Yeast strains and growth conditions

The Saccharomyces cerevisiae strains used in this study are derived from YPH499 (Mata, ade2-101, his3-Δ200, leu2-Δ1, ura3-52, trp1-Δ63, and lys2-801 51. Cells were grown at 24 °C on non-fermentable YPG medium (1% w/v yeast extract, 2% w/v bacto peptone, and 3% w/v glycerol (pH 5.0)). For SILAC-analysis arg4Δ cells17 were grown on minimal medium (0.67% w/v yeast nitrogen base without amino acids, 3% w/v glycerol, 0.77% w/v Complete Supplement Mixture minus lysine and arginine). The medium was supplemented with natural arginine and lysine (light) or [13C 6 /15N 4 ]arginine and [13C 6 /15N 2 ]lysine (heavy).

Isolation and purification of mitochondria

Yeast cells were grown in YPG or minimal medium to an optical density (OD 600 ) of 0.7–1.0 (for the preparation of highly purified mitochondria and OM vesicles). For in organello import experiments cells were harvested at an OD 600 of 0.7–1.5 Cells were pelleted and resuspended in 7 ml g−1 wet weight Zymolyase buffer (1.2 M sorbitol, 20 mM potassium phosphate-HCl (pH 7.4)) containing 3 mg g−1 wet weight Zymolyase-20T (Seikagaku Kogyo, Tokyo, Japan). After incubation for 30 min at 24 °C spheroplasts were homogenized with a glass-Teflon potter (20 strokes on ice) in 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 (fatty acid-free, Sigma)). Crude mitochondrial fractions were obtained from the pellet after centrifugation of the homogenate at 12,000×g for 15 min in the presence of 1x protease inhibitor cocktail (Roche)16, 52. Aliquots were stored in SEM buffer (250 mM sucrose, 1 mM EDTA, 10 mM MOPS-KOH (pH 7.2)) at −80 °C. Mitochondrial fractions were loaded onto a three-step sucrose gradient (1.5 ml 60%, 4 ml 32%, 1.5 ml 23%, and 1.5 ml 15% sucrose in EM buffer (1 mM EDTA, 10 mM MOPS-KOH (pH 7.2)) to obtain highly pure mitochondria. The samples were centrifuged for 1 h at 134,000×g and highly pure mitochondria were recovered from the 32–23% sucrose interface16, 52. Mitochondrial purity was assessed by western blotting against various cellular marker proteins.

To obtain the post-mitochondrial fractions S100 and P100 homogenized yeast cells were centrifuged at 20,000×g for 10 min at 4 °C. The supernatant was subjected to two further identical centrifugation steps followed by centrifugation at 100,000×g for 1 h resulting in the supernatant (S100, containing mainly cytosolic proteins) and the pellet (P100, containing largely microsomal fraction). The pellet was resuspended in EM buffer and fractions were analyzed via SDS–PAGE and immunoblotting.

Generation of submitochondrial fractions

For sonication, 50–500 μg mitochondria were suspended in 1 mL SEM buffer in the presence of 500 mM NaCl (or 50 mM for OM L /TOT H samples). Samples were sonicated for 3 × 30 s on ice and subsequently centrifuged at 4 °C for 1 h at 100,000×g. For carbonate extraction, 50–500 μg mitochondrial proteins were incubated in 400–1000 μL freshly prepared 100 mM sodium carbonate. After incubation on ice for 30 min with occasional vortexing, an ultracentrifugation step was performed at 100,000 g for 45 min at 4 °C. Supernatants of both treatments were precipitated with 10% w/v trichloroacetic acid. Proteins were subjected to LC-MS/MS analysis or solubilized in Lämmli buffer, separated by SDS–PAGE and analyzed by Western blotting.

For swelling, mitochondria were suspended in 400 μL EM buffer and incubated on ice for 30 min with occasional mixing followed by centrifugation for 15 min at 20,000 × g. Obtained supernatants were precipitated with 10% w/v trichloroacetic acid. Pellet and supernatant samples were analyzed by SDS–PAGE and immunodecoration.

