peptido, GRP1, Grp1, Harnstoff, B1, Immune Precipitations, Apaf1, Carmol, ARK, cloruro de hidrogeno, Immune Precipitation, hac-1, peptides, Hydrogen chloride, E927b, PTPSTEP, Co-Immunoprecipitation, dapaf-1, beta-Trypsin, Precipitation, l(2)SH2 0323, chlorure de sodium, arc, dapaf, dark, ark, T1, cloruro sodico, salt, Wasserstoffchlorid, Immune, Neural-specific protein-tyrosine phosphatase, DARK, l(2)k08110, sample, D1, Kochsalz, peptidos, stage, dApaf1, Precipitations, dArk, Dapaf-1, 2-iodo-, PTHB1, GPH, dark/dapaf-1/hac-1, rock salt, D-Apaf-1, dApaf-1, anon-53Fa, l(2)SH0173, stepk, apaf1, chloridohydrogen, classic hairy cell leukemia, ur, Peptide, [HCl], carbamide, APAF1, common salt, Natriumchlorid, carbonyldiamide, Hac-1, C18, 3.1.3.48, Chlorwasserstoff, Hydrochloride, table salt, Dark, NaCl, l(2)SH0323, Dark/Apaf-I, CG6829, dark/hac-1/dapaf-1, CYH1, PRSS, Step, CG11628, uree, HCl, Tripcellim, Dark/Dapaf-1/HAC1, Striatum-enriched protein-tyrosine phosphatase, hac1, natrii chloridum, beta Trypsin, dApaf-1/DARK/HAC-1, sample population, DmelCG11628, dapaf-1S, Trypure, H2NC(O)NH2, apaf-1, dapaf-1L, STEP, Acetamide, Peptid, Polypeptide, chlorane, 1728, hydrochloric acid, dAPAF-1, developmental stage.Breast Carcinoma, Human Mammary Neoplasm, primary breast cancer, Carcinoma, malignant tumor of the breast, Breast Carcinomas, "breast neoplasm" EXACT [MTH:120], Neoplasms, mammary neoplasm, Mammary Cancers, Human Mammary Neoplasms, Mitochondrial., Tumor, "breast tumor" EXACT [NCI2004_11_17:C2910], Mitochondrial Contractions, Human, Breast Malignant Neoplasm, Contractions, Breast Malignant Tumor, "neoplasm of breast (disorder)" EXACT [SNOMEDCT_2005_07_31:126926005], Mammary Carcinomas, Breast Tumor, Neoplasm, Mitochondrion, Human Mammary Carcinoma, "mammary tumor" EXACT [CSP2005:2016-0671], NOS, breast tumor, cancer of the breast, Malignant Neoplasm of Breast, Carcinomas, BC, Mammary Neoplasms, malignant neoplasm of breast, Mitochondrial, Human Mammary, Breast Neoplasm, Mammary Neoplasm, Contraction, Mammary Carcinoma, Human Mammary Carcinomas, Cancers, "mammary neoplasm" RELATED [], Cancer of the Breast, Mitochondrial Contraction, mitochondria, breast cancer, cancer of breast, Malignant Tumor of Breast, Mammary, Mammary Cancer, Breast Tumors, Breast Cancer, Breast Malignant Tumors, Cancer of Breast, Breast, mammary cancer, cancer, mammary tumor, Breast Malignant Neoplasms, breast, Tumors, Cancersodium salt, DmelCG42277, eIF3-S1, IPP2A2, ammonium formate, 13C-labeled, MeCN, cadmium salt, E 920, NCMe, ion, determination, leg, instrument configuration, zinc formate, lead salt, LFQ, magnesium formate, protein, heavy chain disease, zinc salt, l(1)AA33, 5730420M11Rik, peptide, Polypeptides, L-Isomer Methionine, ammonium tetraformate, Methionine, peptido, cobalt(II) formate dihydrate, CH3-C#N, SEC, 1, 2, Min, Software Engineering, 2-amino-3-mercaptopropanoic acid, Cystein, Analysis, protein aggregate, Injectables, Computer Program, WMS, DmelCG42273, C, SET, Divorced, E, Man (Taxonomy), peptides, Analyses, TAF-I, cisteina, M, long, Open, P5.3, sec, min, Computer Programs and Programming, chemical