Reagents

The following chemicals were used in this study: oligomycin (A5588, ApexBio), antimycin A (A8674, Sigma-Aldrich), Q-VD-OPh (A1901, ApexBio), rapalog A/C hetero-dimerizer (635057, Takara), bafilomycin A1 (sc-201550, Santa Cruz Biotech), TBK1 inhibitor GSK8612 (S8872, Selleck Chemicals), TBK1 inhibitor BX795 (ENZ-CHM189-0005, Enzo Life Sciences), ULK1/2 inhibitor (MRT68921, BLDpharm), Vps34-IN1 inhibitor (APE-B6179, ApexBio) and DMSO (D2438, Sigma-Aldrich).

Plasmid construction

The sequences of all cDNAs were obtained by amplifying existing plasmids, HAP1 cDNA, or through gene synthesis (Genscript). For insect cell expressions, the sequences were codon-optimized and gene-synthesized (Genscript). With the exception of the NAP1-6×Ala mutant, which was obtained through gene synthesis (Genscript), all other plasmids were generated by Gibson cloning. Gibson reactions were transformed into DH5-alpha competent E. coli cells (18265017, ThermoFisher). Single colonies were picked, grown overnight in liquid cultures and pelleted for DNA plasmid extraction using the GeneJet Plasmid Miniprep kit (Thermo Fisher). The purified plasmid DNA was submitted for DNA Sanger sequencing (MicroSynth). All insert sequences were verified by Sanger sequencing. Positive clones were further analyzed by whole plasmid sequencing (Plasmidsaurus). A detailed protocol is available (https://doi.org/10.17504/protocols.io.8epv5x11ng1b/v1).

Cell lines

All cell lines were cultured at 37 °C in a humidified 5% CO2 atmosphere. HeLa (RRID: CVCL_0058) and HEK293T (RRID: CVCL_0063) cells were acquired from the American Type Culture Collection (ATCC). HAP1 parental (RRID: CVCL_Y019) cells, HAP1 FIP200 knockout (RRID: CVCL_TI59) and HAP1 ATG5 knockout (RRID: CVCL_SE00) were purchased from Horizon Discovery. HeLa and HEK293T cells were grown in DMEM (Thermo Fisher) supplemented with 10% (v/v) FBS (Thermo Fisher), 25 mM HEPES (15630080, Thermo Fisher), 1% (v/v) non-essential amino acids (11140050, Thermo Fisher) and 1% (v/v) penicillin–streptomycin (15140122, Thermo Fisher). HAP1 cells were cultured in Iscove’s modified Dulbecco’s medium (Thermo Fisher) supplemented with 10% (v/v) FBS (Thermo Fisher) and 1% (v/v) penicillin–streptomycin (15140122, Thermo Fisher). All cell lines were tested regularly for mycoplasma contamination. A detailed protocol is available (https://doi.org/10.17504/protocols.io.n2bvj3y5blk5/v1).

Generation of CRISPR–Cas9 knockout cells

All knockout cell lines were generated using CRISPR–Cas9. Candidate single-guide RNAs (sgRNAs) were identified using CHOPCHOP (RRID: SCR_015723; https://chopchop.cbu.uib.no). The sgRNAs were selected to target all common splicing variants. Using Gibson Cloning, the sgRNAs were ordered as short oligonucleotides (Sigma-Aldrich) and cloned into pSpCas9(BB)-2A-GFP vector (RRID: Addgene_48138). The successful insertion of the sgRNAs was verified by Sanger sequencing. A detailed description of this cloning is available (https://doi.org/10.17504/protocols.io.j8nlkkzo6l5r/v1).

Plasmids containing a sgRNA were transfected into HeLa cells with X-tremeGENE8 (Roche). Single GFP-positive cells were sorted by fluorescence-activated cell sorting (FACS) into 96-well plates. Single-cell colonies were expanded and collected for screening to identify positive clones by immunoblotting. Clones that showed a loss of protein expression for the target of interest were further analyzed by Sanger sequencing of the respective genomic regions. After DNA extraction, the regions of interest surrounding the sgRNA target sequence were amplified by PCR and analyzed by Sanger sequencing. The DNA sequences were compared to sequences from the parental line, and the edits were identified using the Synthego ICE v2 CRISPR Analysis Tool (https://www.synthego.com/products/bioinformatics/crispr-analysis). A detailed protocol is available (https://doi.org/10.17504/protocols.io.8epv59yx5g1b/v1).

For NAP1 and SINTBAD single knockout lines, we transfected sgRNAs for the respective target genes into naive HeLa cells (RRID: CVCL_0058) to obtain NAP1 knockout clones #32 (RRID: CVCL_D2YA) and #62 (RRID: CVCL_D2YB) or SINTBAD knockout clone #7 (RRID: CVCL_D2Y9). In cases in which we generated multiple gene knockouts in the same cell line, we sequentially transfected sgRNAs for the respective target genes. For NAP1/SINTBAD DKO clones #13 (RRID: CVCL_C9DV) and #14 (RRID: CVCL_D2Q0), the cells were first transfected with NAP1 sgRNA-targeting plasmids, and positive clones were then transfected with SINTBAD sgRNA-targeting plasmids. For NAP1/SINTBAD DKO clones #20 (RRID: CVCL_C8QB) and #26 (RRID: CVCL_D2Q1) in the pentaKO background, the pentaKO line (RRID: CVCL_C2VN), first described in a previous publication30, was transfected with NAP1 and SINTBAD sgRNA-targeting plasmids. For NAP1/SINTBAD/ULK1/2 4KO in the pentaKO background, ULK1/2 were first knocked out in the pentaKO line (RRID: CVCL_C2VS), and this cell line was then used further to delete NAP1/SINTBAD (RRID: CVCL_C9DW). An overview of the CRISPR–Cas9 knockout clones generated in this study can be found in Supplementary Table 1.

