Yeast strains and growth conditions
The yeast strains used in this study are derived from S. cerevisiae BY4741 and YPH499 (Supplementary Table 4). The wild-type strain YPH499 as well as the mutant strains Cox4His, Tom20His, Tom40HA, Tom40HA ubx2∆, ubx2∆, pam17∆ and pre9∆pam17∆ were described previously17,42,43. The wild-type strain BY4741 as well as the mutant strains pth2∆, ubx2∆, ubp16∆, rsp5-1, mdm30∆, mfb1∆ and vms1∆ were obtained from EUROSCARF. Tagging and deletion of open reading frames were performed by homologous recombination using DNA cassettes amplified by PCR using Taq and Vent polymerase (NEB)44 or KOD hot-start DNA-Polymerase (Merck Millipore). The genetic information for a triple HA tag was chromosomally introduced before the STOP codon of the open reading frame of TOM40 in pth2∆ and tom70∆ cells using the cassette amplified from pFA6a 3HA-His3MX645. The open reading frame of DSK2 was deleted using the pFA6a KanMX4 cassette46. Deletion of PTH2 in ubp16∆ and ubx2∆, VMS1 in pth2∆ and UBP16 in rsp5-1 was performed using the pFA6A His3MX6 cassette46. Deletion of PAM17 in the pth2∆, dsk2∆, rsp5-1, ubp16∆, mfb1∆, mdm30∆, hrd1∆ and doa10∆ strains, and deletion of TOM70 in the BY4741 and Tom40HA strains was performed using the pFA6a hphNT1 cassette44 (the deletion cassettes used are shown in Supplementary Table 5). Yeast strains were cultured according to standard protocols at temperatures between 24 °C and 37 °C in complete YP-medium (1% (w/v) yeast extract, 2% (w/v) bacto-peptone) or selective minimal medium (SM) (0.67% (w/v) yeast nitrogen base with ammonium sulfate; 0.07% (w/v) amino acid mixture) containing 2% (w/v) glucose (YPD, SMD), 2% (w/v) sucrose (YPS, SMS), 2% (w/v) galactose (YPGal, SMGal) or 3% (w/v) glycerol as carbon source. The cell cultures were grown until the early logarithmic growth phase, on the basis of the optical density at a wavelength of 600 nm (OD600).
Construction of plasmids
Pth2 was cloned with its endogenous promoter (962 bp upstream of the start codon) into pRS41643. The Pth2 D174A and b2-DHFRGGxY L251G/P252G mutations were generated by site-directed mutagenesis. The putative transmembrane domain of Pth2 (amino acids 12–32) was deleted by PCR amplification of the entire plasmid without the region encoding amino acids 12–32 followed by in vitro recombination using HiFi assembly (NEB). A list of the plasmids used is provided in Supplementary Table 5. Plasmids were used for the expression of cytochrome b2 precursor variants containing DHFR. Folding of the DHFR domain prevents the complete translocation of the preproteins through the TOM channel and therefore arrests the N-terminal b2-part in the mitochondrial import site. Two types of b2-DHFR precursors were accumulated in the import site—b2∆-DHFR, which carries a matrix-targeting signal, and b2-DHFR, which contains both a matrix-targeting signal and an inner membrane sorting signal17,47.
Growth analysis of yeast strains
For comparing the growth of different yeast strains, exponentially growing cells were diluted to an OD600 of 1.0 and diluted 1:5 (five times). The dilutions were spotted onto YP or SM plates containing glucose or glycerol as the sole carbon source and incubated at the indicated temperatures. Pictures of plates were taken after 1 to 4 days, depending on growth temperature and carbon source.
