JPOST Repositoryapplication/xmlhttps://storage.jpostdb.org/JPST002501/files/171004_kuma_atg5_tmt10_2_9.xlsxhttps://storage.jpostdb.org/JPST002501/files/177_kuma_atg5_tmt10_5.rawhttps://storage.jpostdb.org/JPST002501/files/181_kuma_atg5_tmt10_7.rawhttps://storage.jpostdb.org/JPST002501/files/183_kuma_atg5_tmt10_8.rawhttps://storage.jpostdb.org/JPST002501/files/171_kuma_atg5_tmt10_2.rawhttps://storage.jpostdb.org/JPST002501/files/185_kuma_atg5_tmt10_9.rawhttps://storage.jpostdb.org/JPST002501/files/173_kuma_atg5_tmt10_3.rawhttps://storage.jpostdb.org/JPST002501/files/175_kuma_atg5_tmt10_4.rawhttps://storage.jpostdb.org/JPST002501/files/179_kuma_atg5_tmt10_6.rawhttps://storage.jpostdb.org/JPST002501/files/171004_kuma_atg5_tmt10_2_9.pdResultprimaryOK200ProteomicsHidetaka KosakoMus Musculus (mouse)https://repository.jpostdb.org/entry/JPST002501Tokushima UniversityjPOSTThe degradation of organelles by autophagy is essential for cellular homeostasis. The Golgi apparatus has recently been demonstrated to be degraded by autophagy, but little is known about how the Golgi is recognized by the forming autophagosome. Using quantitative proteomic analysis and two novel Golgiphagy reporter systems, we found that the five-pass transmembrane Golgi-resident proteins YIPF3 and YIPF4 constitute a Golgiphagy receptor. The interaction of this complex with LC3B, GABARAP, and GABARAPL1 is dependent on a LIR motif within YIPF3 and putative phosphorylation sites immediately upstream; the stability of the complex is governed by YIPF4. Expression of a YIPF3 protein containing a mutated LIR motif caused an elongated Golgi morphology, indicating the importance of Golgi turnover via selective autophagy. The reporter assays reported here may be readily adapted to different experimental contexts to help deepen our understanding of Golgiphagy.YIPF3 and YIPF4 regulate autophagic turnover of the Golgi apparatus.Kitta Shinri S, Kaminishi Tatsuya T, Higashi Momoko M, Shima Takayuki T, Nishino Kohei K, Nakamura Nobuhiro N, Kosako Hidetaka H, Yoshimori Tamotsu T, Kuma Akiko AMethane, MeCN, Raw, Lysine Hydrochloride, Laboratory, atg5, TFA, NINA C, Apaf-1, House Mouse, heavy chain disease, trichloro-, prevention, Long Term, nodus primitivus., 5730420M11Rik, Trifluoroacetate, Polypeptides, KL receptor activity, Lysine Acetate, hydrogen chloride, dorsal marginal zone, Method, CH3-C#N, B1, SCO5, 2, SCO1, 2-amino-3-mercaptopropanoic acid, Gsfsow3, WMS, L Lysine, C79691, SET, C, RNEN3, C79325, hac-1, K, M, W, T6G21.3, ATG5, AI047805, DmelCG4299, Lccp, set, Wasserstoffchlorid, LYS, column, scientific observation, ACN, Lysin, sample, Acn, D1, Bs, Half Cystine, hAPG5, SGS, LC-MS2, NEWBORN (0-27 DAYS), Stars, preventive therapy, Zinc Cysteinate, Trypsin/K, anon-53Fa, Longterm Effect, l(2)SH0173, Guanidine Monohydroiodine, Procedure, not genetically inherited, mice C57BL/6xCBA/CaJ hybrid, ACMICD, DmelCG5125, PBT, TPSG1, Dm1, Chlorwasserstoff, Methionin, Hmet, (2R)-2-amino-3-sulfanylpropanoic