Release of soluble mitochondrial proteins by Bax treatment was carried out as previously described17. Mitochondria were incubated for 1 h at 37 °C in buffer consisting of 250 mM sucrose, 150 mM KCl, and 10 mM MOPS-KOH (pH 7.2) in the presence or absence of 100 nM human Bax. Supernatant and pellet fractions were separated by centrifugation at 16,000×g for 15 min at 4 °C.

For the isolation of OM vesicles, highly pure mitochondria were swollen in hypoosmotic buffer (5 mM potassium phosphate (pH 7.4), 1 mM PMSF) at a concentration of 4 mg ml−1. After incubation on ice for 20 min sample was treated with a glass-Teflon potter (20 strokes). The homogenate was then subjected to two consecutive ultracentrifugation steps on discontinuous sucrose gradients as described31 to recover OM vesicles.

Protease activity assay

Cell-free translated Prd1 and Prd1E502Q (point mutation by site-directed mutagenesis) was generated using the RTS100 wheat germ system (5PRIME). Prd1 was incubated with 15 μM Cox4 presequence peptide (MLSLRQSIRFFKPATRT) or radiolabelled Cox4 precursor protein in import buffer without BSA supplemented with 1× Complete, EDTA-free protease inhibitor cocktail (Roche)40. Samples were separated on Nu-PAGE (Novex) and subjected to immunoblotting.

Sample preparation for mass spectrometry

In total four replicates (two biological replicates with each time one forward (e.g., SN heavy /PEL light ) and one reverse experiment (e.g., SN light /PEL heavy ) respecively; Fig. 1a) were used. Material was isolated from four independent yeast cultures, of which two were grown with supplementation of heavy and two with supplementation of light amino acids (see Yeast strains and growth conditions for details). Sample amounts were equalized based on Bradford protein determination. Highly purified mitochondria were generated and subjected to sonication or carbonate extraction. Next, heavy and light samples were pooled 1:1 to determine the following ratios for four independent replicates: (i) supernatant sonication (SN son ) vs. pellet sonication (PEL son ), (ii) supernatant carbonate (SN carb ) vs. pellet carbonate (PEL carb ). Cysteines were reduced with 10 mM DTT for 30 min at 56 °C, and free sulfhydryl groups were carbamidomethylated using 30 mM iodoacetamide for 30 min at room temperature in the dark. Proteins were precipitated with a 10-fold excess of ethanol for 1 h at −40 °C, followed by centrifugation at 14,000×g at 4 °C for 30 min. Obtained pellets were washed with acetone, followed by centrifugation as above for 15 min. Samples were resuspended in 2 M GuHCl, 50 mM NaH 2 PO 4 (pH 7.8), and diluted 10-fold with 50 mM NH 4 HCO 3 . Acetonitrile (ACN) and CaCl 2 were added to final concentrations of 5% and 1 mM, respectively. Trypsin (Promega, sequencing grade) was added at a ratio of 1:30 and incubated at 37 °C for 12 h. Peptide samples were desalted using SPEC C18 AR tips (Agilent, Waldbronn, Germany) according to the manufacturer’s instructions, and dried under vacuum. Samples were resuspended in 10 mM KH 2 PO 4 (pH 2.7) and fractionated using strong cation exchange chromatography (SCX17). Digests were controlled by monolithic column HPLC53.

Strong cation exchange chromatography

SCX was performed using a self-packed 150 mm × 550 μm PolySULFOETHYL A column (200 Å pore size, 5 μm particle size; PolyLC, Columbia, MD, USA) in combination with an Ultimate 3000 HPLC system (Thermo Fisher, Germering, Germany). Peptides were separated at a flow rate of 20 μL min−1 using a binary gradient (SCX buffer A: 5 mM KH 2 PO 4 (pH 2.7) 20% ACN (pH 2.7); SCX buffer B: 5 mM KH 2 PO 4 , 200 mM KCl, 20% ACN (pH 2.7)) ranging from 0 to 95% B in 50 min. Six fractions were collected in concatenation mode, as previously described17. Fractions were desalted using self-packed Oligo R3 (Thermo Scientific) microcolumns, dried under vacuum and resuspended in 0.1% trifluoroacetic acid (TFA).