Generation of stable cell lines

Stable cell lines were generated using lentiviral or retroviral expression systems. For retroviral transductions, HEK293T cells (RRID: CVCL_0063) were transfected with VSV-G (a kind gift from Richard Youle), Gag-Pol (a kind gift from Richard Youle) and pBMN constructs containing our gene of interest using Lipofectamine 3000 or Lipofectamine LTX (L3000008 or A12621, Thermo Fisher). The next day, the medium was exchanged with fresh media. Viruses were collected 48 h and 72 h after transfection. The retrovirus-containing supernatant was collected and filtered to avoid crossover of HEK293T cells into our HeLa cultures. HeLa cells, seeded at a density of 800,000 per well, were infected by the retrovirus-containing supernatant in the presence of 8 mg ml−1 polybrene (Sigma-Aldrich) for 24 h. The infected HeLa cells were expanded, and 10 days after infection, they were sorted by FACS to match equal expression levels where possible. A detailed protocol is available (https://doi.org/10.17504/protocols.io.81wgbyez1vpk/v1).

The following retroviral vectors were used in this study: pBMN-HA-NAP1 (RRID: Addgene_208868), pBMN-HA-NAP1 delta-TBK1 (L226Q/L233Q) (RRID: Addgene_208869), pBMN-HA-SINTBAD (RRID: Addgene_210209), pBMN-mEGFP-OPTN (RRID: Addgene_188784), pBMN-mEGFP-NDP52 (RRID: Addgene_188785), pBMN-BFP-Parkin (RRID: Addgene_186221) and pCHAC-mito-mKeima (RRID: Addgene_72342). Empty backbones used to generate these retroviral vectors were pBMN-HA-C1 (RRID: Addgene_188645), pBMN-mEGFP (RRID: Addgene_188643) and pBMN-BFP-C1 (RRID: Addgene_188644).

For lentiviral transductions, HEK293T cells (RRID: CVCL_0063) were transfected with VSV-G (a kind gift from Gijs Versteeg), Gag-Pol (a kind gift from Gijs Versteeg) and pHAGE constructs containing our gene-of-interest using Lipofectamine 3000 (L3000008, Thermo Fisher). The next day, the medium was exchanged with fresh media. Viruses were collected 48 h and 72 h after transfection. The lentivirus-containing supernatant was collected and filtered to avoid crossover of HEK293T cells into our HeLa cultures. HeLa cells, seeded at a density of 800,000 per well, were infected by the lentivirus-containing supernatant in the presence of 8 mg ml−1 polybrene (Sigma-Aldrich) for 24 h. The infected HeLa cells were expanded, and 10 days after infection, they were used for experiments. A detailed protocol is available (https://doi.org/10.17504/protocols.io.6qpvr3e5pvmk/v1).

The following lentiviral vectors were used in this study: pHAGE–FKBP–GFP–NDP52 (RRID: Addgene_135296), pHAGE–FKBP–GFP–NAP1 (RRID: Addgene_208862), pHAGE–FKBP–GFP–NAP1 delta-NDP52 (S37K/A44E) (RRID: Addgene_208863), pHAGE–FKBP–GFP–NAP1 delta-FIP200 (I11S/L12S) (RRID: Addgene_208864), pHAGE–FKBP–GFP–NAP1 delta-TBK1 (L226Q/L233Q) (RRID: Addgene_208865), pHAGE–FKBP–GFP–SINTBAD (RRID: Addgene_216840), pHAGE–FKBP–GFP–OPTN (RRID: Addgene_208866), pHAGE–FKBP–GFP–OPTN(2–119) (RRID: Addgene_208867) and pHAGE–mt-mKeima–P2A–FRB–Fis1 (RRID: Addgene_135295).

Mitophagy experiments

To induce mitophagy, cells were treated with 10 µM oligomycin (A5588, ApexBio) and 4 µM antimycin A (A8674, Sigma-Aldrich). In case cells were treated for more than 8 h, we also added 10 µM Q-VD-OPh (A1901, ApexBio) to suppress apoptosis. Samples were then analyzed by SDS–PAGE and western blot or flow cytometry. A detailed protocol is available (https://doi.org/10.17504/protocols.io.n2bvj3yjnlk5/v1).

Nutrient starvation experiments

To induce bulk autophagy, cells were starved by culturing them in Hank’s balanced salt medium (Thermo Fisher). Cells were collected and analyzed by SDS–PAGE and western blot analysis. A detailed protocol is available (https://doi.org/10.17504/protocols.io.4r3l228b3l1y/v1).

Rapalog-induced chemical dimerization experiments

The chemically induced dimerization experiments were performed using the FRB–Fis1 and FKBP fused to our gene-of-interest system. After consecutive lentiviral transduction of HeLa cells with both constructs, in which the FRB–Fis1 also expresses mt-mKeima, cells were treated with the Rapalog A/C hetero-dimerizer rapalog (635057, Takara) for 24 h. Cells were then analyzed by flow cytometry. A detailed protocol is available (https://doi.org/10.17504/protocols.io.n92ldmyynl5b/v1).

Flow cytometry

For mitochondrial flux experiments, 700,000 cells were seeded in six-well plates 1 day before the experiment. Mitophagy was induced by treating the cells for the indicated times with a cocktail of O/A, as described above. Cells were collected by removing the medium, washing with PBS (14190169, Thermo Fisher), trypsinization (T3924, Sigma-Aldrich) and then resuspending in complete DMEM medium (41966052, Thermo Fisher). The cells were then filtered through 35 µm cell-strainer caps (352235, Falcon) and analyzed by an LSR Fortessa Cell Analyzer (BD Biosciences). Lysosomal mt-mKeima was measured using dual excitation ratiometric pH measurements at 405 nm (pH 7) and 561 nm (pH 4) lasers with 710/50 nm and 610/20 nm detection filters, respectively. Additional channels used for fluorescence compensation were BFP and GFP. Single fluorescence vector-expressing cells were prepared to adjust photomultiplier tube voltages to ensure that the signal was within detection limits and to calculate the compensation matrix in BD FACSDiva Software (RRID: SCR_001456; http://www.bdbiosciences.com/instruments/software/facsdiva/index.jsp). Depending on the experiment, we gated for BFP-positive, GFP-positive and mKeima-positive cells with the appropriate compensation. For each sample, 10,000 mKeima-positive events were collected, and data were analyzed in FlowJo (v.10.9.0) (RRID: SCR_008520; https://www.flowjo.com/solutions/flowjo). Our protocol was based on a previously described protocol (https://doi.org/10.17504/protocols.io.q26g74e1qgwz/v1).