acid, CG6829, dark/hac-1/dapaf-1, DRONINAC, Acid, pbt, HLA-DR-associated protein II, Caspase-14 subunit p10, DI-2, TRANSMEMBRANE PROTEIN G5P, I-2Dm, Swiss Mouse, pooled, Guanidine Phosphate, reagent, Caspase-14 subunit p19, MASCOT, Methodological, alpha, beta Trypsin, Paddy, I-2PP1, MKP11.20, TAF-IBETA, mug77, spirit of wood, liquid chromatography-tandem mass spectroscopy, PRSS31, Indicator, MKP11_20, DMZ, Mouse, TAF-Ibeta, methyl cyanide, chlorane, STARS, TMT, Liquimeth, Methyl Alcohol, krk1, Hydroxylamine Hydrochloride, 3.4.22.-, HEL-S-279, Apg5l, 2-amino-4-(methylsulfanyl)butanoic acid, Effects, chlorure d'hydrogene, Biocatalysts, number, Pierce, mouse <Mus musculus>, L-Zystein, mini-ICE, APG5L, CH3OH, precursor, thomson, 17alpha)-isomer, CG7826, mass-to-charge ratio, APG5, Guanidium, AUTOPHAGY 5, LC-MSMS, CG7835, Gene Products, Mus musculus domesticus, CG42273, Lysine, Half-Cystine, NINAC, Technique, CG54125, Cesium Trifluoroacetate, Monohydroiodine, NinaC, Acinus, Hydrogen chloride, Longterm, L Cysteine, E-920, beta-Trypsin, 2pp2a, Sciences, Hcys, Long-Term, ATATG5, CG10574, acinusL, Study, Injectable, mKIAA0989, Enzyme, Monohydrobromide, Guanidinium Chloride, 2PP2A, 2-amino-4-(methylthio)butanoic acid, acinusS, CYSTEINE, dSET, dSet, FREE CYSTEINE, peptidos, 2.2, (2R)-2-amino-3-mercaptopropanoic acid, metionina, PTHB1, measuring, dark/dapaf-1/hac-1, proto-oncogene c-Kit, Guanidine Monohydrate, Guanidine Nitrate, 1-(14)C-labeled, AW319544, dApaf-1, Proteins, 2-phospho-D-glycerate hydro-lyase, Methyl alcohol, chloridohydrogen, backward, acetonitrile, L-Isomer, mKIAA0670, Engine, CD142, polypeptide, APAF1, Temperatures, 3110067M24Rik, Hac-1, I-2PP2A, KIT ligand receptor activity, C18, Acetate, chemical analysis, Long Term Effects, Dm I-2, Nitrate, 5alpha, MFS1, Sodium, MIXL, MeOH, L-Lysine, Met, XKrk1, Guanidine Monohydrobromide, AU020952, Guanidine, Col4a-1, Guanidine Sulfate, underdeveloped, ensemble, prophylaxis, Striated muscle activator of Rho-dependent signaling, Neural enolase, Chloride, Dark/Dapaf-1/HAC1, House Mice, (R)-2-amino-3-mercaptopropanoic acid, ethanenitrile, CD117, 2-Amino-3-mercaptopropionic acid, Longterm Effects, ME-IV, plan specification, Gene Proteins, C-Kit, control, Ssm, C88337, xkl-1, liquid chromatography tandem mass spectrometry, E920, Newborn, Sulfate, Enisyl, hydrochloric acid, Methoxide, reversed, liquid chromatography tandem mass spectroscopy, Hac-1/Dark, IPP2A2, Bru, E 920, NCMe, determination, DmelCG6829, Mus domesticus, Dm NinaC, classic hairy cell leukaemia, Monohydrate, Guanidine Sulfate (2:1), CG5125, CASP-14, Hydrogenchlorid, Xkl-1, peptide, Techniques, L-Isomer Methionine, Gsfsco1, Methionine, peptido, BANF, Min, Apaf1, Cystein, ARK, Gsfsco5, prevention and control, Injectables, Effect, DmelCG42273, MIX, wood alcohol, SOW3, Svc, DmAtg5, 3-hydroxy-5-estrane-17-carbonitrile, LCMSMS, AI837106, peptides, reference sample, TAF-I, cisteina, 2610036I19Rik, 2610510L13Rik, hypoplasia, min, arc, epsilon-diaminocaproic acid, Swiss Mice, mAPC, Phosphate, ark, T1, ASP, preventive