Phosphoproteomic identification of ULK substrates reveals VPS15-dependent ULK/VPS34 interplay in the regulation of autophagy (2021)
Figure 8

Figure 8. Recruitment of autophagy proteins to aberrant structures regulated by both ULK complex and VPS34 complex I activity.

A) HEK293A and VPS15 KOs were treated with vehicle or VPS34-IN1 (1μM; IN1), alone or with MRT68921 (1μM; MRT), for 18 hrs before fixation and visualisation of ULK1 (blue), p62 (green) and ATG9A (red). Dashed boxes show magnified regions of interest.

B) HEK293A were transfected with siRNAs targeting ATG14, UVRAG or p62, or with non-targeting siRNA (RISC-free, RF) and cells were treated with VPS34-IN1 (IN1; 1μM) for 0, 4 or 18 hrs as indicated. Quantification of ATG13 pS318/Total ATG13, mean +/- SEM, n=3. Identical samples were loaded onto 2 separate SDS PAGE gels for Western blot analysis, indicated by dashed line.

C) Cells transfected as in B were treated with IN1 for 18 hours as indicated, fixed and labelled for ubiquitin (green), FIP200 (red) and p62 (blue). FIP200-positive bodies were quantified from 4 independent experiments with mean +/- SEM plotted. Cells transfected with ATG14 siRNA generated significantly more FIP200 bodies per cell than all other conditions tested.

Data information: * p<0.05., *** p<0.001, [one-tailed ANOVA]. Scale bars 10µm.

Materials and Methods

Cell Culture

Buffers used for cell culture were Dulbecco's Modified Eagle Medium-High Glucose (DMEM, Sigma) and 0.25% trypsin-EDTA solution (Sigma), Earle's Balanced Salt Solution (EBSS) phosphate buffered saline (PBS) and versene (0.02% (w/v) EDTA, 11mg/L phenol red in PBS) (produced in house). Cell lines were cultured in DMEM supplemented with 10% foetal bovine serum (FBS, Sigma), 500U/ml Penicillin + 100µg/ml Streptomycin (Pen-Strep, Sigma) and 4.8mM L-glutamine (full medium), and maintained in 10% CO2. Short tandem repeat analysis was used to confirm the identity of every cell line utilised herein.

HEK293A, HEK293T and MEFs were described previously (Chan et al., 2009; Chan et al, 2007; McAlpine et al., 2013). Ulk1-/- Ulk2-/- (ULK DKO) MEFs used in the SILAC screen were described in (McAlpine et al., 2013) and the DKO MEF line used in the TMT screen, VPS15 KO clones and lentivirally rescued VPS15 KO clones were generated as described below. When CRISPR/Cas9-mediated genome engineering was used to generate the VPS15 KO HEK293A clonal cell lines following the Zhang laboratory protocol (Ran et al, 2013). The first coding exon of VPS15 (gene name PIK3R4) was targeted with 2 sgRNAs (sgA, forward 5′-CAA AAA CCT TCA CAA CGA CC-3′; reverse 5′-GGT CGT TGT GAA GGT TTT TG-3′; and sgC, forward 5′-ACA AAT CTG GAG TTC GTC AT-3′; reverse 5′-ATG ACG AAC TCC AGA TTT GT-3′), designed using the Zhang laboratory CRISPR design tool (now offline, previously available at https://zlab.bio/guide-design-resources). sgRNAs were cloned into Bbs1 site of pSpCas9(BB)-2A-GFP plasmid also obtained from the Zhang laboratory (Addgene; 48138). Following single-cell sorting on GFP-positive cells, Western blotting was used to screen colonies for VPS15 deficiency. VPS15 KO clones (sgA-B3, sgA-D6 and sgA-D11) were used interchangeably between exepriments.

Treatments were applied for specified times, diluted in either EBSS or full medium: Torin 1 (100nM, Cayman Chemical), MRT68921 (1µM, Sigma), 991 (1µM, Selleckchem), Oligomycin (1µM, Sigma), Deferiprone (1mM, Sigma), Antimycin (1µM, Sigma), Bafilomycin A1 (100nM, Calbiochem) and VPS34-IN1 (1µM, Cayman Chemical).