For rapalog-induced mitophagy experiments, cells were seeded as described above and treated for 24 h with 500 nM rapalog A/C hetero-dimerizer (Takara). Cells were collected as described above, and the mt-mKeima ratio (561/405) was quantified by an LSR Fortessa Cell Analyzer (BD Biosciences). The cells were gated for GFP/mt-mKeima double-positive cells. Data were analyzed using FlowJo (v.10.9.0). A detailed protocol is available (https://doi.org/10.17504/protocols.io.n92ldmyynl5b/v1).

Cellular fractionation and mitochondrial isolation

Mitochondria were isolated as described previously80. In brief, cells were collected from 15 cm dishes by trypsinization, after treatment with DMSO or O/A where indicated, and centrifuged at 300×g for 5 min at 4 °C. The cell pellet was washed with PBS, and a fraction of the PBS-washed cell pellet was transferred to a new tube and lysed in RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% NP-40) supplemented by cOmplete EDTA-free protease inhibitors (11836170001, Roche) and phosphatase inhibitors (Phospho-STOP; 4906837001, Roche). This sample served as a whole cell lysate reference. The remaining PBS-washed cells were processed further for mitochondrial isolation. The PBS-washed cell pellet was resuspended in 1 ml mitochondrial isolation buffer (250 mM mannitol, 0.5 mM EGTA, and 5 mM HEPES-KOH pH 7.4) and lysed by 15 strokes with a 26.5 gauge needle (303800, Becton Dickinson). The homogenate was centrifuged twice at 600×g for 10 min at 4 °C to pellet cell debris, nuclei and intact cells. The supernatant was collected and centrifuged twice at 7,000×g for 10 min at 4 °C to pellet mitochondria. The supernatant was removed, and the mitochondrial pellet was resuspended in 1 ml of mitochondrial isolation buffer. The resuspended mitochondrial pellets were centrifuged two more times at 10,000×g for 10 min at 4 °C. After removal of the supernatant, the pellets were resuspended in the mitochondrial isolation buffer. The final mitochondrial pellet was lysed in RIPA buffer and processed further for western blot analysis. A detailed protocol is available (https://doi.org/10.17504/protocols.io.kqdg3x4zzg25/v1).

SDS–PAGE and western blot analysis

For SDS–PAGE and western blot analysis, we collected cells by trypsinization and centrifugation. Cell pellets were washed with PBS and lysed in RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% NP-40) supplemented by cOmplete EDTA-free protease inhibitors (11836170001, Roche) and phosphatase inhibitors (Phospho-STOP; 4906837001, Roche). After incubating for 20 min on ice, samples were cleared by centrifugation and soluble fractions were collected. Protein concentrations were adjusted for equal loading and loaded on 4–12% SDS–PAGE gels (Thermo Fisher) with PageRuler Prestained protein marker (Thermo Fisher). Proteins were transferred onto nitrocellulose membranes (RPN132D, GE Healthcare) for 1 h at 4 °C using the Mini Trans-Blot Cell (Bio-Rad). After the transfer, membranes were blocked with 5% milk powder dissolved in PBS-Tween (0.1% Tween 20) for 1 h at 20–25 °C. The membranes were incubated overnight at 4 °C with primary antibodies dissolved in the blocking buffer, washed and incubated with species-matched secondary horseradish-peroxidase-coupled antibodies diluted 1:10,000 for 1 h at room temperature. Membranes were processed for western blot detection with SuperSignal West Femto Maximum Sensitivity Substrate (34096, Thermo Fisher) and imaged with a ChemiDoc MP Imaging system (Bio-Rad). Images were analyzed with ImageJ81 (RRID: SCR_003070; https://imagej.net/). A detailed protocol is available (https://doi.org/10.17504/protocols.io.eq2lyj33plx9/v1). Details on primary and secondary antibodies can be found in the Reporting Summary.

Immunofluorescence and confocal microscopy

Cells were seeded on HistoGrip (Thermo Fisher) coated glass coverslips in 24-well plates. Cells were allowed to adhere overnight and treated as indicated before fixation. Cells were fixed in 4% (w/v) paraformaldehyde and diluted in 100 mM phosphate buffer for 10 min at room temperature. The paraformaldehyde was removed and samples were washed three times with 1× PBS. Cells were permeabilized with 0.1% (v/v) Triton X-100 and diluted in 1× PBS for 10 min. After permeabilization, samples were blocked for 15 min with 3% (v/v) goat serum diluted in 1× PBS with 0.1% Triton X-100. Primary antibodies, diluted in blocking buffer, were incubated with the samples for 90 min. Unbound antibodies were removed in three washing steps with 1× PBS. Secondary antibodies, diluted in blocking buffer, were incubated with the samples for 60 min. Unbound secondary antibodies were removed by three washes with 1× PBS before coverslips were mounted onto glass slides with DABCO-glycerol mounting medium. Coverslips were imaged with an inverted Leica SP8 confocal laser scanning microscope equipped with an HC Plan Apochromat CS2 ×63/1.40 oil immersion objective (Leica Microsystems). Images were acquired in three dimensions using z-stacks, with a minimum range of 1.8 µM and a maximum voxel size of 90 nm laterally (x, y) and 300 nm axially (z), using a Leica HyD Hybrid detector (Leica Microsystems) and the Leica Application Suite X (LASX v.2.0.1) (RRID: SCR_013673; https://www.leica-microsystems.com/products/microscope-software/details/product/leica-las-x-ls). The z-stack images are displayed as maximum-intensity projections. Three images were taken for each sample. A detailed protocol is available (https://doi.org/10.17504/protocols.io.5qpvobz99l4o/v1). Details on primary and secondary antibodies can be found in the Reporting Summary.

