Import of precursor proteins and peptides
Precursor proteins were generated in the presence of [35S]methionine in reticulocyte lysate (Promega). Import into mitochondria was performed in import buffer (250 mM sucrose, 10 mM MOPS/KOH, pH 7.2, 80 mM KCl, 2 mM KH2PO4, 5 mM MgCl2, 5 mM methionine, and 1% fatty-acid free BSA) at 25°C in the presence of 2 mM NADH and 2 mM ATP. 1 µM valinomycin, 8 µM antimycin A, and 20 µM oligomycin were used to dissipate the membrane potential. Mitochondria were proteinase K treated, washed with SEM buffer (250 mM sucrose, 20 mM MOPS/KOH, pH 7.2, and 1 mM EDTA) and analyzed by BN-PAGE or SDS-PAGE in conjunction with autoradiography or Western blot.
For import competition experiments, mitochondria were preincubated with the indicated concentration of peptide for 2 min at 25°C.
For antibody inhibition, mitochondria were incubated for 20 min on ice in 10 mM MOPS, pH 7.4, to convert them to mitoplasts. After addition of the respective antibodies they were incubated for 15 min on ice, re-isolated, and resuspended in import buffer.
Arrest of radiolabeled Su9-DHFR was performed at 0°C, essentially as described in Kanamori et al. (1999)
. In brief, isolated mitochondria (0.5 mg/ml) were incubated in 10 mM MOPS/KOH, pH 7.2, 250 mM sucrose, 10 mM KCl, 5 mM MgCl2
, 2 mM methionine, and 10 µM carbonyl cyanid 3-chlorophenylhydrazon (CCCP). After an incubation with radiolabeled Su9-DHFR precursor for 15 min on ice, mitochondria were diluted 1:5 using 10 mM MOPS/KOH, pH 7.2, 250 mM sucrose, 10 mM KCl, 20 µM CCCP, 0.5 µM methotrexate, and 1 mM NADPH, and re-isolated. After resuspension in the same buffer (0.5 mg/ml) the sample was split into two; one half was proteinase K treated, the other one not. The supernatant of the proteinase K–treated sample was precipitated using 15% TCA while the mitochondrial pellet of the untreated sample was analyzed.
Cross-linking analyses and purification of photo-adducts
For photo cross-linking, mitochondria were suspended in import buffer lacking BSA to 1 µg/µl, supplemented with 2 µM photo-peptide, and incubated 10 min on ice. UV irradiation was performed for 30 min on ice with a self-made device containing a halogen metal vapor lamp and a glass screen to filter protein-damaging wavelengths below 300 nm (Jahn et al., 2002
). Subsequently, mitochondria were washed with SEM buffer and analyzed by Western blotting. For isolation of photo-adducts, mitochondria were resuspended (10 µg/µl) in lysis buffer (100 mM Tris/HCl, pH 8.0, 8 M urea, 1% SDS, 2% Triton X-100, 1 mM EDTA, 200 mM NaCl, and 1mM PMSF) and incubated for 10 min at room temperature. Samples were diluted to 1 µg/µl with dilution buffer (lysis buffer lacking SDS and containing 0.8 M urea, 2 µg/ml leupeptin, and 2 mM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride), and incubated for 10 min at 4°C. After removal of insoluble particles the sample was loaded onto streptavidin agarose (Thermo Fisher Scientific). Washing of the column material was performed using wash buffer A (as lysis buffer, but with 2% SDS), wash buffer B (as lysis buffer, but with 0.2% SDS and 1 M NaCl), and wash buffer C (100 mM Tris/HCl, pH 8.0, 0.2% Triton X-100, and 1 mM EDTA). Samples were eluted by incubation at 95°C for 15 min in protein loading buffer (2% SDS, 10% glycerol, 60 mM Tris/HCl, pH 6.8, 0.01% bromphenole blue, and 1% 2-mercaptoethanol).
Photo cross-linking of purified proteins was performed at a 0.5–1.5 molar ratio of protein to peptide for 30 min on ice.
Chemical cross-linking was performed using purified protein (1 µM) mixed with the indicated amount of peptide or additional protein for 15 min on ice in buffer containing 20 mM Hepes, pH 7.2, and 100 mM NaCl. 100 µM DFDNB was used to cross-link for 30 min on ice, after which the reaction was quenched for 15 min with 140 mM Tris/HCl, pH 7.5, and 5% β-mercaptoethanol.