Isolation of mitochondria
Purification of mitochondria was performed by differential centrifugation48. Yeast cells were collected at an early logarithmic growth phase (5,500g, 8 min, 24 °C). Cells were washed with distilled H2O and resuspended in DTT buffer (100 mM Tris/HCl pH 9.4, 10 mM dithiothreitol (DTT)) at a concentration of 2 ml per g wet weight of the cell pellet, followed by incubation for 30–45 min at growth temperature under constant shaking. Cells were then washed in zymolyase buffer (1.2 M sorbitol, 20 mM KPi pH 7,4) and resuspended in zymolyase buffer at a concentration of 7 ml per g of cells. Subsequently, cells were incubated with 4 mg zymolyase per g of cells under constant shaking for 30–45 min at growth temperature to digest the cell wall. Next, cells were pelleted (2,500g, 5 min, 24 °C) and washed once with zymolyase buffer. The obtained spheroplasts were resuspended in ice-cold homogenization buffer (0.6 M sorbitol, 10 mM Tris/HCl pH 7.4, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM phenylmethylsulfonylfluoride (PMSF), 0.2% (w/v) bovine serum albumin) using 6.5 ml of buffer per g of cells. Cells were homogenized using a glass potter with 15 strokes up and down. Subsequently, cell debris and large organelles like the nucleus were removed (2,500g, 5 min, 4 °C). The supernatant was centrifuged to isolate the mitochondria (17,000g, 15 min, 4 °C). The mitochondrial pellet was resuspended in SEM buffer (250 mM sucrose, 10 mM MOPS/KOH pH 7.2, 1 mM EDTA) and washed again in SEM buffer. The isolated mitochondria were resuspended in SEM buffer. The protein concentration was determined using the Bradford assay and mitochondria were aliquoted at a protein concentration of 10 mg ml−1. Mitochondria were frozen in liquid nitrogen and stored at −80 °C.
Cryo-slicing blue native gel electrophoresis
For high-resolution complexome profiling, a blue native gradient gel (2–13% (w/v) acrylamide, 0.06–0.40% (w/v) bis-acrylamide, 67 mM ε-amino n-caproic acid, 50 mM Bis-Tris/HCl, pH 7.0) was used. Mitochondria corresponding to 1 mg protein amount were pelleted and solubilized in 0.8 ml lysis buffer (20 mM Tris/HCl pH 7.4, 0.1 mM EDTA, 50 mM NaCl, 10% (v/v) glycerol) containing 1% (w/v) digitonin for 30 min on ice (we used optimized conditions for a mild and efficient lysis of the yeast mitochondrial preparation by applying 8 mg purified digitonin to 1 mg mitochondrial protein; the protein to digitonin ratio of 1:8 enables efficient extraction of yeast mitochondrial membrane protein complexes, yet is milder than the 1:10 protein:digitonin ratio that is often used for yeast mitochondria49 or the application of other detergents8; this is illustrated by the predominant presence of the physiological, fully assembled dimer of the F1FO-ATP synthase in comparison to the monomer (Fig. 1b and Extended Data Fig. 8d), whereas the typical 1:10 ratio conditions lead to an about equal distribution between dimer and monomer on blue native gels49). Subsequently, the sample was loaded onto a sucrose gradient consisting of 50% (w/v) sucrose and 20% (w/v) sucrose. After centrifugation, the upper phase was removed and the remaining supernatant was mixed with loading dye (0.5% (w/v) Coomassie G-250, 50 mM ε-amino n-caproic acid, 10 mM Bis-Tris/HCl, pH 7.0). The sample was applied to a loading zone of 5 cm width. Electrophoresis was performed at 15 mA in the presence of BN cathode buffer (0.02% (w/v) Coomassie G-250, 50 mM Tricine, 15 mM Bis-Tris/HCl, pH 7.0) and anode buffer (50 mM Bis-Tris/HCl, pH 7.0). After 1 h, the BN cathode buffer was replaced by a cathode buffer lacking Coomassie G-250 and the electrophoresis was continued for 2.5 h at 15 mA. A 2.5 cm lane was then excised, fixed in 30% ethanol/15% acetic acid, embedded in tissue embedding medium (Leica) and subjected to cryo-slicing50. Using a step size of 0.3 mm along the gel lane, 245 slices were obtained, extensively washed and separately digested with trypsin50.