Protein expression in bacteria

After the transformation of the pGEX-4T1 or pETDuet vectors encoding our proteins of interest in E. coli Rosetta pLysS cells (70956-4, Novagen), cells were grown in LB medium at 37 °C until an optical density at 600 nm of 0.4 and then continued at 18 °C; once the cells reached an optical density of 0.8, protein expression was induced with 100 µM isopropyl β-d-1-thiogalactopyranoside for 16 h at 18 °C. Cells were collected by centrifugation, resuspended in lysis buffer and stored at −80 °C until purification.

Baculovirus generation and protein expression in insect cells

To purify recombinant proteins from insect cells, we purchased gene-synthesized codon-optimized sequences from Genscript in pFastBac-Dual vectors. Constructs were used to generate bacmid DNA, using the Bac-to-Bac system, by amplification in DH10BacY cells82. After the bacmid DNA was verified by PCR for insertion of the transgene, we purified bacmid DNA for transfection into Sf9 insect cells (12659017, Thermo Fisher; RRID: CVCL_0549). To this end, we mixed 2,500 ng of plasmid DNA with FuGene transfection reagent (Promega) and transfected 1 million Sf9 cells seeded in a six-well plate. About 7 days after transfection, the V0 virus was collected and used to infect 40 ml of 1 million cells per ml of Sf9 cells. The viability of the cultures was closely monitored, and upon the decrease in viability and confirmation of yellow fluorescence, we collected the supernatant after centrifugation and stored this as V1 virus. For expressions, we infected 1 l of Sf9 cells, at 1 million cells per ml, with 1 ml of V1 virus. When the viability of the cells decreased to 90–95%, cells were collected by centrifugation, washed with 1× PBS and flash-frozen in liquid nitrogen. Pellets were stored at −80 °C until purification.

Protein expression in HEK293 cells

Proteins were expressed in FreeStyle HEK293-F cells (RRID: CVCL_6642), grown at 37 °C in FreeStyle 293 Expression Medium (12338-026, Thermo). The day before transfection, cells were seeded at a density of 0.7 × 106 cells per ml. On the day of transfection, a 400 ml culture was transfected with 400 µg of the MAXI-prep DNA, diluted in 13 ml of Opti-MEMR I Reduced Serum Medium (31985-062, Thermo) and 800 µg Polyethylenimine (PEI 25K; 23966-1, Polysciences), also diluted in 13 ml of Opti-MEM media. At 1 day post transfection, the culture was supplemented with 100 ml EXCELL R 293 Serum-Free Medium (14571C-1000 ml, Sigma-Aldrich). Another 24 h later, cells were collected by centrifugation at 270×g for 20 min. The pellet was washed with PBS to remove medium and then flash-frozen in liquid nitrogen. Pellets were stored at −80 °C until purification.

Protein purification

Linear tetra-ubiquitin fused to GST (GST–4×Ub) was cloned into a pGEX-4T1 vector (RRID: Addgene_199779), expressed in E. coli as described above and resuspended in lysis buffer (50 mM Tris-HCl pH 7.4, 300 mM NaCl, 2 mM MgCl2, 1 mM dithiothreitol (DTT), cOmplete EDTA-free protease inhibitors (Roche) and DNase (Sigma-Aldrich)). Cell lysates were sonicated twice for 30 s. Lysates were cleared by centrifugation at 45,000×g for 45 min at 4 °C in a SORVAL RC6+ centrifuge with an F21S-8x50Y rotor (Thermo Scientific). The supernatant was collected and incubated with pre-equilibrated Glutathione Sepharose 4B beads (GE Healthcare) for 2 h at 4 °C with gentle shaking to bind GST–4×Ub. Samples were centrifuged to pellet the beads and remove the unbound lysate. Beads were then washed twice with wash buffer (50 mM Tris-HCl pH 7.4, 300 mM NaCl, 1 mM DTT), once with high-salt wash buffer (50 mM Tris-HCl pH 7.4, 700 mM NaCl, 1 mM DTT) and two more times with wash buffer (50 mM Tris-HCl pH 7.4, 300 mM NaCl, 1 mM DTT). Beads were incubated overnight with 4 ml of 50 mM reduced glutathione dissolved in wash buffer (50 mM Tris-HCl pH 7.4, 300 mM NaCl, 1 mM DTT) at 4 °C to elute GST–4×Ub from the beads. To collect the supernatant, the beads were collected by centrifugation. The beads were washed twice with 4 ml of wash buffer and the supernatant was collected. The supernatant fractions were pooled, filtered through a 0.45 µm syringe filter, concentrated with a 10 kDa cut-off Amicon filter (Merck Millipore) and loaded onto a pre-equilibrated Superdex 200 Increase 10/300 GL column (Cytiva). Proteins were eluted with SEC buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM DTT). Fractions were analyzed by SDS–PAGE and Coomassie staining. Fractions containing purified GST–4×Ub were pooled. After concentrating the purified protein, the protein was aliquoted and snap-frozen in liquid nitrogen. Proteins were stored at −80 °C. A detailed protocol is available (https://doi.org/10.17504/protocols.io.q26g7pbo1gwz/v1).

For GST–LC3B, as previously described83, we inserted human LC3B cDNA in a pGEX-4T1 vector (RRID: Addgene_216836). The last five amino acids of LC3B were deleted to mimic the cleavage by ATG4. Proteins were expressed in E. coli as described above and purified using a similar GST-batch protocol as described for linear GST–4×Ub. A detailed protocol is available (https://doi.org/10.17504/protocols.io.3byl4qnbjvo5/v1).