For in organello DFDNB cross-linking 1 µg/µl mitochondria were suspended in 20 mM Hepes, pH 7.2, and 100 mM NaCl and incubated with 20 µM presequence peptide for 15 min at 25°C. Subsequent to cross-linking with 1 mM DFDNB for 30 min on ice the reaction was stopped by 250 mM Tris/HCl, pH 7.4, and washing of mitochondria with 20 mM Hepes, pH 7.2, 80 mM KCl, and 600 mM sorbitol.
Protein purifications, immunoprecipitation, and in vitro binding
Based on secondary structure prediction software included in Geneious Pro (Drummond et al., 2010
) and the predicted transmembrane span, we generated Tim50IMS
(aa 132–476), Tim50ΔPBD
(aa 132–365), and Tim50PBD
(aa 395–476), which were cloned into pPROEX HTc. These constructs, as well as Tom20CD
(Brix et al., 1999
), were purified via immobilized-metal affinity chromatography. GST was purified via glutathione–Sepharose 4B (GE Healthcare). For in vitro photo cross-linking, constructs were dialyzed against 10 mM MOPS/NaOH, pH 7.2, 5 mM MgCl2
, 2 mM KH2
, and 20 mM KCl.
Tim23 pull-down was performed as described previously (Geissler et al., 2002
). In brief, purified His-tagged Tim23IMS
(Truscott et al., 2001
) was immobilized on Ni-NTA agarose (QIAGEN). Mitochondria were solubilized (1 µg/µl) using 20 mM Hepes/KOH, pH 7.4, 100 mM KOAc, 10 mM Mg(OAc)2
, 10% (vol/vol) glycerol, 20 mM imidazol, and 0.5% (vol/vol) Triton X-100 for 30 min at 4°C. Binding of the solubilized mitochondrial proteins to the immobilized protein was performed at 4°C for 1 h. Subsequent to washing with solubilization buffer (0.25% [vol/vol] Triton X-100), bound proteins were eluted with 8 M urea containing SDS-PAGE loading buffer.
Immunoprecipitation used HA and 6xHis antibodies, immobilized on Dynabeads ProtG. Mitochondria were solubilized (2 µg/µl) in 50 mM sodium phosphate, pH 7.4, 100 mM NaCl, 10% glycerol, and 1% digitonin before incubation with the antibody beads. Subsequent to washing, bound proteins were eluted by incubation in protein loading buffer (without β-mercaptoethanol) at 60°C for 10 min.
Peptidic photo probes were synthesized with a peptide synthesizer (model 433A; Applied Biosystems) using standard fluorenylmethoxycarbonyl (Fmoc) chemistry in the 0.1 mmol scale. The photophore was directly introduced into the polypeptide chain by using the Fmoc derivative of the photoreactive amino acid para-benzoyl-Phe-OH (BPA; Bachem). The biotin moiety was introduced through biotinylated Lys. Due to the limited solubility of the corresponding amino acid derivative Fmoc-Lys(biotin)-OH (Novabiochem), its coupling was performed manually under visual inspection in a pear-shaped flask. For this purpose, the dried N-terminally deprotected resin was resuspended with 0.5 mmol Fmoc-Lys(biotin)-OH in N-methyl-2-pyrrolidon (5 ml), 0.9 mmol 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate in dimethylformamide (2 ml), 2 mmol diisopropylethylamine in N-methyl-2-pyrrolidon (1 ml), and rotated for 45 min at room temperature. The resin was washed with N-methyl-2-pyrrolidon (3x) and the coupling step was repeated as before. The resin was then transferred back into the synthesizer and automated synthesis was resumed with an Fmoc-deprotection cycle. The completed peptides were deprotected and cleaved from the resin via incubation in solution containing 95% trifluoroacetic acid, 2.5% triisopropylsilane, and 2.5% water for 4 h at room temperature. Purified products were characterized by reversed-phase HPLC and mass spectrometry. Biotin-labeled pALDH was synthesized in the same way as the photo-probes, but without the incorporation of the BPA.
pCox4 (MLSLRQSIRFFKPATRTLCSSRYLL), SynB2 (MLSRQQSQRQSRQQSQRQSRYLL), and scrambled pALDH-s (MLRGKQPTKSLLPQRSPKLSAAA) were purchased from JPT Peptide Technologies as an N-terminal amine and a C-terminal amide.