Nano-LC-MS/MS analysis

Nano-LC-MS/MS was performed on an LTQ-Orbitrap Elite mass spectrometer coupled to an Ultimate 3000 RSLC (both Thermo Fisher Scientific). Briefly, peptides were preconcentrated on a C18 trapping column (Acclaim PepMap, 100 μm × 2 cm, 5 μm particle size, 100 Å pore size, Thermo Fisher Scientific) in 0.1% TFA and separated on a C18 main column (Acclaim PepMap, 75 μm × 50 cm, 2 μm particle size, 100 Å pore size, Thermo Fisher Scientific) using a binary gradient (solvent A: 0.1% formic acid (FA); solvent B: 0.1% FA, 84% ACN) ranging from 3 to 42% B in 185 min, at a flow rate of 250 nL min−1. MS survey scans were acquired in the Orbitrap from 300 to 2000 m/z at a resolution of 60,000 using the polysiloxane m/z 371.1012 as a lock mass. The 20 most intense signals above an intensity threshold of 104 and with charge states 2–5 were subjected to collision induced dissociation in the ion trap with a normalized collision energy of 35%, taking into account a dynamic exclusion of 10 s. Automatic gain control (AGC) target values and maximum injection times were set to 106 and 50 ms for MS and 104 and 100 ms for MS2.

Data interpretation and protein assignments

Data interpretation was conducted with the help of MaxQuant (v 1.305) using Andromeda and the Saccharomyces Genome Database (February 2011; 6750 target sequences) and the following settings: (i) trypsin without missed cleavages; (ii) carbamidomethylation of cysteine as fixed and (iii) oxidation of methionine, acetylation of protein N-termini, 13C 6 15N 2 Lys and 13C 6 15N 4 Arg as variable modifications; and (iv) MS and MS/MS tolerances of 10 ppm and 0.5 Da, respectively. Only unique peptides were considered for quantification at a false discovery rate of <1% (peptides and proteins). The eight data sets were merged to a master table and only proteins for which at least two unique peptides were quantified for the sonication or the carbonate data sets were considered to determine average ratios from the four SN sonication vs. PEL sonication ratios (SN son /PEL son ) and the four SN carbonate vs. PEL carbonate ratios (SN carb /PEL carb ). The reference proteomes of resident proteins of the OM and IMS were extracted from ref. 31 including three novel OM proteins Mim2, Caf4 and Atg32, and ref. 17, respectively (Supplementary Data 5). For the reference set of Supplementary Fig. 2 each protein was manually reviewed using the original literature containing biochemical data on their sublocalization. The most frequent contaminant in mitochondrial proteomic studies, Plasma membrane ATPases PMA1 and PMA26, 31 were not further assigned in this study. Novel mitochondrial proteins were identified by their absence in both, the PROMITO data set6, 7 and SGD (manually curated annotations; Version July 2016)8.

Statistical model and prediction

We set up a statistical model describing the distribution of proteins. We used our reference set of proteins with well-known localization (Supplementary Fig. 2) as protein list P L, whereas all others represented list P U . We created a model using solely P L and applied this model on P U . Therefore, we used the ratios s = log 10 (SN son /PEL son ) and c = log 10 (SN carb /PEL carb ) for sonication (S) and carbonate extraction (C). The two-dimensional multivariate normal distribution was taken as the model with parameters

$$\mu = \left( {{\mu _{\rm{S}}},{\mu _{\rm{C}}}} \right),\,\Sigma = \left( {\begin{array}{*{20}{c}}\\ {\sigma _{\rm{S}}^2} & {\rho {\sigma _{\rm{S}}}{\sigma _{\rm{C}}}} \\ \\ {\rho {\sigma _{\rm{S}}}{\sigma _{\rm{C}}}} & {\sigma _{\rm{C}}^2} \end{array}} \right)$$

where ρ is the correlation between S and C. The probability density function is defined as