Transfections/Transductions

For all experiments other than VPS15 KO transient rescue experiments, in which JetPRIME (Polyplus) was used, Lipofectamine 2000 (Life Technologies) was used for plasmid transfections, with both reagents used as per the manufacturer's instructions. For knockdown experiments, 20µM siRNA stocks were made up in siRNA dilution buffer (Dharmacon) before transfection at 50nM final concentration using Lipofectamine 2000 (Life Technologies). Cells were reverse transfected with siRNA on day 1 and again on day 2 before preparation for analysis by Western blot or immunofluorescence on day 4.

Primary ULK DKO MEFs derived from day 13 embryos were cultured in full media supplemented with 20% FBS before immortalisation via retroviral transduction of SV40 large T antigen. When stably rescuing VPS15 KO clones, lentiviral transfer vectors (3rd generation) encoding wild type, S861A, S861E, VPS15-HA_IRES_EGFP, or GFP alone were used. Pen-Strep was excluded from culture media and was replenished only after successful transduction. Retro- and lentiviruses were produced in HEK293T. Alongside lentiviral transfer plasmids listed above, psPAX2 gag-pol and VSVG plasmids were transfected at a 4:3:1 ratio. To generate retroviruses pBABE SV40T Puro, MMLVgag-pol and VSVG plasmids were transfected at a 3:2:1 ratio. Full medium was replenished after transfection and after 24 hrs, virus-containing media were collected and supplemented with Polybrene (8µg/ml, Sigma). The virus-containing media were centrifuged (4000rcf, 5 mins) and filtered (0.45μM Millex HV filter (Millipore)) before addition to target cells. If lentiviral transduction efficiency was poor, this process was repeated ≤3 times, with virus-producing HEK293T discarded at 3 days-post transfection. Fluorescence activated cell sorting (FACS) was used to isolate the lowest EGFP expressing lentivirus-transfected cells 5-7 days after transduction. Puromycin (1µg/ml, Sigma) was added to growth media of retrovirally-immortalised cells for 7 days to isolate successfully transduced cells.

Constructs and siRNAs

Constructs encoding VPS15-ZZ, VPS34, ATG14+Beclin1, VPS34 complex I (multicassette complex encoding VPS34, VPS15, Beclin1 and ATG14-ZZ), VPS34 complex II (multicassette complex encoding VPS34, VPS15, Beclin1 and UVRAG-ZZ) and GST-PX were kindly provided by Roger Williams PhD. Phosphomutant and HA-tagged VPS15 constructs were generated in this study. The expression plasmid for UVRAG was provided by Christian Behrends PhD and the myc tag added by Harold Jefferies PhD. Myc-ULK1 1-427 kinase inactive was generated in this study, and all other myc-ULK constructs were reported previously (Chan et al., 2009; Chan et al., 2007). FLAG-PRKAG2 was from Grahame Hardie PhD whilst FLAG-PRKAG2 Δ124 and all PRKAG2-GFP constructs were generated in this study. PRKAA1-myc and PRKAB2 were kindly provided by David Carling, PhD. GFP-2xFYVE was a gift of George Banting PhD was from Roger Williams PhD. The lentiviral transfer vector encoding WT VPS15 was purchased from VectorBuilder, with all variants generated in this study. RISC-free siRNAs (D-001220-01), or siGENOME SMARTpools targeting ATG14 (M-020438-01), UVRAG (M-015465-01) or p62 (M-010230-00) were ordered from Dharmacon.