The trypsin-digested peptides were dissolved in 20 µl sample buffer (0.5% (v/v) trifluoroacetic acid in H2O) and 1 µl aliquots (or less) were taken for LC–MS/MS analysis. Loading onto the precolumn (PepMap 100, C18 stationary phase) was achieved through an autosampler of a split-free UltiMate 3000 RSLCnano HPLC (Dionex/Thermo Fisher Scientific). Subsequent elution and separation on the SilicaTip column emitter (inner diameter, 75 µm; tip, 8 µm; New Objective,; packed 23 cm with ReproSil-Pur 120 ODS-3 (C18 stationary phase; Dr. Maisch HPLC)) occurred during a three-step linear gradient generated from eluent A (0.5% (v/v) acetic acid) and eluent B (0.5% (v/v) acetic acid in 80% (v/v) acetonitrile): after 5 min equilibration in 3% B, 90 min from 3% B to 30% B; 20 min from 30% B to 50% B; and 10 min from 50% B to 99% B. Subsequent column washing/regeneration comprised 5 min 99% B; 5 min from 99% B to 3% B; 10 min 3% B. The flow rate was set to 300 nl min−1. Electrospray parameters were positive ion mode, spray voltage 2.3 kV, transfer capillary temperature 300 °C. Data were acquired on the QExactive HF-X mass spectrometer (Thermo Fisher Scientific) with the following settings: maximum MS/MS injection time = 200 ms; dynamic exclusion time = 45 s; minimum signal intensity threshold = 40,000 (counts), fragmentation = 15 top precursors; mass isolation width = 1.0 m/z.
Primary MS data were processed using msconvert (https://proteowizard.sourceforge.io; v.3.0.11098; settings: Mascot generic format, filter options ‘peakPicking true 1’, ‘threshold count 500 most-intense’). Obtained peak lists were then m/z-calibrated using the MaxQuant raw file processor (v.1.6.17, https://www.maxquant.org)51,52 and used as input for a Mascot (v.2.7, Matrix Science) database search against the UniProtKB/SwissProt yeast database (SwissProt_YEAST_20201007) with general contaminations added (GPM cRAP database; cRAP_20190304). Match parameters were as follows: precursor mass tolerance = ±2.5 ppm, variable modifications = acetyl (protein N-term), carbamidomethyl (C), formyl (N-term, S, T), Gln->pyro-Glu (N-term Q), Glu->pyro-Glu (N-term E) and oxidation (M), fragment mass tolerance = ± 20 mmu, missed tryptic cleavage(s) = 1. Export filter settings were as follows: peptide-spectrum-match (PSM) FDR = 3%, minimum ion score = 0.5, grouping of related protein hits used the name of the predominant member. Exogenous contaminants (for example, keratins, trypsin, IgG chains) or protein identifications based on only one specific peptide in less than three slice samples were not considered further.
For label-free quantification of proteins, LC–MS data were processed as previously described9,50,53,54 with some crucial improvements. MaxQuant (v.1.6.17; https://www.maxquant.org)51,52 was used to determine and mass-calibrate peptide signal intensities (peak volumes) from recorded FT full scans. Systematic variations in peptide elution times were corrected by LOESS regression after pairwise alignment of the datasets (median peptide elution times over all of the aligned datasets were used as a reference). Matching of peak volumes and peptide identities (obtained either directly or indirectly from MS/MS database matches) was achieved using custom developed software53 (m/z and elution time matching tolerances were ±1.5–2 ppm and ±1 min, respectively). Global offsets in peptide intensity between runs were corrected by normalization to the local median of the relative peptide intensities (consistently assigned peptides within a window of 40 slices). For effectively eliminating the impact of missing, non-consistent or incorrectly assigned peptide intensity values, we applied an additional procedure consisting of four steps. First, the accuracy of all assigned peptide intensity values was determined by analysing matrices (peptides versus runs) of protein-specific peptide intensity values for their internal consistency. Within these matrices, the pairwise relationships of peptide intensity values between and within MS-runs (in all possible combinations) provided distributions of predicted intensities for connected matrix cells from which expected intensity values (EPVs) could be calculated (by kernel density estimation), which served as measure of consistency of the respective peak volume values and were later used as weighting factors. If insufficient data precluded determination of EPVs, values interpolated from EPV-validated intensities in neighbouring (window of 5) slices/datasets were used alternatively. Second, a time- and run-dependent detectability threshold was estimated for each of the matrix cells (peptides versus runs) using the third percentile of intensity values from peptides co-eluting within a 3 min time window. Third, for each protein, peptide intensity values from qualified runs (that is, consistent protein-specific peak volume values of respective columns in each protein matrix) were merged (EPV-weighted least squares fits to the dataset with the highest number of peptide intensities assigned to the respective protein) into a single intensity value vector, termed protein reference ridge. These vectors reflected the maximum protein coverage of MS/MS-identified and quantified peptides with their relative ionization efficiencies and were used to determine molecular abundances (abundancenorm spec values) as described previously53. Fourth, protein quantification was achieved by a weighted fitting of its measured peptide intensities in five consecutive slices (equivalent to a sliding average) to its reference ridge (Extended Data Fig. 2). Quantification details for each data point in a protein profile (Supplementary Table 2) are provided in the ‘peptide details’ feature of the expert viewer tool (Extended Data Fig. 3; https://www.complexomics.org/datasets/mitcom).