Genetic manipulation of yeast and growth conditions
YPH499 or BY4743 were cultured in YP medium (1% yeast extract, 2% peptone) containing 2% glucose (YPD) or 3% glycerol (YPG) or 3% lactate (YPL) at 30°C. TIM50+/ΔPBD
(in BY4743; yCS1) was generated through transformation of a C-terminal HA tagging cassette, resulting in the deletion of a region encoding amino acids 366–476 (Janke et al., 2004
Sporulation of the BY4743 TIM50+/ΔPBD
HA3 strain was achieved using a stationary culture from YPD, which had been washed in sporulation medium (2% KAc, 0.2% yeast extract, 0.1% glucose, 0.2% leucine, 0.04% histidine, lysine, and uracil) and cultivated at 24°C for 5 d in sporulation medium. Subsequently, tetrads were dissected on YPD plates and grown at 30°C.
For in vivo expression of different Tim50 constructs, full-length Tim50 (1–476) or Tim50ΔPBD
(1–365) was cloned into pME2780 without (pCS22 and pCS20) or with a single HA tag (pCS23 and pCS21; Mumberg et al., 1994
). Constructed plasmids were then used to replace the wild-type protein encoding plasmid within the gene deletion strain to yield strains yCS5-8 (Chacinska et al., 2005
The strain containing TIM50
under the control of the GAL1
promoter (Geissler et al., 2002
) was transformed with plasmids pCS27 and pCS26, encoding Tim50 (1–476) or Tim50ΔPBD
(1–365), both of which contained a single C-terminal HA tag, under the control of the MET25
promoter (Mumberg et al., 1994
) to give yCS2 and yCS3. Strains were precultured in selective lactate medium (0.67% YNB, 3% lactate, 0.003% lysine, 0.002% adenine, histidine, tryptophane, and uracil, pH 5.0) supplemented with 1% galactose and 1% raffinose. Subsequently, cells were grown in equivalent selective medium containing additional 0.01% glucose for 38 h at 30°C.
was cloned into pME2804 under the control of the GALS
promoter (Mumberg et al., 1994
). The resulting plasmid (pCS28) was introduced into MB29 (Bömer et al., 1997
) by plasmid shuffling (yCS4). Cultures were grown essentially as described previously (Geissler et al., 2002
). In brief, cells were precultured in YPL containing 1% raffinose and 1% galactose for 28 h. The main culture was grown for 31 h in YPL with 0.1% glucose.
To isolate mitochondria, yeast cells were treated with 7 mg zymolase/g cell wet weight and opened in a Dounce Homogenizer. Mitochondria were isolated by differential centrifugation and were pelleted at 17,000 g
as described previously (Stojanovski et al., 2007
). Appropriate aliquots were stored in SEM buffer at −80°C.
Photo-adducts purified by colloidal Coomassie-stained (Neuhoff et al., 1988
) SDS-PAGE were subjected to an in-gel digest. In brief, gel pieces were cut out, washed with 25 mM NH4
/water, 25 mM NH4
/50% acetonitrile, and 100% acetonitrile, followed by reduction with 10 mM dithiothreitol, 25 mM NH4
/water at 56°C for 1 h, washing (see above) and carbamidomethylation with 25 mM iodoacetamide in 25 mM NH4
/water. After digestion with 120 ng trypsin at 37°C overnight, peptides were extracted from the gel with 0.1% trifluoroacetic acid (TFA) and separated by reverse-phase chromatography (EASY-nLC; Bruker Daltonics) using a PepMap100 C18 nano-column (Dionex) and an elution gradient from 9.5–90.5% acetonitrile in 0.1% TFA for 80 min. The eluate from the column was mixed with α-cyano-4-hydroxycinnamic acid (HCCA) as matrix (4.5% of saturated HCCA in 90% acetonitrile, 0.1% TFA, 1 mM NH4
) and spotted onto an anchorchip target using a robot (Proteineer fc II; Bruker Daltonics). Samples on the target were analyzed in a MALDI-TOF/TOF mass spectrometer (Ultraflextreme; Bruker Daltonics) recording MS and post-source decay MS/MS spectra using the software WARP-LC, AutoXecute, and FlexAnalysis (Bruker Daltonics). MS/MS spectra with precursor masses, which match any of the masses calculated for candidate cross-linked peptides, were selected for further manual evaluation supported by the software Biotools (Bruker Daltonics) to verify peptide identities and to identify the cross-linked amino acids.