Antibodies

Anti-LC3-B (ab48394) and Beta Tubulin (ab6046) were from Abcam and anti-actin AC40 (A4700) was from Sigma-Aldrich. Anti ATG14 (M184-3) was from MBL Life Science and anti-VPS15 (NBP130463) was from Novus Biologicals. Anti-GM130 (610822), LAMP1 (CD107a), p62 (for use in Western blotting, 610833) and TIM23 (611223) were from BD Biosciences and anti-p62 (for use in immunofluorescence, GP62-C) was from Progen Biotechnik. Anti-ULK1 (sc-33182) was from Santa Cruz Biotech. Anti-Beclin1 (3738), Phospho-PRKAA1 T172, VPS34 (3811), Phospho-VPS34 Serine249 (13857), PRKAB1/2 (4150) and Phospho-PRKAB2 Ser39 (82791) were from Cell Signalling Technology. Anti-Phospho-ATG13 Ser318 (600-401-C49) was from Rockland. HRP-conjugated anti GST (RPN1236) was from GE Healthcare and anti-Ferritin (ABIN99122) was from Antibodies-Online. Anti FIP200 (17250-1-AP) was from Protein Tech. Anti Phospho-VPS15 Serine861 was generated during this study, as was Phospho-PRKAG2 Serine124. Anti-WIPI2 mouse monoclonal (Polson et al, 2010), anti mATG9 Armenian hamster monoclonal (Webber & Tooze, 2010) and anti-ATG13 (Chan et al., 2009) were previously described and both mouse monoclonal myc (9E10) and GFP (3E1) was from the Francis Crick Institute.

Secondary antibodies for immunofluorescence (anti-rabbit IgG Alexa Fluor 488, 555, and 647; anti-mouse IgG Alexa Fluor 488, 555, and 647) were from Life Technologies and anti-hamster Cy3 was from Jackson ImmunoResearch. For use in Western blotting, HRP-conjugated secondary antibodies were from GE Healthcare.

Generation of Lysates for Western Blotting

PBS was used to wash cells twice before lysis in TNTE buffer (20mM Tris pH6.8, 150mM NaCl, 5mM EDTA, 1% w/v Triton X-100) containing 1X EDTA-Free Complete Protease Inhibitor cocktail (Roche) and 1X PhosSTOP (Roche). To prevent cell detachment, HEK293A that were treated with EBSS were not washed. Lysates were cleared by centrifugation at 13,200rcf for 5 mins. Post-nuclear supernatants were mixed with 5X sample buffer (213.5mM Tris-HCl pH6.8, 50% w/v glycerol, 16% β-mercaptoethanol, 15% w/v SDS, bromophenol blue) to a final concentration of 1X before incubation at 100°C for 5 mins. Lysates were then analysed by SDS PAGE as described previously (Judith et al., 2019).

Immunoprecipitation

For immunoprecipitation experiments, 10cm dishes were washed twice with cold PBS before harvesting in 500µl TNTE. Post-nuclear supernatants (PNS) were prepared as described above. A sample of PNS was retained and mixed with sample buffer comprising the loading control/input sample. The antibody-conjugated resins used are: myc-Trap_A (ChromoTek), GFP-Trap A (ChromoTek), anti-FLAG M2 Affinity Agarose Gel (Sigma) and IgG Sepharose 6 Fast Flow affinity resin (GE Healthcare). To immunoprecipitate ULK complexes for peptide array-based in vitro kinase assays, protein G sepharose beads (Sigma) were bound with anti-myc 9E10 by incubating 50µl protein G sepharose slurry per 10cm dish with 15µg antibody at 4°C, turning end over end for 60 mins. All beads were washed twice in PBS and twice in TNTE before use.

Immunoprecipitation experiments were performed by rotating clarified lysates with washed beads at 4°C end over end for 2-4 hrs, after which beads were pelleted by centrifuging the lysate-resin mixture at 7000rcf. The supernatant (unbound sample) was retained to test pulldown efficiency. The pelleted beads were washed in TNTE containing 0.1% Triton X-100 (3-5 x 1ml). If no further processing was required, the beads were aspirated and prepared for Western blot analysis.

To improve sample purity when purifying ULK complexes and PRKAG2, the wash buffer NaCl concentration was increased from 150mM to 300mM. Where expression levels of target proteins differed, immunoprecipitates sometimes had to be normalised prior to analysis. In these cases, plasmid amounts used for transfection were altered empirically until expression levels were within the same range. Where required, pcDNA3.1 (+) empty vector was added proportionately such that the total amount of DNA used for transfection was maintained.