For OPTN-GST, we cloned human OPTN cDNA in a pETDuet-1 vector with a carboxy-terminal GST-tag (RRID: Addgene_216843). Proteins were expressed in E. coli as described above and purified using a similar GST-batch protocol as described for linear GST–4×Ub. A detailed protocol is available (https://doi.org/10.17504/protocols.io.dm6gp3nb8vzp/v1).

For mCherry–OPTN, we cloned human OPTN cDNA in a pETDuet-1 vector with an amino-terminal 6×His tag followed by a tobacco etch virus (TEV) cleavage site (RRID: Addgene_190191). Proteins were expressed in E. coli as described above and cells were resuspended in lysis buffer (50 mM Tris-HCl pH 7.4, 300 mM NaCl, 2 mM MgCl2, 5% glycerol, 10 mM Imidazole, 2 mM β-mercaptoethanol, cOmplete EDTA-free protease inhibitors (Roche), CIP protease inhibitor (Sigma-Aldrich) and DNase (Sigma-Aldrich)). Cell lysates were sonicated twice for 30 s. Lysates were cleared by centrifugation at 45,000×g for 45 min at 4 °C in a SORVAL RC6+ centrifuge with an F21S-8x50Y rotor (Thermo Scientific). The supernatant was filtered through a 0.45 µm filter and loaded onto a pre-equilibrated 5 ml His-Trap HP column (Cytiva). After His-tagged proteins were bound to the column, the column was washed with three column volumes of wash buffer (50 mM Tris-HCl pH 7.4, 300 mM NaCl, 10 mM imidazole, 2 mM β-mercaptoethanol). Proteins were then eluted with a stepwise imidazole gradient (30, 75, 100, 150, 225 and 300 mM). Fractions at 75–100 mM imidazole contained the 6×His-TEV–mCherry–OPTN and were pooled. The pooled samples were incubated overnight with TEV protease at 4 °C. After the 6×His tag was cleaved off, the protein was concentrated using a 50 kDa cut-off Amicon filter (Merck Millipore) and loaded onto a pre-equilibrated Superdex 200 Increase 10/300 GL column (Cytiva). Proteins were eluted with SEC buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM DTT). Fractions were analyzed by SDS–PAGE and Coomassie staining. Fractions containing purified mCherry–OPTN were pooled. After concentrating the purified protein, the protein was aliquoted and snap-frozen in liquid nitrogen. Proteins were stored at −80 °C. A detailed protocol is available (https://doi.org/10.17504/protocols.io.4r3l225djl1y/v1).

For mCherry–NDP52, we cloned human NDP52 cDNA in a pETDuet-1 vector with an N-terminal 6×His tag followed by a TEV cleavage site (RRID: Addgene_187829). Proteins were expressed in E. coli as described above and purified using a similar His-Trap protocol as described for mCherry–OPTN. A detailed protocol is available (https://doi.org/10.17504/protocols.io.5qpvobdr9l4o/v1).

Human NDP52 cDNA was cloned into a pGST2 vector with an N-terminal GST tag followed by a TEV cleavage site (RRID: Addgene_187828). Proteins were expressed in E. coli as described above and purified using a similar GST-batch protocol as for linear GST–4×Ub. A detailed protocol is available (https://doi.org/10.17504/protocols.io.36wgq35xklk5/v1).

To purify NAP1 or GST–NAP1, human NAP1 cDNA was synthesized and cloned in a pcDNA3.1 vector (RRID: Addgene_216837), after which it was subcloned into a pGEX-4T1 vector with an N-terminal GST tag followed by a TEV cleavage site (RRID: Addgene_217610). After expression of unlabeled NAP1 in E. coli (which we used in Figs. 5b and 7e and Extended Data Fig. 5) or GST–NAP1 (which we used in Fig. 6e and Extended Data Fig. 4) as described above, proteins were purified using a similar GST-batch protocol as described for linear GST–4×Ub. A detailed protocol is available (https://doi.org/10.17504/protocols.io.kqdg3xk41g25/v1).

To purify NAP1–mCherry, as described previously40, human NAP1 cDNA was subcloned together with N-terminal GST–TEV and mCherry tags into a pCAGG by the Vienna BioCenter Core Facilities Protech Facility (RRID: Addgene_198036). Proteins were expressed in HEK293 cells as described above and purified using a similar GST-batch protocol as described for linear GST–4×Ub. A detailed protocol is available (https://doi.org/10.17504/protocols.io.5jyl8jw6dg2w/v1).

To purify MBP–NAP1, human NAP1 cDNA was gene-synthesized (by Genscript) and subcloned into a pGEX-4T1 vector with an N-terminal MBP-tag followed by a TEV cleavage site before wild-type NAP1 (RRID: Addgene_208871), NAP1 delta-NDP52 (S37K/A44E) (RRID: Addgene_208872) or NAP1 delta-TBK1 (L226Q/L233Q) (RRID: Addgene_208873). After expression of MBP–TEV–NAP1 in E. coli (which we used for Fig. 7d) as described above, proteins were purified using a similar protocol as described for linear GST–4×Ub, except using Amylose Beads (Biolabs) instead of Glutathione Sepharose 4B Beads (GE Healthcare). A detailed protocol is available (https://doi.org/10.17504/protocols.io.ewov1q2ykgr2/v1).

To purify SINTBAD–GFP and SINTBAD–mCherry from insect cells, we purchased gene-synthesized codon-optimized GST–TEV–SINTBAD–EGFP and GST–TEV–SINTBAD–mCherry in a pFastBac-Dual vector from Genscript (RRID: Addgene_198035 and RRID: Addgene_208874). Proteins were expressed in insect cells as described above and purified using a similar GST-batch protocol as for linear GST–4×Ub. A detailed protocol is available (https://doi.org/10.17504/protocols.io.rm7vzb1o8vx1/v1).