measures, reaction, Hydroxylammonium Chloride, IGAAD, fSAP152, Nina C, Methodological Studies, DmelCG10574, ppm, trifluoro-, TF, Guanidine Sulfate (1:1), dApaf1, TEAB, Sodium Methoxide, house mouse, Reagents, Sl, GPHYSD2, Racemethionine, Long-Term Effects, reagents, CT16120, APG5-LIKE, phapii, Monohydrochloride, Alcohol, D-Apaf-1, SAC domain-containing protein 8, mouse, LC-MS/MS, 2-Amino-4-(methylthio)butyric acid, Dmel_CG7826, StF-IT-1, Tr-kit, Th, Search, classic hairy cell leukemia, Cys, S-adenosyl-L-methionine:thiol S-methyltransferase activity, HCD, Mini-ICE, lysine, kl1-A, Henson's node, KIT, reactif, Carbinol, fixed, Dmel_CG7835, Acetic acid, carbinol, Mnb, MNB, parent ion, tyrosine-protein kinase Kit, Mus musculus, MILD1, MS/MS, mice, Xylella fastidiosa str. Temecula1, Sonications, HCl, Trifluoroacetic, Guanidine Monohydrochloride, L-Cysteine, hac1, kit, MASS, CG4299, AW124434, MICE, Methodological Study, L-Cystein, domesticus, Trichloromethane, dapaf-1S, nanospray, Wood, apaf-1, dapaf-1L, precursor ion, Cesium, Acetamide, Biocatalyst, Polypeptide, i2pp2a, 2010107M05Rik, Cysteine Hydrochloride, Dapaf-1/HAC-1, Procedures, AW549739, Reagent, SCF receptor activity, L Isomer, FBN, Eno-2, Gene, Methyl, ACINUS, Methylalkohol, presence, PHAPII, LC-MS-MS, NSE, Dark/Hac-1/dApaf1, method, Hac1, Dark/Hac-1/dApaf-1, DmelCG1643, reduced, scfr, House, Ms1, ECTOL1, Injection, method used in an experiment, Studies, L-Methionine, Wood Alcohol, Del(8)44H, tiny, Mice, SCFR, cloruro de hidrogeno, Reagents and Indicators, Pedameth, 4.2.1.11, Pro-Mega, Guanidium Chloride, Search Engines, Fdc, reactivo, MS1, ninac, Swiss, MS2, ipp2a2, dapaf-1, dapaf, dark, Indicators, CG 5125, Xylella fastidiosa (strain Temecula1 / ATCC 700964), OCTD, 10^[-6], DARK, wood naphtha, taf-ibeta, stem node, male sterility 1, Long-Term Effect, dArk, Dapaf-1, Guanidine Hydrochloride, 2-iodo-, thiol methyltransferase activity, Controlled, small, Atg5l, Neonatal, DYRK1, Controlling, 3H-labeled, DL-Methionine, igaad, apaf1, D6Ertd375e, CG1643, Guanidinium, Peptide, group, Mus muscaris, [HCl], count in organism, LC/MS/MS, Mus, Protein, I2PP2A, Hydrochloride, Guanidine Sulfite (1:1), CT42491, Dyrk1, Dark, (3alpha, Dark/Apaf-I, ACETONITRILE, tandem MS, Zystein, cyanomethane, WMS2, Data Base, DDA, Enolase 2, c-KIT, PRSS, nodal stem, MALE STERILITY 1 PROTEIN, 2-amino-3-sulfanylpropanoic acid, CC1, Tripcellim, L-2-Amino-3-mercaptopropionic acid, dApaf-1/DARK/HAC-1, sample population, Laboratory Mice, Protein Gene Products, Trypure, dSET/TAF-Ibeta, c-kit, 2610030F17Rik, wood spirit, SSKS, alpha-amino-gamma-methylmercaptobutyric acid, Peptid, assay, SCH, AA407739, 6-diaminohexanoic acid, Laboratory Mouse, dAPAF-1, Neuron-specific enolase, 3-OHECNGolgi ribbon, D17Wsu94e, Golgi Complex, KLIP1, finger4, Complex, RGD1310157, LRRGT00110, C6orf109, Golgi complex, FinGER3, Golgi, FinGER4, Apparatus, 2310034L04Rik, RGD1308185, dJ337H4.3, Golgi.2010107M05Rik, APG5-LIKE, the brain, Atg5l, suprasegmental