The oversampling of the blue native gel separation (0.3 mm step size) was used to provide robust protein quantification without compromising effective size resolution. Thus, each protein abundance value, that is, each data point in an abundance mass profile, integrated the results from five consecutive LC–MS/MS analyses. Moreover, the processing of LC–MS input data described above provided objective measures for reliability and accuracy of protein quantification: (1) the number of protein-specific peptide intensities used and (2) the deviation of these peptide intensities from the expected peptide intensities. This information was integrated into a ‘reliability score’ (ranging from 0 (not significant) to 1 (maximum reliability)) for each protein abundance value (Supplementary Table 2).
Evaluation, accession and visualization of data
A total of 1,891 proteins with a required minimal number of two protein-specific peptides were identified in the entire csBN–MS dataset. Of these, 906 proteins were considered to be bona fide mitochondrial proteins on the basis of respective annotations in either the UniProtKB/SwissProt database, the Yeast Genome Database (SGB) or ref. 6. Together, the 906 mitochondrial proteins represent the core of the complexome dataset termed the MitCOM (Supplementary Tables 1 and 2). Information on (1) molecular mass and membrane-spanning helices was inferred from the UniProtKB/SwissProt database, (2) localization in submitochondrial compartments was extracted from the respective SGD GO terms, and (3) functional classification was taken from ref. 6 supplemented by GO annotations and literature for the proteins not already classified therein (Fig. 2, Supplementary Table 1 and Extended Data Fig. 6). Among the proteins identified with one protein-specific peptide only, an additional 49 mitochondrial proteins were found to be potentially significant and were separately added to the list of MitCOM proteins in Supplementary Table 1. The remaining 985 proteins, all identified by at least two protein-specific peptides, were classified as non-mitochondrial proteins predominantly localized in the endoplasmic reticulum and cytosol (Fig. 1, Extended Data Fig. 7 and Supplementary Table 3). Information on their molecular mass and membrane integration, their preferred subcellular localization and their functional classification was obtained as described for the mitochondrial proteins above.
The principles of mass estimation of protein complexes using blue native electrophoresis were outlined in ref. 55. For converting slice numbers to apparent molecular masses of proteins/complexes we used a set of marker proteins/complexes with a defined migration pattern (that is, sharply focused profile peaks) and molecular masses broadly distributed over the sampled blue native gel range (name/subunits with references; predicted molecular mass, log[molecular mass], slice maximum): oxoglutarate dehydrogenase/ketoglutarate dehydrogenase complex (Kgd1/Odo1–Kgd2/Odo2–Lpd1/Dldh–Kgd456,57,58,59,60; 3,020 kDa, 3.48, 21.4); pre-60S ribosome large subunit (RL3, RLP24, NOG1; PDB: 3JCT (ref. 61); 2,680 kDa, 3.43, 11.5); dimer of F1FO-ATP synthase (complex V dimer8; 1,250 kDa, 3.10, 54.1); respiratory III2IV2 supercomplex (ref. 8; 1,000 kDa, 3.00, 62.4); respiratory III2IV1 supercomplex (ref. 8; 750 kDa, 2.88, 71.6); monomer of F1FO-ATP synthase (complex V monomer8; 600 kDa, 2.78, 85,6); SAM–Mdm10 complex (PDB: 7BTX (ref. 62); 185.5 kDa, 2.27, 143.8); TIM22 complex (PDB: 6LO8 (ref. 63); 174 kDa, 2.24, 136.2); ATM1 (ABC transporter mitochondrial; PDB: 4MYC (ref. 64); 155 kDa, 2.19, 157); SAMcore complex (Sam50, Sam37, Sam3562,65; 129.4 kDa, 2.11, 173.5); succinate dehydrogenase (complex II8; 130 kDa, 2.11, 182); NADP-cytochrome P450 reductase (Ncp1/NCPR66; 77 kDa, 1.88, 239). The resulting relationship of molecular mass over slice numbers was fitted with a sigmoidal function (IGOR Pro 9 WaveMetrics) and the resulting fit-line was used for calibration (Extended Data Fig. 2d).