To purify TBK1, GFP–TBK1 or mCherry–TBK1, we purchased gene-synthesized codon-optimized GST–TEV–TBK1, GST–TEV–EGFP–TBK1 and GST–TEV–mCherry–TBK1 in a pFastBac-Dual vector from Genscript (RRID: Addgene_208875, RRID: Addgene_187830, RRID: Addgene_198033) for expression in insect cells. Proteins were expressed in insect cells as described above and purified using a similar GST-batch protocol as for linear GST–4×Ub. A detailed protocol is available (https://doi.org/10.17504/protocols.io.81wgb6wy1lpk/v1).

To purify FIP200–GFP from insect cells, we purchased gene-synthesized codon-optimized GST–3C-FIP200–EGFP in a pGB-02-03 vector from Genscript (RRID: Addgene_187832). Proteins were expressed in insect cells as described above and purified using a similar GST-batch protocol as for linear GST–4×Ub. A detailed protocol is available (https://doi.org/10.17504/protocols.io.dm6gpbkq5lzp/v1).

To purify the MBP–ULK1 from HEK293 GnTI cells (RRID: CVCL_A785), we expressed the ULK1 kinase from a pCAG backbone encoding MBP–TSF–TEV–ULK1 (RRID: Addgene_171416). The protein was expressed in HEK293 cells as described above, collected 48 h post transfection and the cell pellet was resuspended in 25 ml lysis buffer (50 mM Tris-HCl pH 7.4, 200 mM NaCl, 1 mM MgCl2, 10% glycerol, 0.5% CHAPS, 1 mM TCEP, 1 µl benzonase (Sigma-Aldrich), cOmplete EDTA-free protease inhibitors (Roche), CIP protease inhibitor (Sigma-Aldrich)). Cells were homogenized with a douncer. Cell lysates were cleared by centrifugation at 10,000×g for 45 min at 4 °C with a SORVAL RC6+ centrifuge with an F21S-8x50Y rotor (Thermo Scientific). The soluble supernatant was collected and loaded on a StrepTrap 5 ml HP column for binding of the Twin-Strep-tagged ULK1 protein, washed with six column volumes of wash buffer (50 mM Tris-HCl pH 7.4, 200 mM NaCl, 1 mM DTT) and eluted with elution buffer (50 mM Tris-HCl pH 7.4, 200 mM NaCl, 1 mM DTT and 2.5 mM desthiobiotin). Fractions were analyzed by SDS–PAGE and Coomassie staining. Fractions containing MBP–ULK1 were pooled and concentrated with a 50 kDa cut-off Amicon filter (Merck Millipore). The proteins were loaded onto a pre-equilibrated Superose 6 Increase 10/300 GL column (Cytiva). Proteins were eluted with SEC buffer (25 mM HEPES pH 7.5, 150 mM NaCl, 1 mM DTT). Fractions were analyzed by SDS–PAGE and Coomassie staining. Fractions containing purified MBP–ULK1 were pooled. After concentrating the purified protein, the protein was aliquoted and snap-frozen in liquid nitrogen. Proteins were stored at −80 °C. A detailed protocol is available (https://doi.org/10.17504/protocols.io.bvn2n5ge).

Microscopy-based bead assay

Glutathione Sepharose 4B beads (GE Healthcare) were used to bind GST-tagged bait proteins. To this end, 20 µl of beads were washed twice with distilled water (dH2O) and equilibrated with bead assay buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM DTT). Beads were then resuspended in 40 µl bead assay buffer, to which bait proteins were added at a final concentration of 5 µM. Beads were incubated with the bait proteins for 1 h at 4 °C at a horizontal tube roller. Beads were then washed three times to remove unbound GST-tagged bait proteins and resuspended in 30 µl bead assay buffer. Where indicated, we also added MgCl2 and ATP to the buffer to allow the phosphorylation of targets by TBK1. Glass-bottom 384-well microplates (Greiner Bio-One) were prepared with 20 µl samples containing prey proteins at the concentrations described below and diluted in bead assay buffer, and 3 µl of beads were added per well. For the experiments in Fig. 3d, NDP52 was used at a final concentration of 50 nM, FIP200–GFP, SINTBAD–mCherry and TBK1 were used at a final concentration of 100 nM. For Fig. 5b, mCherry–OPTN and GFP–TBK1 were used at a final concentration of 250 nM, and NAP1 was used from 100 nM to 10 µM. For Fig. 6e, GFP–TBK1 was used at a final concentration of 250 nM. For Fig. 7d, mCherry–OPTN, GFP–TBK1 and unlabeled NDP52 were used at a final concentration of 250 nM, while wild-type and mutant forms of NAP1 were used from 100 nM to 10 µM. For Fig. 7f, mCherry–OPTN, GFP–TBK1 and unlabeled NDP52 were used at a final concentration of 250 nM, while wild-type MBP–NAP1 was used at 10 µM. For Extended Data Fig. 3b, NDP52–mCherry, FIP200–GFP and mCherry–TBK1 were used at a final concentration of 250 nM. For Extended Data Fig. 9b, unlabeled OPTN, GFP–TBK1 and unlabeled NDP52 were used at a final concentration of 250 nM, while wild-type mCherry–SINTBAD was used from 100 nM to 10 µM. For Extended Data Fig. 10, GFP–TBK1 was used at a final concentration of 250 nM. The beads were incubated with the prey proteins for 30 min before imaging, with the exception of Fig. 3d and Extended Data Fig. 3, in which proteins were co-incubated for 4 h and 1 h, respectively before imaging. Samples were imaged with a Zeiss LSM 700 confocal microscope equipped with Plan Apochromat ×20/0.8 WD 0.55 mm objective and using the Zeiss ZEN Microscopy software (v.2022; RRID: SCR_013672). Three biological replicates were performed for each experimental condition.