MALDI-TOF-MS was also used to co-detect recombinant presequence receptor domains and their respective photo-adducts at the level of intact proteins. For this purpose, the photoreaction mixtures were desalted by RP C4 ZipTips (Millipore) and bound material was eluted with 80% acetonitrile/0.1% TFA. For dried droplet preparation, 1 µl eluate was mixed with 2 µl α-cyano-4-hydropxycinnamic acid (HCCA) matrix solution and spotted to a ground steel sample support. The matrix solution was prepared from a saturated HCCA solution (in 0.1% TFA/acetonitrile 2:1) by diluting the supernatant 1:6 with 70% acetonitrile/0.1% TFA. Mass spectra were acquired on a MALDI-TOF/TOF mass spectrometer (Ultraflex; Bruker Daltonics) operated in the linear mode as described previously (Dimova et al., 2009
). In brief, positively charged ions in the mass-to-charge (m/z) range 5,000–25,000 were analyzed in the linear mode and a mixture of three standard proteins (Protein Calibration Mix 2; LaserBio Laboratories; m/z range 6181–23982) was used for external calibration with the post-processing software FlexAnalysis 3.3 (Bruker Daltonics).
Homology modeling and peptide docking
The cytoplasmic domain of the yeast Tom20 has been modeled using the homology modeling protocol as implemented in ROSETTA (Misura et al., 2006
). Subsequent to screening the query sequence for regions that possess an experimentally characterized homologue, secondary structure prediction and preparation of the sequence alignments have been performed using HHpred server
(Söding et al., 2005
). The fragment libraries were obtained from the Rosetta Server. The solution NMR structure of rat Tom20 (Protein Data Bank accession no. 1OM2
) was used as the template (for the modeled 74 residue–long fragment the compared sequences share 25 and 43% sequence identity and similarity, respectively; Abe et al., 2000
). Generated models (2,500) have been sorted based on their energies (ROSETTA score) and compared with the template molecule by calculating the root mean square deviation (RMSD) on Cα positions in the LSQMAN program (Kleywegt and Jones, 1994
). The best model was identified as the fourth on the list of top ROSETTA scores, exhibiting a calculated RMSD of 1.23 Å between 66 superposed Cα positions.
Docking of pALDH to the modeled yeast Tom20 fragment has been performed using the FlexPepDock protocol (Raveh et al., 2010
) implemented in the ROSETTA package. The initial position of the peptide was obtained based on the superposition with the structure of rat Tom20 (Protein Data Bank accession no. 1OM2
) in complex with the same peptide. Four different conformations of the peptide (models 5, 8, 11, and 12 out of 20 presented in the solution NMR structure) have been selected for the docking experiments and 2,500 decoys for each of them have been calculated. Docking of peptide model 11 gave the best ROSETTA scores (over 900 top scores out of 10,000) and the model with the lowest energy was selected.
BN-PAGE, SDS-PAGE, and Western blotting were performed by standard procedures. Proteins were detected using fluorescent dye coupled to secondary antibodies (LI-COR) using a fluorescence scanner (FLA-9000; Fujifilm) and the ImageReader FLA-9000 software or by enhanced chemiluminescence (GE Healthcare). Contrast adjustments were performed using Photoshop CS4 (Adobe). Quantifications were performed using ImageQuant TL (GE Healthcare).
Online supplemental material
Fig. S1 shows the validation of the photo cross-linking approach using the Tom20CD
. Fig. S2 shows import, import block, and photo cross-linking experiments concerning the characterization of the photo-peptides. Fig. S3 shows the mass spectrometric identification of photo cross-linking sites. Fig. S4 shows the determination of the photo cross-linking stoichiometry. Fig. S5 presents further data on the characterization of the Tim23↓ and the Tim50ΔPBD-HA1
mutant mitochondria. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.201105098/DC1