Protein Elution for Peptide Array-Based In Vitro Kinase Assays

To generate kinase samples for use in peptide array-based in vitro kinase assays, HEK293A cells were cotransfected with ATG13-FLAG, FLAG-FIP200 and myc-Ulk1 or myc-Ulk2 in 10cm dish format. Transfected cells were starved for 1 hr +/- MRT68921 as indicated and complexes were immunoprecipitated as described above. To elute ULK complexes, 100µl kinase reaction buffer (KRB - 20mM HEPES pH7.4, 20mM MgCl2, 25mM beta-glycerophosphate, 2mM dithiothreitol, 100µM sodium orthovanadate) per 10cm dish supplemented with 0.5mg/ml myc peptide (EQKLISEEDL) was added to fully aspirated beads before shaking for 20 mins at 25°C. Equal protein amounts were confirmed by immunoblot. The elution buffer was supplemented with MRT68921 where appropriate, to a final concentration in the in vitro kinase assay reaction mixture of 1µM.

In Vitro Kinase Assays

HEK293A expressing kinase (myc-Ulk1 WT/KI) or substrates (VPS34 complex I components, VPS15-ZZ alone or FLAG-PRKAG2 fragments) were lysed and proteins immunoprecipitated as described above. Kinase-expressing dishes were starved for 30-60 mins before lysis. Washed beads were equilibrated in KRB before kinase- and substrate-bound beads were mixed or left unmixed as indicated. The reaction mixture (Radioactive Assays: 32µl KRB with 100µM ATP and 2µCi ATP[γ-32P], (Easy Tide Lead, Perkin Elmer); Non-Radioactive Assays: 32µl KRB with 1.8mM ATP]) was added to fully aspirated beads, before incubating for 30 minutes at 30°C. Sample buffer was used to terminate reactions, and samples were electrophoresed by SDS PAGE. For radioactive assays, gels were fixed using Instant Blue Coomassie staining (Expedeon) as per manufacturer's instructions and gels were dried before using to expose autoradiography/chemiluminescence film (Amersham Hyperfilm ECL, GE). For non-radioactive assays, the reaction mixtures were analysed by immunoblot.

Eluted ULK complexes were used to phosphorylate peptides in array format (see above and Fig EV2). For each experiment, 2 identical array membranes (produced in house) were activated via 2 mins incubation in methanol, rinsed well in wash buffer (20mM HEPES pH7.4, 0.02% Triton X-100) before 4x10 minute incubations with wash buffer rocking at room temperature. Membranes were rocked overnight in blocking buffer (20mM HEPES pH7.4, 0.02%Triton X-100, 0.2mg/ml BSA) at 4°C. The following day, reaction mixtures were generated by diluting kinase eluates (see above) to 5ml with protein dilution buffer (0.05mg/ml BSA in KRB) before mixing 1:1 with ATP solution (200µM ATP, 200µCi ATP[γ-32P] in KRB) and adding to arrays. The peptide array membranes were rocked gently for 15 mins at room temperature in 10ml reaction mixtures before rinsing thoroughly in PBS. Membranes were then washed extensively by rocking at room temperature for 10 mins in: PBS x2, 30% Glacial Acetic Acid (Fisher) x3, dH2O x2, 0.1N NaOH x1, dH2O x2, 30% Glacial Acetic Acid x2. Phosphorylated peptide arrays were visualised using autoradiography/ chemiluminescence film (Amersham Hyperfilm ECL, GE).

Phosphoantibody Generation

15mer peptides centred on VPS15 serine 861 encoding a phosphoserine in the central position (NVNEEWK(pS)MFGSLDC) were generated in house and used to immunize rabbits. The antibodies were affinity purified with the phosphoS861 peptide. The affinity purified phosphoantibody was used in the presence of the non-phosphorylated peptide (NVNEEWKSMFGSLDC) at 5X molar ratio. The PRKAG2 phosphoserine 124 was were produced and purified by Covalab, FR.