For quantification, we used an artificial intelligence script that automatically quantifies signal intensities from microscopy images by drawing line profiles across beads and recording the difference between the minimum and maximum gray values along the lines. The artificial intelligence was trained to recognize beads using cellpose84. Processing is composed of two parts, with the first operating in batch mode. Multichannel input images are split into individual TIFF images and passed to cellpose (running in a Python environment) (RRID: SCR_008394; http://www.python.org). The labeled images produced by cellpose are re-assembled into multichannel images. Circular regions of interest (ROIs) are fitted to the segmented particles, and a pre-defined number of line profiles (here set to 20) are drawn automatically, starting at the center of the ROI and extending beyond the border of the circular ROI. This results in line profiles from the center of the bead into the inter-bead space of the well, allowing us to quantify the signal intensities at the rim of the beads. To prevent line profiles from protruding into adjacent beads, a combined ROI containing all beads was used. The results generated by artificial intelligence were inspected manually for undetected beads, incorrect line profiles or false-assigned bead structures. For each bead, a mean fluorescence and standard deviation are obtained based on the 20 line profiles per bead. Beads with standard deviations equal to or greater than half the mean value were either excluded or subjected to manual inspection for correction. To correct for inter-experiment variability in absolute values, the mean values for each bead were divided by the average bead intensity of the control condition. These values are then plotted and subjected to statistical significance calculations. A detailed protocol is available (https://doi.org/10.17504/protocols.io.14egn38pzl5d/v1).

GFP pull-down assay

GFP-tagged TBK1 was mixed with 20 µl of GFP-Trap agarose beads (Chromotek) at a final concentration of 1 µM. To this end, 20 µl of beads were washed twice with dH2O and equilibrated with bead assay buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM DTT). Beads were then resuspended in 40 µl bead assay buffer, to which GFP–TBK1 was added at a final concentration of 5 µM. Beads were incubated with GFP–TBK1 for 1 h at 4 °C at a horizontal tube roller. Beads were washed three times to remove unbound GFP-tagged bait protein. Protein master mixes with prey protein were prepared in bead assay buffer at the following concentrations: mCherry–OPTN (1 µM); mCherry–NDP52 (1 µM); GST–NAP1 (1–10 µM). The protein master mixes were added to the beads and incubated for 1 h at 4 °C at a horizontal tube roller. Beads were washed three times to remove unbound proteins, diluted in 60 µl of 1× protein loading dye and heat-inactivated at 95 °C for 5 min. Samples were analyzed by SDS–PAGE and Coomassie staining as described above. A detailed protocol is available (https://doi.org/10.17504/protocols.io.e6nvwd6x2lmk/v1).

In vitro kinase assay

Recombinant proteins TBK1 and NAP1 or MBP–ULK1 and NAP1 were mixed in kinase buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM DTT). The kinases were used at 50 nM and mixed with 250 nM NAP1. The kinase reactions were started by the addition of 2× ATP/MgCl2 kinase buffer to a final concentration of 10 mM MgCl2 and 100 mM ATP. Protein mixtures were prepared as master mixes and divided over the number of time points. To control for potential protein instability, we induced the latest time point first and then went gradually to the shortest time point. In this way, all protein mixtures were kept at room temperature for the same time, and reactions could be terminated together. Termination of reactions was achieved by the addition of 6× protein loading dye and heat inactivation at 95 °C for 5 min. Samples were separated on 4–12% SDS–PAGE gels (NP0321BOX, NP0322BOX or NP0323BOX, Thermo Fisher) with PageRuler Prestained protein marker (Thermo Fisher). After the run, the SDS–PAGE gel was either stained with Coomassie or transferred to nitrocellulose membranes for western blot analysis. In the case of Coomassie staining, the gel was incubated for 10 min in Coomassie solution, fixed for 10 min with fixation solution (40% ethanol, 10% acetic acid, 50% dH2O) and then destained overnight in dH2O. The band corresponding to NAP1 was cut from the gel with a fresh scalpel and submitted for mass spectrometry analysis. In the case of western blotting, the proteins were transferred onto nitrocellulose membranes (RPN132D, GE Healthcare) for 1 h at 4 °C using the Mini Trans-Blot Cell (Bio-Rad). The membranes were then processed further for western blot analysis, as described above. A detailed protocol for in vitro kinase assays is available (https://doi.org/10.17504/protocols.io.4r3l225xjl1y/v1).

Immunoprecipitation

FIP200 knockout HAP1 cells were transiently transfected with pcDNA3.1 NAP1–EGFP (RRID: Addgene_216837) or empty pcDNA3.1 vector as a negative control, using Lipofectamine 2000 (Thermo Fisher). This cell line was selected as FIP200 deletion results in TBK1 hyperactivation and thus increased NAP1 phosphorylation. After 48 h, cells were collected by trypsinization and the cell pellet was washed with PBS once before cells were lysed in lysis buffer (100 mM KCl, 2.5 mM MgCl2, 20 mM Tris-HCl pH 7.4, 0.5% NP-40). Samples were lysed for 20 min on ice before cell lysates were cleared by centrifugation at 20,000×g for 10 min at 4 °C. Protein concentrations of the cleared protein lysates were then determined with the Pierce Detergent Compatible Bradford Assay Kit (23246, Thermo Fisher). For both samples, negative control and NAP1–EGFP lysates, 12 mg of cell lysate was incubated overnight with 20 µl or GFP-Trap agarose beads (GTA-20, Chromotek). In the morning, samples were washed three times in lysis buffer before the beads were resuspended in protein loading dye, supplemented with 100 mM DTT and boiled for 5 min at 95 °C. Samples were loaded on 4–12% SDS–PAGE gels (NP0322BOX, Thermo Fisher) with PageRuler Prestained protein marker (Thermo Fisher). After the run, the SDS–PAGE gel was stained with Coomassie and destained overnight. The band corresponding to NAP1–EGFP was cut from the gel with a fresh scalpel and submitted for mass spectrometry analysis. A detailed protocol is available (https://doi.org/10.17504/protocols.io.5jyl8pmndg2w/v1).