structures, Apg5l, Laboratory, atg5, AW319544, SAC domain-containing protein 8, Mus domesticus, mouse, APG5L, CG1643, House Mouse, encephalon, APG5, TPSG1, S-adenosyl-L-methionine:thiol S-methyltransferase activity, 3110067M24Rik, DmelCG1643, House, Mus, AUTOPHAGY 5, proteomic analysis, Mus musculus domesticus, synganglion, Mice, DmAtg5, Mus musculus, Swiss, mice, TRANSMEMBRANE PROTEIN G5P, Swiss Mouse, House Mice, Swiss Mice, suprasegmental levels of nervous system, Paddy, ATATG5, Encephalon., ASP, ATG5, domesticus, Laboratory Mice, MKP11.20, mug77, PRSS31, MKP11_20, C88337, Mouse, house mouse, TMT, Laboratory Mouse, thiol methyltransferase activity, hAPG5biochemical pathways, single-organism catabolic process, multicellular organismal catabolic process, ShakB, Ghrfr, LC3B, MM46, ER-Phagy, MAP1A/1BLC3, Map1lc3, CG1321, C6orf109, histology, CG32508, protein, Organelle, sci, phosphorylation, CMRF35A1, protein polypeptide chains, Readability, pass, initial autophagic vacuole, GEF2, HOW, How, cellular degradation, 2, 3, 6, protein aggregate, anatomy and histology, CMRF-35, l(3)j5D5, Autophagosome, 2310034L04Rik, 24B, D17Wsu94e, Shak B, catabolism, membrane region, stru, proteins, l(3)S053606, Golgi, l(1)TH73, ATG8, CG10293, Atg8, CG34358, l(3)j5B5, LC3b, ER Phagy, Nucleophagy, MAP1LC3B-a, biotransformation, l(1)LB21, R-9-29, LIR, shkB, l(1)W3b, CLM-6, RKS10, ELG, Pas, 0904/17, l(1)W3, shB, 1010001C15Rik, integral to membrane, GABARAP-a, inx8, lit, Ribophagy, 9130422N19Rik, nj-156, pas, Reticulophagy, SZ1, ELONGATED, KLIP1, Autophagocytosis, Cellular, secretion, APG8L, W3, little, AI196471, GEF-2, breakdown, Golgi Complex, Autophagy, finger4, l(1)R-10-3, GATE16, BRI1-associated receptor kinase, l(1)R-10-7, Shak-B, Apg8l, Dmel_CG12678, SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE 3, anon-EST:Liang-2.39, CMRF-35A, Understanding., CMRF35A, P62, shak-B, number, Lipophagy, Gene, protein-containing complex, presence, cellular catabolism, F17M5.190, morphology, polypeptide chain, integral component of membrane, Gene Products, Cellular Autophagy, R9-29, ATG8L, ATG8F, SERK3, dJ337H4.3, 3110025G09Rik, CMRF35-A1, anatomy, l(3)s2612, breakdown of chemical, ATSERK3, Complex, Phagophores, RGD1308185, l(1)R-9-29, Atg8l, Golgi ribbon, nj156, Dmel_CG32508, DmelCG10293, region of membrane, DmelCG34358, IGSF16, CG15451, F17M5_190, cellular breakdown, degradation, clone 2.39, protein complex, GEC1, Gec1, Proteins, RGD1310157, Phosphorylations, Golgi complex, selective autophagy, qkr, Apparatus, l(3)S090417, motif, count in organism, native protein, natural protein, KH93F, Protein, proteomic analysis, whole membrane, CMRF35, FinGER3, FinGER4, autophagosome, breakdown of molecule, who, CG12678, APG8-LIKE, ATG8C, RECEPTOR KINASES LIKE SERK 10, transmembrane, ATG8B, ATG8A, biodegradation, ATBAK1, Who/How, l(1)19Eb, Phagophore, Protein Gene Products, Gene Proteins, GATE-16, breakdown of substance, GECI, MAP1A|1BLC3, qkr[93F], l(1)R-10-14, LRRGT00110, E81falseTMT-based proteomic