Confocal Microscopy

For immunofluorescence analysis, cells were seeded on coverslips before fixation in 3% PFA. 5-minute incubations in either methanol or 0.2% Triton X‐100 in PBS were used to permeabilise cells, which were then blocked in 5% BSA in PBS. Cells were incubated for 1 hr in in 5% BSA with the addition of appropriate primary and secondary antibodies, before mounting on slides and analysis using either a Zeiss Upright 710 confocal microscope with a 63× objective lens or, for data shown in Figs 7 and 8, a Zeiss Invert 880 confocal microscope with Airyscan.

High Throughput Screening Image Acquisition and Analysis

For the HTS experiments, cells were grown in 96-well plates (Greiner Bio One Ltd 655090) before fixation, permeabilisation and antibody staining as described above. Imagining was performed using an Opera Phoenix High Content Screening System (PerkinElmer) with 40x/NA 1.1 water-immersion lens. Z-stacks from 0 to 1µm with a step size of 0.5µm were acquired using excitation lasers at 405, 488, and 555nm, and emission filters at 450, 525, and 580nm, respectively. Cell segmentation and quantification analysis were performed using Harmony software 4.9 detecting the parameters indicated in Appendix Table S3.

Mass Spectrometry

WT and ULK DKO MEFs were grown in DMEM supplemented with 10% dialyzed FCS (Invitrogen) supplemented with a combination of either 100 mg per litre of light (14N,12C) or heavy (15N,13C) lysine and arginine (CK Isotopes), with full label incorporation confirmed by mass spectrometry before use. Cells were treated with EBSS or with SILAC media supplemented with Torin 1 (100nM) for 2 hrs to induce ULK activation. Cells were detached and mixed in a 1:1 ratio heavy:light and 5mg total protein per mixture was used per experiment. Proteins were digested using a combination of trypsin and LysC and peptides were fractionated using Strong Cation Exchange chromatography using a Poly LC PolySULFOETHYL A 100 x 4.6mm 5um 200A column. The flow rate was 1ml/min and the gradients used volatile buffers A: 10 mM Ammonium Formate, 25% ©, pH 3.0 and B: 500 mM Ammonium Formate, 25% ACN, pH 6.8. All fractions collected were taken to dryness by vacuum centrifugation. 5mg of Titansphere titanium dioxide beads were used per SCX fraction for phosphopeptide enrichment as describe previously (Swaffer et al, 2016). Dried phosphopeptides from the titanium dioxide enrichment were cleaned up in preparation for LC-MS/MS analysis using C18 Stage Tips and taken to dryness by vacuum centrifugation. Each sample was resuspended in 35ul 1% TFA, sonicated for 15 mins and injected 3 times (10ul per injection). Peptide mixtures were separated on a 50 cm, 75um I.D. Pepmap column over a 3hr gradient and eluted directly into an LTQ-Orbitrap Velos mass spectrometer. The instrument ran in data dependent acquisition mode with the top 10 most abundant peptides selected for MS/MS by either CID, MSA or HCD fragmentation techniques (one fragmentation technique per injection). MaxQuant and Perseus were used for data processing with false discovery rate (FDR) of 1% set at the protein, peptide and phosphosite level.