analysis., HardwareType, mAPC, Divorces, purification, copper, Estimated, AI047805, DmelCG4299, isolation and purification, IGAAD, set, Dmel_CG32467, Dmel_CG32466, lithium salt, column, l(1)B2/13.1, DmelCG10574, RUN, Run, Half Cystine, GPHYSD2, Racemethionine, ammonium (2:1) salt, CG10040, nickel salt, SGS, phapii, l(1)LB9, PLATEST, cobalt (+2) salt, Zinc Cysteinate, Modern, acid, 2-Amino-4-(methylthio)butyric acid, Dmel_CG7826, StF-IT-1, Th, CG12131, not genetically inherited, fragmented, Thermo Scientific, Cys, Source Softwares, AA33, Software Tools, Programs, ACMICD, Program, Computer Applications, sodium (4:1:1) salt, HCD, Dm1, Ionen, magnesium salt, l(2)k13906, Algorithm, Computer Applications Software, Computer Applications Softwares, Methionin, Softwares, Hmet, nickel (+2) salt, (2R)-2-amino-3-sulfanylpropanoic acid, 5.3, Dmel_CG7835, Mnb, MNB, parent ion, Acid, ammonium salt, Software Applications, CHARGES, HLA-DR-associated protein II, instrument, DI-2, MS/MS, Source Software, acide, I-2Dm, cracked, formate, cobaltous formate, acids, cromium (+3), L-Cysteine, MASS, acido, CG4299, AW124434, human, L-Cystein, I-2PP1, data analysis, Applications, TAF-IBETA, Charge, precursor ion, Charges and Fees, copper salt, ammonium (4:1) salt, TAF-Ibeta, Polypeptide, methyl cyanide, lead (+2) salt, Liquimeth, i2pp2a, Proteomes, Platelets, Computer Software Applications, CG1849, human being, Cysteine Hydrochloride, 2-amino-4-(methylsulfanyl)butanoic acid, nickel formate dihydrate, lead formate, fractured, aluminum salt, cesium salt, L-Zystein, L Isomer, FBN, Gene, precursor, Computer, thomson, protein-containing complex, CG32467, CG7826, CG32466, PHAPII, hardware, mass-to-charge ratio, formic acid, Human, potassium salt, Homo sapiens, isolation, Injection, ECTOL1, 14C-labeled, CG42277, CG7835, Gene Products, CG42273, L-Methionine, Half-Cystine, Separated, Man, Pedameth, Application, T6K12_14, Open Source Softwares, Dipr, thallium (+1) salt, MS2, iones, Software Application, L Cysteine, Runt, E-920, Selb, ipp2a2, rubidium salt, Open Source Software, 2pp2a, Hcys, calcium formate, methanoic acid, Charges, ions, CG10574, Computer Software Application, OCTD, Injectable, Fee, data processing, 2-amino-4-(methylthio)butanoic acid, 2PP2A, Tools, CYSTEINE, taf-ibeta, dSET, dSet, FREE CYSTEINE, peptidos, cromium (+3) salt, (2R)-2-amino-3-mercaptopropanoic acid, metionina, Saeure, sodium formate, LB5, Applications Software, Open Source, DYRK1, Computer Software, 1-(14)C-labeled, nickel formate, 3H-labeled, DL-Methionine, protein complex, Proteins, DmelCG1849, igaad, split, strontium formate, total expressed protein, potassium formate, copper (+2) salt, Rnt, acetonitrile, strontium salt, L-Isomer, Peptide, Tool, group, lLB5, polypeptide, Software Tool, secret agent, native protein, I-2PP2A, DmelCG12131, cupric formate, chemical analysis, Dm I-2, Protein, I2PP2A, hemorrhaged, MFS1, Dyrk1, aluminum formate, Software, ACETONITRILE, tandem MS, Zystein, cyanomethane, WMS2, Met, T6K12.14, AU020952, torn, Separation, lithium formate, roe, ensemble, 2-amino-3-sulfanylpropanoic acid, Saeuren, Separations, CC1, Engineering, L-2-Amino-3-mercaptopropionic