$$\begin{array}{ccccc}{f\left( s,c\,|\,\mu ,\Sigma \right)} = \frac{1}{{2\pi {\sigma _{\rm{S}}}{\sigma _{\rm{C}}}\sqrt {1 - {\rho ^2}} }}\, {\rm{exp}}\left( {\frac{{ - 1}}{{2\left( {1 - {\rho ^2}} \right)}}\left[ {\frac{{{{\left( {{s} - {\mu _{\rm{S}}}} \right)}^2}}}{{\sigma _{\rm{S}}^2}} + \frac{{{{\left( {c - {\mu _{\rm{C}}}} \right)}^2}}}{{\sigma _{\rm{C}}^2}} - \frac{{2\rho \left( {{{s}} - {\mu _{\rm{S}}}} \right)\left( {{{c}} - {\mu _{\rm{C}}}} \right)}}{{{\sigma _{\rm{S}}}{\sigma _{\rm{C}}}}}} \right]} \right).\end{array}$$

To estimate the parameters, we utilized the maximum-likelihood estimators, that is

$$\hat \mu = \frac{1}{{\left| {{P_{\rm{L}}}} \right|}}{\sum} {\left( {s,c} \right)} ,\hat \Sigma = \frac{1}{{\left| {{P_{\rm{L}}}} \right|}}{\sum} {\left( {\left( {s,c} \right) - \hat \mu } \right) \cdot {{\left( {\left( {s,c} \right) - \hat \mu } \right)}^T}} $$

We obtained the following parameters for the three stages:

$$\begin{array}{ccccc}& {\rm{Soluble}}:\,\hat \mu \,\left( {1.0273,\;1.2561} \right),\,\hat \Sigma \left( {\begin{array}{*{20}{c}}\\ {0.0640} & {0.0054} \\ \\ {0.0054} & {0.0289} \\ \end{array}} \right);\\ \\ & {\rm{Peripheral}}:\,\hat \mu \,\left( { - 0.0351,\;1.1155} \right),\,\hat \Sigma \left( {\begin{array}{*{20}{c}}\\ {0.1355} & {0.0649} \\ \\ {0.0649} & {0.0797} \\ \end{array}} \right);\\ \\ & {\rm{Integral}}:\,\hat \mu \,\left( { - 0.5041,\; - 0.6293} \right),\,\hat \Sigma \left( {\begin{array}{*{20}{c}}\\ {0.0373} & {0.0179} \\ \\ {0.0179} & {0.2459} \\ \end{array}} \right).\end{array}$$

To determine the probability p that a protein of P U belongs to a certain state, we computed the average over all three states adding an equally distributed background model b for proteins that cannot be described by any state, let

$$\begin{array}{*{20}{l}}{p_{i,{\rm{soluble}}}} = \frac{{f\left( {{{\left( {s,c} \right)}_i}{\rm{|}}{\mu _{{\rm{soluble}}}},\,{\Sigma _{{\rm{soluble}}}}} \right)}}{{f\left( {\left( {s,c} \right)\,{\mu _{{\rm{soluble}}}},\,{\Sigma _{{\rm{soluble}}}}} \right) \,+\, f\left( {{{\left( {s,c} \right)}_i}{\rm{|}}{\mu _{{\rm{peripheral}}}},\,{\Sigma _{{\rm{peripheral}}}}} \right) \,+ \,f\left( {{{\left( {s},{c} \right)}_i}{\rm{|}}{\mu _{{\rm{integral}}}},\,{\Sigma _{{\rm{integral}}}}} \right) \,+ \,b}}, \hfill \end{array}$$

$$\begin{array}{*{20}{l}}{p_{i,{\rm{peripheral}}}} = \frac{{f\left( {{{\left( {s,c} \right)}_i}{\rm{|}}{\mu _{{\rm{peripheral}}}},\,{\Sigma _{{\rm{peripheral}}}}} \right)}}{{f\left( {{{\left( {s,c} \right)}_i}{\rm{|}}{\mu _{{\rm{soluble}}}},\,{\Sigma _{{\rm{soluble}}}}} \right) \,+\, f\left( {{{\left( {s,c} \right)}_i}{\rm{|}}{\mu _{{\rm{peripheral}}}},\,{\Sigma _{{\rm{peripheral}}}}} \right) \,+\, f\left( {{{\left( {s,c} \right)}_i}{\rm{|}}{\mu _{{\rm{integral}}}},\,{\Sigma _{{\rm{integral}}}}} \right) \,+ \,b}}, \hfill \end{array}$$