To generate samples for TMT analysis, SV40 immortalized WT and ULK DKO MEFs were starved for various time points as indicated before trypsinisation, washing in 0.1% (w/v) BSA PBS and pelleting via centrifugation (3000rcf, 1 minute). Cell pellets were resuspended in ice cold PBS, then pelleted again for lysis in 200µl 8M Urea lysis buffer (8M Urea, 50mM HEPES pH8.2, 75mM NaCl, 1X PhosStop (Roche), 1X Complete EDTA-Free Protease Inhibitor). Lysates were sonicated and protein concentration was measured before 220µg aliquots of each sample were digested in a combination of trypsin and LysC. Next, individual digests were cleaned and concentrated using Nest Group C18 MacroSpin columns. Peptide labelling with TMT reagent was performed according to the manufacturer's protocol (TMT 10plexTM, #90110, Thermo). For peptide enrichment the Thermo Scientific High-Select Fe-NTA phosphopeptide enrichment kit (A32992) was used as described here (https://star-protocols.cell.com/protocols/66). Phosphopeptides eluted from the procedure were dried by vacuum centrifugation and subjected to fractionation using the High pH Reversed Phase Fractionation kit (Pierce, 84868). The resulting dried TMT labelled phosphopeptides were resuspended in 1% TFA and separated on a 50 cm, 75um I.D. Pepmap column over a 2-4 hr gradient at 40°C and eluted directly into the mass spectrometer (Orbitrap Fusion Lumos). The instrument was run in data dependent acquisition mode with the most abundant peptides selected for MS/MS by HCD fragmentation. MaxQuant v1.6.x was used to process the raw data acquired with a reporter ion quantification method. A protein, peptide and phosphosite estimated false discovery rate (FDR) of 1% was used with the mus musculus Uniprot KB database used for database searching. Reporter ion intensities were imported into Perseus from the Phospho STY Sites table for further downstream processing.

To measure changes in VPS15 phosphorylation upon Ulk1 overexpression, HEK293A were cotransfected with the multicassette VPS34 complex I construct (see above) alone, with myc-Ulk1 WT or with myc-Ulk1 KI. After lysing cells, IgG Sepharose 6 Fast Flow affinity resin was used to coimmuniprecipitate VPS34 complex I via ATG14-ZZ. Samples were electrophoresed on a 4-12% mini gel (NuPAGE) and gels were fixed (Instant Blue, Expedeon) before VPS15 bands were excised and subjected to overnight trypsin digestion. Dried peptides were resuspended in 0.1% fluoracetic acid and separated on a 50 cm, 75um I.D. EasySpray C18 column over a 30-minute gradient and eluted directly onto a Q-Exactive mass spectrometer. The instrument was run in data dependent acquisition mode. MaxQuant and Perseus were used to process the data. Phosphorylation of serine, threonine and tyrosine and oxidation of methionine were used as variable modifications in the search settings. Potential ULK substrates were highly enriched in myc-Ulk1 WT-expressing cells only.

Phosphoproteomal Analysis

Analytical approaches for SILAC screen are described in the text. Bioinformatics analyses for TMT were performed as follows: 3 variables were defined to describe both the starvation- and the ULK-dependence of the phosphorylation events as shown in Fig EV1F. The comparisons selected were: WT 0 mins-WT 60 mins (increase in phosphorylation after 60 mins starvation in WT MEFs), WT 60 mins-DKO 60mins (decrease in phosphorylation in DKO compared to WT MEFs at 60 mins starvation) and WT 60 mins-WT 60 mins Refed (decrease in phosphorylation when full media was replenished in WT MEFs). Threshold values for each variable were established by trial and error and phosphopeptides simultaneously satisfying all three were brought forward preferentially.

A fourth variable was used to annotate the dataset, statistical similarity to Prkab2 S38. As ULK substrates may display varied phosphorylation dynamics, peptides with a correlation coefficient of >0.7 were brought forward to maximise substrate identification (Pearson correlation coefficients of >0.8 are considered significant). Phosphopeptides satisfying the first three variables were given precedence when shortlisting putative ULK substrates as this was more stringent. However, as many of the sites shortlisted using the four variables did not display profiles indicative of direct ULK phosphorylation the dataset was curated manually with clear false positives removed.

Protein Purification

GST-PX for use in the GST overlay assays were purified from E. coli as described in (Wirth et al, 2019). Purification protocols of human complexes I and II were described previously (Ohashi et al., 2016; Ohashi et al, 2019). The derivative mutants were purified essentially in the same way as the WT complexes.