acid, l(1)19Ea, (R)-2-amino-3-mercaptopropanoic acid, ethanenitrile, l(2)46Cf, chromic formate, 2-Amino-3-mercaptopropionic acid, ME-IV, Protein Gene Products, P235, Computer Programs, Gene Proteins, dSET/TAF-Ibeta, CG14601, 2610030F17Rik, CG14603, Applications Softwares, Ion, Data, Fees, Modern Man, SSKS, calcium salt, alpha-amino-gamma-methylmercaptobutyric acid, E920, Peptid, assay, variable, AA407739, CG14600, Data Analyses, l(2)46CFdProtein Gene Products, Mitochondrial Contraction, Gene Proteins, Contractions, Mitochondrial, mitochondria, native protein, protein complex, Protein, Contraction, Gene Products, Proteins, Mitochondrion, Gene, protein, protein aggregate, Mitochondrial., protein-containing complex, Mitochondrial ContractionsNetworks, malignant tumor of the breast, outer mitochondrial membrane, Kinship, ion, Conflict Resolution, Inner, postnatal development, mammary neoplasm, PORIN, histology, Human Mammary Neoplasms, growth and development, protein, AL033343, Tumor, Mitochondrial Contractions, dmnt, Negotiation, Mitochondrial Membranes, ATVDAC1, Roles, "neoplasm of breast (disorder)" EXACT [SNOMEDCT_2005_07_31:126926005], mKIAA0929, GRP1, Grp1, Vdac5, symptoms, Concepts, Life Cycle, 26Fe, cancer of the breast, Pro Oxidant, protein aggregate, anatomy and histology, EG:114E2.2, IKKg, Coactivator independent of AF-2, mitochondrion outer membrane, Family Life Cycle, KEY, Key, A, BC, Mammary Neoplasms, malignant neoplasm of breast, MINT, T22N4.9, Kinship Network, Moods, AU043990, PTPSTEP, x11Lalpha, Human Mammary Carcinomas, Eucarya, c-ros-1, eucaryotes, Mints, dMad, Oxygen, CG13316, iron-sulfur cluster biosynthesis, VDAC-1, Inner Mitochondrial Membrane, cancer of breast, 6430513E09Rik, l(2)k08110, Role Concepts, Dmint1, Malignancies, D10Ertd214e, Family Life Cycles, Urop11, mammary cancer, X11, outer mitochondrion membrane, DmMnt, Breast Malignant Neoplasms, GPH, accepteur, Tumors, screening, Carcinoma, "breast neoplasm" EXACT [MTH:120], Oxygen Radical, dX11L, CIA, C10orf70, 5076, bHLHd3, DmIKKgamma, Mint, Role Concept, Iron 56, Benign, ATP synthase D chain, positive regulation of homeostatic process, Ionen, Playthings and Play, dIKK, Inner Mitochondrial Membranes, Kenny, 3.1.3.48, Role, Plaything, Anti-silencing function protein 1 homolog A, NOS, Conflict, T22N4_9, Filiation, cia, Malignant Neoplasm of Breast, Oxygen Radicals, Carcinomas, Hydrogen-acceptor, Mitochondrial, Mad-3E, Iron, growth pattern, Outer, Step, CG11628, Radical, non-developmental growth, RBM15C, IKK-gamma, MNT, Toys, Eukarya, signs, Benign Neoplasms, CCG1-interacting factor A, Striatum-enriched protein-tyrosine phosphatase, voltage dependent anion channel 1, "mammary neoplasm" RELATED [], Fe, Malignant Neoplasms, Life Cycles, Acceptor, Eisen, DmelCG16910, MCF3, breast cancer, STEP, Breast Tumors, Breast Malignant Tumors, Cytosols, mnt, NCoA-5, Negotiations, iron, other neoplasm, cancer, hCIA, primary breast cancer, Outer Mitochondrial, Arbitrating, MitoNEET, Plays, Akzeptor, Family Member, GRP1/cytohesin 1, Outer Mitochondrial Membranes, conformation, Neoplasms, Benign