Abundance–mass profiles of all MitCOM proteins (Supplementary Table 2) were analysed for their composition of individual components (peaks) using custom-developed software (complexomics-mitcom v.1.0; released as a Python package under the MIT license, available at Zenodo (https://doi.org/10.5281/zenodo.7355040)). First, locations of apparent peaks were determined by local maxima search with subsequent filtering (minimum relative height 0.1, minimum relative prominence 0.5, maximum width 50). Then, a multicomponent Gaussian model was initialized with the number and locations of the identified peaks. The model was iteratively adjusted and fitted to the profile. Up to 12 Gaussian components were added preferably at locations with large residuals, resulting in an improved fitting of highly overlapping peaks, manifesting as ‘shoulders’. Sensible limits and stop conditions were applied to avoid overfitting. A total of 818 MitCOM proteins was accessible to automated analysis. From a total number of 5,224 peaks (manually curated), 4,070 peaks were adequately fitted by our algorithm providing parameters for their apparent mass, half-width and molecular abundance (Fig. 1c). The total peak count (manually curated) was used for statistical analysis in Fig. 2 and Extended Data Fig. 6. For the non-mitochondrial proteins, the number of apparent peaks was counted manually (Extended Data Fig. 7).
Abundance–mass profiles of all MitCOM proteins were deposited in the openly accessible interactive resource platform Complexome Profiling Data Resource (CEDAR)67, where they can be accessed through an interactive online visualization tool (https://www3.cmbi.umcn.nl/cedar/browse/experiments/crx36). Protein profile normalization, filtering, baseline subtraction and magnification/scaling for convenient display and evaluation are integrated functions of this viewer (Extended Data Fig. 3). Moreover, using custom Pearson correlation analysis, it offers a basic method to search for proteins with similar abundance profile peaks or patterns. An extended version of the viewer with additional features enabling the inspection of the peptide intensity information underlying each datapoint of the protein profiles (Extended Data Fig. 3), an integrated help function explaining the use of the available features and a supplemental viewer containing abundance–mass profiles of the quantified non-mitochondrial proteins (that passed a quality check) are available online (https://www.complexomics.org/datasets/mitcom).
For a global view on the complexome organization of MitCOM (Extended Data Figs. 4 and 5), unsupervised protein profile matching was performed as follows (similar to the approach in ref. 11). Successfully fitted protein profile components (Fig. 1c) were filtered by width (max 20 s.d.) and location (peak full width at half maximum fully inside gel slice range) and clipped at 2.5 s.d. These defined the boundaries of 3,263 profile segments as seed regions of interest (ROIs). Comparison of each ROI to its corresponding segment in other profiles using Pearson correlation yielded 82,268 high-correlating segments (r ≥ 0.95) as additional ROIs. Finally, out of the total of 85,531 ROIs, the ones that originated from the same profile and had similar boundaries (within 3 slices) were merged. The resulting 49,112 ROIs were used to assess similarity of protein components. To this end, a distance metric was designed that incorporated the following ROI-specific values: (1) slice index of left boundary; (2) slice index of right boundary; (3) maximum abundance; (4) r value from correlation with seed ROI (average if ROIs were merged). All of the values were minimum/maximum-normalized, except for boundaries, which were square-root-transformed, giving the highest weight to component locations and quickly penalizing differences. The distance of any two ROIs is determined by the Euclidean distance of their respective value vectors and the coefficient r of their mutual correlation. With the maximum theoretical distance being a dataset-specific fixed value, distances could easily be converted to a normalized similarity score ranging from 0 (most dissimilar) to 1 (identical location and abundance values). On the basis of the custom distance metric, pairwise distances of all protein ROIs were calculated and used for visualizing component similarity using a t-SNE plot (Extended Data Fig. 5).