Thermostability Assays

Purified VPS34 complex I (WT or mutant) were diluted to 0.5 mg/mL in dilution buffer (20 mM HEPES 8.0, 150 mM NaCl, and 0.5 mM TCEP for complex I). For each sample, 10 μl of the diluted sample was absorbed in a glass capillary, then set on a holder for a differential scanning fluorimetry reader (Prometheus, NanoTemper). The thermal stability was measured in a range from 15˚ C to 95˚ C following the manufacturer's instruction.

GUV In Vitro Kinase Assays

The lipid composition for the GUVs in vitro kinase assays was 18% liver PI, 10% DOPS, 17% DOPE, 55% DOPC, 0.03% DSPE-PEG-Biotinyl, 0.017% Liss-Rhodamine. GUV generation, activity measurement, and image analysis were described previously (Ohashi et al, 2020).

LUV In Vitro Kinase Assays

The lipid composition for LUV in vitro kinase assays was 18% liver PI, 10% DOPS, 17% DOPE, 55% DOPC. For Fig EV4A, 800nm LUVs generated via extrusion were incubated in lipid kinase reaction buffer (described previously (Ohashi et al., 2020)) containing 200μM ATP with VPS34 complex I incorporating WT, 6SA or 6SE VPS15-ZZ, coimmunoprecipitated via VPS15-ZZ. Reactions were shaken at room temperature for up to 30 minutes before spotting on PVDF. Membranes were blocked in PBST 3% BSA and PI3P visualised using the GST-PX probe (2µg/ml, PBST 3% BSA). Bound GST-PX was detected via immunoblot using HRP-conjugated anti GST antibodies. For Fig EV4C, LUVs were generated as described previously (Ohashi et al., 2020). 100nM of complex I (WT, 6SA or 6SE) were used for the assays. The activity was measured in the presence of 50 μM ATP using ADP-Glo assay kit (Promega). For each experiment, ATP-ADP mixtures were included to generate an ATP-ADP conversion line. The luminescence values were recorded by a microplate reader (PHERAstar, BMG Labtech). The values were subtracted by the intercept of the ATP-ADP conversion line (background subtraction). Specific activity was calculated as: background-subtracted luminescence value/slope of the conversion line/protein concentration/reaction time (min).

Lipid Flotation Assays

Lipid flotation assays using LUVs were described previously (Ohashi et al., 2020).

Western Blot and Immunofluorescence Quantification

Image J (NIH) was used to quantify Western blots. For immunofluorescence analysis, WIPI2 puncta number were quantified using Imaris 8x64 software (Bitplane), with cell numbers counted manually.

Statistical Analysis

Experiments were repeated independently ≥ 3 times when quantifications are shown and ≥ 2 times if not, unless otherwise stated. Where WIPI2 puncta were counted, 8 images (around 100 cells total) were analysed per condition per repeat. Mass spectrometry experiments were performed once only. GraphPad Prism 8.4.1 was used to plot graphs and calculate statistics. One-tailed ANOVAs were performed in all cases other than Fig 4F, EV4C and EV4D in which student's T tests were employed.

Use of the ULK1 Motif

In Fig 1B, the ULK1 motif (Egan et al., 2015) was rendered as follows, beginning at position -3 and ending at position +2 relative to the phosphoacceptor at position 0 (serine or threonine), with conforming residues in square brackets and non-conforming residues in braces: [M/L/Q/F]-{P}-{P}-[S/T]-[Y/I/M/S/F/V/W]{P}-[Y/S/H/I/W/M]{P}.

Comment on Nomenclature

Human protein/gene names are represented in uppercase, with the first letter along capitalised for non-human homologs (murine unless stated). Exceptions are human proteins Beclin1 and p62, spelled thus to correspond with the prevalent naming convention. 'ULK' is used to refer to ULK homologs generally, typically ULK1/Ulk1 and ULK2/Ulk2. When referring to S. cerevisiae proteins, their gene names are given with the first letter capitalised and followed by the letter p.

Data Availability

The SILAC and TMT proteomics datasets are deposited at PRIDE hosted at the EBI. Data are available via ProteomeXchange with identifier PXD022228.

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