Sample preparation for mass spectrometry analysis

Coomassie-stained gel bands were prepared and analyzed by mass spectrometry as previously described40. In brief, bands were cut out and destained with a mixture of acetonitrile and 50 mM ammonium bicarbonate. Disulfide bridges were reduced using DTT, and free SH-groups were subsequently alkylated by iodoacetamide. The digestion with trypsin (Trypsin Gold, Mass Spec Grade; Promega, V5280) was carried out overnight at 37 °C, while the digestion with chymotrypsin was carried out at 25 °C for 5 h. Then the digestion was stopped by adding 10% formic acid to an end concentration of approximately 5%. Peptides were extracted from the gel with 5% formic acid by repeated sonication.

Liquid chromatography–mass spectrometry analysis

Peptides were separated on an Ultimate 3000 RSLC nano-HPLC system using a pre-column for sample loading (Acclaim PepMap C18, 2 cm × 0.1 mm, 5 μm), and a C18 analytical column (Acclaim PepMap C18, 50 cm × 0.75 mm, 2 μm; all HPLC parts Thermo Fisher Scientific), applying a linear gradient from 2% to 35% solvent B (80% acetonitrile, 0.08 % formic acid; solvent A, 0.1 % formic acid) at a flow rate of 230 nl min−1 over 60 min. A Q Exactive HF-X Orbitrap mass spectrometer (Thermo Fisher) coupled to the HPLC via Proxeon nano-spray-source (all Thermo Fisher Scientific) equipped with coated emitter tips (New Objective) was used with the following settings. The mass spectrometer was operated in data-dependent acquisition mode. Survey scans were obtained in a mass range of 375–1500 m/z with lock mass on, at a resolution of 120,000 at 200 m/z and a normalized AGC target of 3E6. The 15 most intense ions were selected with an isolation width of 1.2 m/z, for a maximum of 100 ms at a normalized AGC target of 1E5 and then fragmented in the HCD cell at 28% normalized collision energy. Spectra were recorded at a resolution of 30,000. Peptides with a charge of +1 or >+7 were excluded from fragmentation, the peptide match feature was set to ‘preferred’ and the exclude isotope feature was enabled. Selected precursors were dynamically excluded from repeated sampling for 20 s. An Exploris 480 Orbitrap mass spectrometer (Thermo Fisher) coupled to the column with a FAIMS Pro ion source (Thermo-Fisher) using coated emitter tips (PepSep, MSWil) was used with the following settings. The mass spectrometer was operated in data-dependent acquisition mode with four FAIMS compensation voltages set to −35, −45, −60 or −70 and 0.8 s cycle time per compensation voltage. The survey scans were obtained in a mass range of 350–1500 m/z, at a resolution of 60,000 at 200 m/z and a normalized AGC target at 100%. The most intense ions were selected with an isolation width of 1.2 m/z, fragmented in the HCD cell at 28% collision energy and the spectra recorded for a maximum of 100 ms at a normalized AGC target of 100% and a resolution of 30,000. Peptides with a charge of +2 to +6 were included for fragmentation, the peptide match feature was set to preferred, the exclude isotope feature was enabled and selected precursors were dynamically excluded from repeated sampling for 20 s. In addition, four doubly charged phosphorylated peptides with m/z values of 909.8989, 956.4566, 797.3509 and 898.8982 were added to an inclusion list.

Mass spectrometry data analysis

HFx raw files were directly used, while Exploris raw files were first split according to compensation voltages (−35, −45, −60, −70) using FreeStyle v.1.7 software (Thermo Scientific; https://www.thermofisher.com/us/en/home/technical-resources/technical-reference-library/mass-spectrometry-support-center/liquid-chromatography-mass-spectrometry-software-support/freestyle-software-support/freestyle-software-support-troubleshooting.html#:~:text=FreeStyle%20is%20free%20of%20charge). They were searched using MaxQuant85 software v.1.6.17.0 (RRID: SCR_014485; http://www.biochem.mpg.de/5111795/maxquant) against the Uniprot human proteome database (release 2021_03) and a database of common laboratory contaminants. Enzyme specificity was set to ‘Trypsin/P’ with two miss cleavages or ‘chymotrypsin+’ with four miss cleavages and the minimal peptide length was set to seven. Carbamidomethylation of cysteine was searched as a fixed modification. ‘Acetyl (Protein N-term)’, ‘Oxidation (M)’, ‘Phospho (STY)’ were set as variable modifications. All data were filtered at 1% PSM+protein+site FDR; reverse hits were removed. The identification and quantification information of sites and proteins was obtained from the MaxQuant ‘Phospho (STY) Sites’ and ‘ProteinGroups’ tables.

Quantification and statistical analysis

For the quantification of immunoblots, we performed a densitometric analysis using Fiji software (RRID: SCR_002285; http://fiji.sc). Graphs were plotted using GraphPad Prism v.9.5.1 (RRID: SCR_002798). For the quantification of microscopy-based bead assays, we used an in-house developed artificial intelligence tool to automate the recognition and quantification of the signal intensity for each bead, which resulted in a mean bead intensity value. These values were plotted and subjected to statistical testing. Depending on the number of samples, and as specified in the figure legends, we used either a Student’s t-test or a one-way or two-way ANOVA test with appropriate multiple comparison tests. Statistical significance is indicated as *P < 0.05; **P < 0.005; ***P < 0.001; ****P < 0.0001; ns, not significant. Error bars are reported as means ± s.d. To ensure the reproducibility of experiments not quantified or subjected to statistical analysis, we showed one representative replicate in the paper of at least three replicates with similar outcomes for the main figures or at least two replicates for supplementary figures, as indicated in figure legends.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.