Preparation of yeast cell extracts
Whole yeast cell lysates were obtained by post-alkaline extraction68. Exponentially growing yeast cells (OD600 of 2.5) were pelleted (3,000g, 5 min, 20 °C) and resuspended in distilled H2O. Subsequently, the samples were mixed with the same volume of 0.2 M NaOH and incubated for 5 min at 24 °C. Cells were pelleted (3,000g, 5 min, 4 °C), resuspended in sample buffer (8 M urea, 5% (w/v) SDS, 1 mM EDTA, 1.5% (w/v) DTT, 0.025% (w/v) bromophenol blue, 200 mM Tris/HCl pH 6.8) and denatured for 10 min at 65 °C shaking at 1,400 rpm.
Yeast extracts for large-scale affinity purification were generated by collecting cells in the early logarithmic growth phase (5,500g, 8 min, 24 °C). Cells were then washed with distilled H2O lysis buffer (0.1 M EDTA, 50 mM NaCl, 10% (v/v) glycerol, 20 mM Tris/HCl pH 7.4). Cells resuspended in lysis buffer were then frozen in liquid nitrogen and cell disruption was achieved by cryo-grinding at 25 Hz for 10 min in a Cryo Mill (Retsch). The obtained lysates were stored at −80 °C until further use.
To obtain small amounts of cell extracts for affinity purifications, cells from exponentially growing cells (OD600 of 100–200) were resuspended in lysis buffer with protease inhibitors (1 mM PMSF, 1× HALT protease inhibitor cocktail (Thermo Fisher Scientific)). Cells were then ruptured with silica beads 6 times 30 s with a 1 min break in between at 4 °C on a cell disruptor (Vortex Disruptor Genie). Cell extracts were cleared by centrifugation (2,000g, 5 min, 4 °C) and directly further processed for affinity purification.
Affinity purification of tagged proteins
For affinity purification of His-tagged proteins17,42,69, isolated mitochondria were solubilized in lysis buffer (20 mM Tris pH 7.4, 0.1 mM EDTA, 50 mM NaCl, 10% (v/v) glycerol, 2 mM PMSF, 1× protease inhibitor cocktail without EDTA) containing 1% (w/v) digitonin and 10 mM imidazole and incubated for 15 min at 4 °C. The solubilized sample was cleared by centrifugation (10 min, 17,000g, 4 °C) and incubated with Ni-NTA agarose beads (Qiagen) that were pre-equilibrated in lysis buffer with 0.1% (w/v) digitonin and 10 mM imidazole. After 1 h incubation at 4 °C under constant rotation, unbound proteins were removed and the affinity matrix was washed with excess amount of lysis buffer containing 0.1% (w/v) digitonin and 40 mM imidazole. Bound proteins were eluted with lysis buffer containing 0.1% (w/v) digitonin and 250 mM imidazole. After addition of sample buffer proteins were denatured for 10 min at 60 °C (Cox4–His) or 5 min at 96 °C.
For affinity purification of HA-tagged proteins17,69, isolated mitochondria or cell extracts were solubilized in lysis buffer containing 1% (w/v) digitonin. After solubilization for 15 min (purified mitochondria) or 30 min (cell extracts), the samples were cleared by centrifugation (17,000g, 10 min, 4 °C). The supernatant was incubated for 1 h at 4 °C under constant rotation with an anti-HA affinity matrix (Roche) that was pre-equilibrated with 0.5 M acetate followed by washing with lysis buffer containing 0.1% (w/v) digitonin. Unbound proteins were removed and the beads were washed with an excess amount of lysis buffer containing 0.1% (w/v) digitonin. Proteins were eluted by incubation with sample buffer at 95 °C.
For purification of Phb1–protein A, mitochondria were solubilized in lysis buffer containing 1% (w/v) digitonin for 1 h at 4 °C. The lysate was cleared by centrifugation (17,000g, 10 min, 4 °C) and incubated for 1.5 h at 4 °C with IgG Sepharose (Cytiva). The IgG Sepharose was washed 10 times with wash buffer (20 mM Tris-HCl pH 7.4, 60 mM NaCl, 10% (v/v) glycerol, 0.1 mM EDTA, 2 mM PMSF, 0.3% (w/v) digitonin). Phb1 and bound proteins were eluted by cleavage of the protein A tag using TEV protease (Thermo Fisher Scientific) in wash buffer at 24 °C for 2.5 h. Subsequently, the protease was removed through its His tag by incubating the eluates with equilibrated Ni-NTA for 30 min at 4 °C.