$$\begin{array}{*{20}{l}}{p_{i,{\rm{integral}}}} = \frac{{f\left( {{{\left( {s,c} \right)}_i}{\rm{|}}{\mu _{{\rm{integral}}}},{\Sigma _{{\rm{integral}}}}} \right)}}{{f\left( {{{\left( {s,c} \right)}_i}{\rm{|}}{\mu _{{\rm{soluble}}}},\,{\Sigma _{{\rm{soluble}}}}} \right) \,+ \,f\left( {{{\left( {s,c} \right)}_i}{\rm{|}}{\mu _{{\rm{peripheral}}}},\,{\Sigma _{{\rm{peripheral}}}}} \right) \,+ \,f\left( {{{\left( {s,c} \right)}_i}{\rm{|}}{\mu _{{\rm{integral}}}},\,{\Sigma _{{\rm{integral}}}}} \right) \,+ \,b}} \hfill \end{array}$$

for every \({1} {\leq} {i} {\leq} {\left| {{P_{\rm U}}} \right|}.\)

Here, we set b = (0.5 ⋅ (max(s) − min(s))(max(c) − min(c)))−1 = 19.482.

Since we assumed that all proteins are equally distributed among all three states, we did not weight the three models. In case a certain state described a protein with a probability >0.5, the protein was discretely assigned to this state. The same holds true in case the probability was 0.4–0.5 by model A (without loss of generality) while concurrently the probability by model B was at least 0.15 smaller. Proteins passing neither criteria were considered as ambiguous. Thus, the model confirmed 94.1% of the original assignments with clear s and c ratios (200/214 soluble, 253/255 peripheral, 292/302 integral, and 152/182 ambiguous). Supplementary Fig. 3 represents the distribution of the reference set P L , whereas Supplementary Fig. 4 illustrates the distribution of all proteins P L + P U . The color coding is red = peripheral, green = integral, and soluble = blue. For every protein, all three color channels RGB were multiplied by their corresponding probabilities to indicate memberships. The model boundaries indicate 80% of each density.

Comparison of total yeast vs. total mitochondrial proteomes

To assess the likelihood that new mitochondrial proteins are also located in other cellular compartments or might represent contaminants, we quantified the complete yeast and mitochondrial proteomes derived from arg4Δ cells grown under non-fermentative conditions (see above). Yeast cells and highly pure mitochondria were lysed in 10% w/v SDS, 150 mM NaCl, 50 mM Tris (pH 8.0). The protein content was determined using the bicinchoninic acid assay (BCA, Thermo Scientific) according to the manufacturer’s instructions. Proteins were carbamidomethylated, precipitated, digested, and samples desalted as described above. 50 µg of digest (mitochondria and yeast) were resuspended in 10 mM ammonium acetate (pH 6.5) and fractionated on a C18 column (Zorbax, I.D. 0.5 mm × 150 mm, Agilent) at pH 6.5 (solvent A: 10 mM ammonium acetate (pH 6.5); solvent B: 10 mM ammonium acetate, 84% ACN) using a 90 min gradient ranging from 3 to 50% B. 20 fractions were collected in a concatenated manner, dried under vacuum, resolubilized in 0.1% TFA. Per fraction 50% was analyzed by nano-LC-MS/MS on Q Exactive Plus coupled to a U3000 RSLC system (both Thermo Fisher Scientific) using LC parameters as above (2 h gradient). MS scans were acquired at a resolution of 70,000, the 15 most abundant ions were fragmented by higher energy collisional dissociation with a normalized collision energy 30% and MS/MS were acquired at a resolution of 17,500. AGC and maximum injection times were set to 3 × 106 and 120 ms for MS and 2 × 105 and 250 ms for MS/MS scans. Raw data were converted into mascot generic files using Proteowizard (Version 2.2.2954) and searched with Mascot, OMSSA, and X!Tandem using SearchGUI 1.12.254. Data were analyzed using PeptideShaker version 0.20.155 at a false discovery rate of 1% on the protein, peptide and peptide-spectrum-match (PSM) levels. For the mitochondrial proteome, normalized spectral abundance factors (NSAF)56 were calculated for estimating the relative amounts of all identified proteins across the mitochondrial proteome. To assess, whether new candidate proteins are located exclusively in mitochondria or also in other cellular compartments, we furthermore calculated NSAF of these proteins in the whole yeast sample, i.e., only considering PSM of mitochondrial proteins. Per protein a reference yeast/mito ratio was calculated by dividing the corresponding yeast NSAF by the mitochondrial NSAF. Thus, proteins that have dual (multiple) subcellular localizations or represent potential contamination should have high yeast/mito ratios, corresponding to a relative enrichment in the yeast proteome as compared to the mitochondrial proteome.