Neoplasm, bHLHe43, Eukaryotes, Gene, Mammary Cancers, Reactive Oxygen Intermediates, Network, protein-containing complex, mitochondrial, Mitochondrial Membrane, Malignant, dIKK-gamma, hAsf1, Human, CG11633, cytohesin/GRP1, Breast Malignant Tumor, morphology, MAD6, Cytosols., Breast Tumor, DmIKK-gamma, Gene Products, Mitochondrion, Mad, "mammary tumor" EXACT [CSP2005:2016-0671], breast tumor, Mood, dmIKKgamma, IKK[[gamma]], Reactive, Toy, DmelCG5675, xmnt, hierro, Human Mammary, anatomy, anatomical systems, Playthings, Malignancy, Research, Mammary Neoplasm, iones, Contraction, Mammary Carcinoma, ARABIDOPSIS THALIANA VOLTAGE DEPENDENT ANION CHANNEL 1, Lin-10, Voltage Dependent Anion Channel 1, l(2)SH2 0323, Dmint, ions, Oxidized donor, Oxygen Species, Cancer of the Breast, Neoplasias, Arbitration, Puppets, Neural-specific protein-tyrosine phosphatase, IKK, Active Oxygen Species, Malignant Tumor of Breast, Mammary, Mammary Cancer, negative regulation of homeostatic process, Play, DmelCG13316, Kinship Networks, Pro-Oxidant, Breast, X11alpha, SHARP, MXD6, Family, Active Oxygen, mammary tumor, Zcd1, Puppet, hAsf1a, ZCD1, Cancer, Autoregulation, Mint1, Menthas, Breast Carcinoma, Human Mammary Neoplasm, lumen, Pro Oxidants, Breast Carcinomas, findings, Eukaryote, Conflict Resolutions, Malignant Neoplasm, Family Research, protein complex, space, Affects, Proteins, fer, stepk, Eukaryotas, function, "breast tumor" EXACT [NCI2004_11_17:C2910], HSPC146, Cell, CG2856, Concept, eukaryotes, Family Members, Breast Malignant Neoplasm, development, IKKgamma, Contractions, Mediating, native protein, Mammary Carcinomas, Protein, VDAC1 Protein, Neoplasm, Active, Human Mammary Carcinoma, ROS, anatomical spaces, regulation of homeostatic process, dX11, ROX, Rox, l(2)SH0323, mitoNEET, Resolution, CYH1, dMnt, Membranes, ferrum, lumen space, Breast Neoplasm, Dmikkgamma, Mediation, postnatal growth, Cancers, Iron-56, CGI-98, Membrane, rox, CG16910, DmelCG11628, HIAA0929, Outer Mitochondrial Membrane, Protein Gene Products, Mitochondrial Contraction, X11Lalpha, Gene Proteins, Eukaryotae, mitochondria, Ion, Families, AW743335, Inner Mitochondrial, Breast Cancer, Pro-Oxidants, CG5678, Cancer of Breast, CG5675, Eucaryotae, Relatives, growth, Neoplasia, breast, bA465L10.6Dynamic, Mitochondrial, T22N4.9, Mitochondrial Fusion, protein complex, Contraction, Fission, ARABIDOPSIS THALIANA VOLTAGE DEPENDENT ANION CHANNEL 1, Proteins, Mitochondrial Fusions, PORIN, Gene, Dynamics, voltage dependent anion channel 1, protein, AL033343, Mitochondrial Fusion and Fission, Mitochondrial., protein-containing complex, Mitochondrial Contractions, Protein Gene Products, Mitochondrial Contraction, 5076, Gene Proteins, Mitochondrial Fission and Fusion, Mitochondrial Dynamic, Contractions, mitochondria, Mitochondrial Fission, native protein, VDAC-1, ATVDAC1, Fissions, Protein, Fusions, Vdac5, Mitochondrial Fissions, Gene Products, Mitochondrion, Cytosols, T22N4_9, protein aggregate, Fusionfalsebreast cancer mitochondria pulldownpull-down of protein extracted from isolated mitochondria 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