For tandem purification of the TOM complex70,71 through Tom22–His and Tom40–Strep, isolated mitochondria were solubilized in tandem buffer (100 mM Tris/HCl pH 8.0, 150 mM NaCl, 10% (v/v) glycerol, 1× protease inhibitor cocktail) supplemented with 5 mM imidazole and 3% (w/v) digitonin for 1 h rotating. The samples were cleared (17,000g, 10 min, 4 °C) and the subsequent purification steps were performed using the ÄKTA Explorer 100 system (Cytiva). In the first steps, Tom22–His was purified using the HisTrap HP column pre-equilibrated with tandem buffer containing 5 mM imidazole and 0.1% (w/v) digitonin. The column was washed with tandem buffer containing 5 mM imidazole and 0.1% (w/v) digitonin and bound proteins were eluted with tandem buffer containing 250 mM imidazole and 0.1% (w/v) digitonin. In the second purification step via Tom40–Strep, the elution sample was applied onto a Strep-Tactin HP column pre-equilibrated with tandem buffer containing 0.1% (w/v) digitonin. The column was washed with excess amount of tandem buffer containing 0.1% (w/v) digitonin and bound protein was eluted with tandem buffer containing 0.1% (w/v) digitonin and 10 mM biotin.
Purification of ubiquitin-modified proteins
Proteins conjugated to His-tagged ubiquitin were purified through Ni-NTA agarose under denaturing conditions41,72. Cells expressing His-tagged ubiquitin were grown to logarithmic growth phase. Cells corresponding to an OD600 of 200 were collected (2,500g, 4 min, 4 °C) and washed with distilled H2O. Cells were resuspended under denaturing conditions to inactivate proteases including deubiquitylating enzymes in 1 ml buffer A (6 M guanidinium hydrochloride, 100 mM NaH2PO4, 10 mM Tris-HCl, pH 8.0). Silica beads (diameter 0.5 mm) were added to the samples and cells were disrupted using a cell disruptor (Vortex Disruptor Genie). Cellular lysates were cleared (500g, 5 min, 4 °C) to remove residual beads. Subsequently, the samples were diluted 1:10 in the presence of 0.05% (v/v) Tween-20 and incubated for 1 h at 24 °C under constant rotation. Insoluble material was removed (3,500g; 10 min; 4 °C) and the remaining supernatants were incubated in the presence of 20 mM imidazole with Ni-NTA agarose beads overnight at 4 °C under constant rotation. After removal of unbound proteins, the beads were washed twice with buffer A containing 20 mM imidazole and 0.05% Tween-20, followed by five washing steps with buffer C (8 M urea, 100 mM NaH2PO4, 10 mM Tris-HCl, pH 6.3, 0.05% Tween-20, 20 mM imidazole). Bound proteins were eluted with HU sample buffer (8 M urea, 5% (w/v) SDS, 1 mM EDTA, 1.5% (w/v) DTT, 0.025% (w/v) bromophenol blue, 200 mM Tris-HCl pH 6.8) for 10 min at 65 °C.
Cells were fractionated by differential centrifugation17. Cells were grown to an early logarithmic growth phase. Cells corresponding to an OD600 of 100 were resuspended in DTT buffer (100 mM Tris pH 9.4, 10 mM DTT) and incubated at 30 °C (20 min, 900 rpm). Cells were then resuspended in zymolyase buffer (1.2 M sorbitol, 20 mM KPi pH 7.4), zymolyase was added at a final concentration of 100 mg ml−1 and cells were incubated at 30 °C (45 min, 900 rpm). Cells were washed with zymolyase buffer and resuspended in ice cold homogenization buffer (0.6 M sorbitol, 10 mM Tris/HCl pH 7.4, 1 mM EDTA, 1 mM PMSF, 0.2% (w/v) bovine serum albumin). Cells were homogenized using a glass potter with 20 strokes up and down. Subsequently, cell debris and large organelles such as the nucleus were removed (1,500g, 5 min, 4 °C). A fraction of the supernatant was collected as post-nuclear supernatant, the remaining supernatant was subjected to centrifugation to isolate mitochondria (17,000g, 15 min, 4 °C). The mitochondrial pellet was resuspended in SEM buffer (250 mM sucrose; 10 mM MOPS/KOH pH 7.2; 1 mM EDTA), mitochondria were purified twice by centrifugation through a sucrose cushion (500 mM sucrose; 10 mM MOPS/KOH pH 7.2; 1 mM EDTA) and mitochondria were resuspended in SEM buffer (P13 fraction). The supernatant of the first 17,000g centrifugation was ultracentrifuged (100,000g, 1 h, 4 °C) and the supernatant was collected as the S100 fraction.