Identification of integral inner and outer membrane proteins

To specifically distinguish integral membrane proteins from the inner and outer mitochondrial membranes, we compared the proteomes of carbonate extracted (i) highly purified OM vesicles (OM Light ) and (ii) total membranes (TOT Heavy ), containing both organellar membranes. Carbonate resistant pellets from OM Light and TOT Heavy samples, according to BCA both ~3 μg, were ultracentrifuged in SEM buffer at 100,000×g and 4 °C for 1 h. Pellets were resolubilized in 2 M GuHCl, 50 mM NaH 2 PO 4 (pH 7.8) and processed as described above. After digestion, samples were pooled in three different proportions: 1 µL OM Light + 10 µL TOT Heavy (1:10), 2:10 and 3:10. Pooled samples were analyzed on a Q Exactive Plus as described above. Data analysis was conducted using MaxQuant57 as described above. As several proteins were absent in either light/heavy samples, OM L and TOT H SILAC intensities were used to calculate per protein its relative intensity in the OM fraction. Values of all three samples were used to determine the average relative intensities in the OM fraction. For integration of the data into the landscape only identified proteins from this study and resident OM proteins of the OM reference proteome (see above) were considered.

In organello import of mitochondrial precursor proteins

Radiolabeled precursors were generated in vitro with the transcription and translation rabbit reticulocyte lysate system (Promega) supplemented with [35S]methionine. Mitochondria (80 μg) and precursor proteins were incubated at 30 °C for 45 min in import buffer (10 mM MOPS-KOH (pH 7.2), 3% w/v bovine serum albumin, 250 mM sucrose, 5 mM MgCl 2 , 80 mM KCl, and 5 mM KP i ). Samples were supplied with 2 mM ATP and 2 mM NADH. Import reactions were abolished or terminated by disruption of the membrane potential by addition of 8 μM antimycin A, 1 μM valinomycin, and 20 μM oligomycin (AVO). Non-imported precursors were digested with 50 μg ml−1 Proteinase K or 25 μg ml−1 Trypsin and incubation for 10–15 min on ice. Mitochondria were reisolated by centrifugation at 16,000×g for 10 min at 4 °C and washed with SEM buffer. Analysis was performed by SDS–PAGE and digital autoradiography using the FLA 9000 image scanner (Fujifilm) and the freeware ImageJ version 1.40 g (National Institutes of Health).

Miscellaneous

Antibodies were generated by immunization of rabbits using synthetic peptides (Supplementary Table 1). Antibodies against the candidate proteins with multiple localizations were raised against the following peptides: Lap3, KEEPIVLPIWDPMGALAK; Gsf2, GEDLKKFRKIRKEQDPDN; Ape4, FKEFFERYTSIESEIVV; Ape2, NRDRDVVNKYLKENGYY; Lsp1, ADHHVSQNGHTSGS ENI; and Zeo1, EKKETKKEGGFLKKLNRK. Peptides were coupled to keyhole limpet hemocyanin via N-terminal cysteines. Western blotting was performed according to standard protocols.

Data processing

Photoshop CS5 (Adobe) was used to process images and the figures were compiled using Illustrator CS5 (Adobe). To show regions of interest blots and autoradiography scans were digitally processed. Uncropped versions of immunoblots and autoradiographs are shown in Supplementary Figs. 8–10.

Data availability

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository58 with the data set identifiers PXD005463 and PXD005541. Further relevant data can be obtained from the authors upon request.