Autophagy is a conserved catabolic process that utilizes a defined series of membrane trafficking events to generate a double-membrane vesicle termed the autophagosome, which matures by fusing to the lysosome. in autophagy regulation. in the cytoplasm and sequesters bulk cytoplasmic components. This vesicle matures by fusing to the lysosome, leading to the degradation of the contents inside (2, 3). Upon nutrient starvation, autophagy is induced to promote cell survival (4, Panaxadiol 5). Yeast genetic and follow-up studies identified over 30 autophagy-related genes (ATG)2 (5, 6), some of which facilitate different forms of selective autophagy while others constitute the core machinery of autophagy (7). Components of the core machinery of autophagy can be segmented into multiple separate functional units (8, 9). In this study, we focus on the Atg1/ULK1 initiator complex. Atg1, and its mammalian counterpart ULK1, are the only protein kinase among the more than 36 Rabbit Polyclonal to WIPF1 ATG proteins. In mouse cDNA was purchased from ATCC (clone ID 5359944). Serine to alanine, aspartic acid, and glutamic acid mutation were generated by PCR-mediated site-directed mutagenesis. Panaxadiol The residue of interest was mutated alongside adjacent acceptor residues, in order to prevent compensatory phosphorylation: S224A (S222A/S223A/S224A); S258A (S256A/T257A/S258A); S224E (S222E/S223E/S224E); S258E (S256E/T257E/S258E). All mutations were subsequently confirmed by direct sequencing. For expression in MEF and HEK293T cells, was subcloned into pBabe/puro containing an N-terminal FLAG-S tag or HA tag. Tandem Affinity Purification FLAG-S-ATG13 was stably expressed to near endogenous levels in MEF cells. Cells were either starved in amino acid and serum-free DMEM media for 1.5 h or kept in full-medium and stimulated with insulin at 1 g/ml for 15 min before harvesting. We used 24 15-cm plates for each condition. Cells were lysed in 600 l of IP lysis buffer (50 mm HEPES pH 7.4, 10 mm KCl, 1 mm EDTA, 1 mm MgCl2, 10% glycerol, and 0.5% Triton X-100) per 15-cm plate, supplemented with protease inhibitors and a mixture of phosphatase inhibitors (Sigma, no P5726 and P0044). Lysates Panaxadiol were incubated on ice for 20 min and then centrifuged for 15 min at 16, 000 and then followed by an additional spin at 100,000 trypsin digestion of polypeptides in each gel slice was performed as described (21). The tryptic peptides were purified using a 2-l bed volume of Poros 50 R2 (Applied Biosystems) reversed-phase beads packed in Eppendorf gel-loading tips (22). The purified peptides were diluted to 0.1% formic acid and then subjected to nano-liquid chromatography coupled to tandem mass spectrometry (nano-LC-MS/MS) analysis as follows. Peptide mixtures (in 20 l) were loaded onto a trapping guard column (0.3 5 mm Acclaim PepMap 100 C18 cartridge from LC Packings, Sunnyvale, CA) using an Eksigent nano MDLC system (Eksigent Technologies, Inc. Dublin, CA) at a flow rate of 20 l/min. After washing, the flow was reversed through the guard column and the peptides eluted with a 5C45% acetonitrile gradient over 85 min at a flow rate of 200 nl/min, onto and over a 75-micron x 15-cm fused silica capillary PepMap 100 C18 column (LC Packings, Sunnyvale, CA). The eluent was directed to a 75-micron (with 10-micron orifice) fused silica nano-electrospray needle (New Panaxadiol Objective, Woburn, MA). The electrospray ionization needle was set at 1800 V. A linear ion quadrupole trap-Orbitrap hybrid analyzer (LTQ-Orbitrap, ThermoFisher, San Jose, CA) was operated in automatic, data-dependent MS/MS acquisition mode with one MS full scan (450C2000 segment of Uniprot protein database (3,363 sequences; European Bioinformatics Institute, Swiss Institute of Bioinformatics and Protein Information Source). The search guidelines were as follows: (i) two missed cleavage Panaxadiol tryptic sites were allowed; (ii) precursor ion mass threshold = 10 ppm; (iii) fragment ion mass threshold = 0.8 Da; and (iv) variable protein modifications were allowed for methionine oxidation, cysteine acrylamide derivatization and protein N-terminal acetylation, mono- and di-methylated lysine and arginine, and tri-methylated Lysine. MudPit.