Blue native gel electrophoresis for standard analysis
For blue native gel separation48, mitochondria were solubilized in lysis buffer (20 mM Tris/HCl pH 7.4, 0.1 mM EDTA, 50 mM NaCl, 10% (v/v) glycerol) containing 1% (w/v) digitonin. The samples were cleared by centrifugation (17,000g, 15 min, 4 °C) and loading dye was added to a final concentration of 0.5% (w/v) Coomassie Brilliant Blue G-250, 10 mM Bis-Tris pH 7.0 and 50 mM 6-aminocaproic acid before loading onto the blue native gel. The prime symbols at the molecular mass markers of the blue native gels in Fig. 3b and Extended Data Fig. 10b,h indicate the correlation between the migration of water-soluble markers and membrane-protein markers according to ref. 62.
Proteins separated on polyacrylamide gels were transferred by semi-dry blotting to a polyvinylidene fluoride membrane (Milipore) with blotting buffer (20% (v/v) methanol, 150 mM glycine, 0.02% (w/v) SDS, 20 mM Tris base) for 2 h at 250 mA. After blotting, membranes were blocked with 5% (w/v) skimmed milk powder in TBS-T (12.5 mM NaCl, 20 mM Tris/HCl, pH 7.4, 0.1% (v/v) Tween-20) or RotiBlock (Roth) for 1 h. The membranes were incubated with primary antibodies for 1–2 h at room temperature or overnight at 4 °C. The membranes were washed with an excess of TBS-T and incubated with secondary antibodies at room temperature for 1 h. Different types of secondary antibodies were used. For detection of immune signals using the Licor system, secondary antibodies against rabbit or mouse were coupled to fluorescent labels (IRDye 800CW, anti-mouse; IRDye 800CW, anti-rabbit; IRDye 680RD, anti-mouse). For detection with the image analyser or X-ray films, an anti-rabbit antibody coupled to horseradish peroxidase was used. Unbound antibodies were removed by washing with excess of TBS-T. The signal of horseradish peroxidase coupled secondary antibodies was detected after incubating the membrane with enhanced chemiluminescence solution73 using either an Amersham Imager 680 (Cytiva) or the LAS3000 image reader (FujiFilm). Fluorescent secondary antibodies were detected on Odyssey CLx Infrared Imaging System (Li-Cor) and analysed using Image Studio (v.5.2.5; Li-Cor). The specificity of the immunosignals was confirmed by their absence in cells or mitochondria from deletion strains. In case of essential genes, the size shift of the band in cells expressing tagged-proteins confirmed the specificity of the immunosignal. The separating white lanes indicate where irrelevant gel lanes were digitally removed. A list of the antibodies used is provided in Supplementary Table 6. Experiments were typically run on several gels, which were analysed in parallel by western blotting, including control western blots. Representative blots were selected and processed from the original files (Supplementary Fig. 1) using ImageJ (v.2.1.0), Adobe Photoshop 2021 and Adobe Illustrator 2021.
Reproducibility and image processing
Representative images are shown for growth and biochemical assays/western blotting, including analysis of yeast growth (wild-type and mutants), total cell extracts, affinity purification from cell extracts, subcellular fractionation, protein steady state levels, blue native electrophoresis and affinity purification from isolated mitochondria. The findings were confirmed by independent experiments for the following figures (minimum number of independent experiments in parentheses): Figs. 3b (2), 3d (3), 3e (2), 4b (2), 4c (2), 4d (2), 4e (2), 4f (2), 4g (2), 5a (2), 5b (3), 5c (2), 5d (3), 5e (2), 5f (3) and 5g (2) and Extended Data Figs. 8f (3), 8h (2), 9b (2), 9c (2), 9d (2), 9e (2), 9f (5), 9g (3), 9h (2), 10a (2), 10b (2), 10c (3), 10d (2), 10e (2), 10f (2), 10g (2) and 10h (2). Images of western blots and growth assays were processed using Adobe Photoshop 2021 and figures were assembled in Adobe Illustrator 2021.
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