analysis of Atg5-/- mouse brainThe brains of neonatal mice (Atg5+/+ in triplicate, Atg5-/- in quadruplicate, and Atg5-/-;NSE-Atg5 in triplicate) were lysed in 500 µL of 6 M guanidine-HCl containing 100 mM Tris-HCl, pH8.0, and 2 mM DTT. The lysates were dissolved by heating and sonication, followed by centrifugation at 20,000 g for 15 min at 4 ºC. Proteins (100 µg each) were reduced in 5 mM DTT at room temperature for 30 min, alkylated in 27.5 mM iodoacetamide at room temperature for 30 min in the dark, and subjected to methanol/chloroform precipitation. After solubilization with 25 µL of 0.1% RapiGest SF (Waters) in 50 mM triethylammonium bicarbonate, the proteins were digested with 1 µg of trypsin/Lys-C mix (Promega) for 16 h at 37 ºC. The peptide concentrations were determined using the Pierce quantitative colorimetric peptide assay. Approximately 25 µg of peptides for each sample was labeled with 0.2 mg of TMT-10plex reagents (Thermo Fisher Scientific) for 1 h at 25 ºC. After the reaction was quenched with hydroxylamine, all the TMT-labeled samples were pooled, acidified with trifluoroacetic acid (TFA), and fractionated using the Pierce high pH reversed-phase peptide fractionation kit. Eight fractions were collected using 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, and 50% acetonitrile (ACN). Each fraction was evaporated in a SpeedVac concentrator and dissolved in 0.1% TFA. LC-MS/MS analysis of the resultant peptides (1 µg each) was performed on an EASY-nLC 1200 UHPLC connected to a Q Exactive Plus mass spectrometer through a nanoelectrospray ion source. The peptides were separated on a 75 µm inner diameter X 150 mm C18 reversed-phase column (Nikkyo Technos) with a linear gradient of 4–20% ACN for 0–180 min and 20–32% ACN for 180–220 min, followed by an increase to 80% ACN for 220–230 min. The mass spectrometer was operated in a data-dependent acquisition mode with a top 15 MS/MS method. MS1 spectra were measured with a resolution of 70,000, an automatic gain control (AGC) target of 3e6, and a mass range of 375–1400 m/z. HCD MS/MS spectra were acquired at a resolution of 35,000, an AGC target of 1e5, an isolation window of 0.4 m/z, a maximum injection time of 100 ms, and a normalized collision energy of 32. Dynamic exclusion was set to 30 s. Raw data were directly analyzed against the SwissProt database restricted to Mus musculus using Proteome Discoverer version 2.2 (Thermo Fisher Scientific) with Mascot search engine version 2.5 (Matrix Science) for identification and TMT quantification. The search parameters were as follows: (a) trypsin as an enzyme with up to two missed cleavages; (b) precursor mass tolerance of 10 ppm; (c) fragment mass tolerance of 0.02 Da; (d) TMT of lysine and peptide N-terminus and carbamidomethylation of cysteine as fixed modifications; and (e) oxidation of methionine as a variable modification. Peptides were filtered at a false-discovery rate of 1% using the percolator node.Sat Jun 01 00:00:00 BST 2024PXD0492201009038822137