Purification of mitochondria Saccharomyces cerevisiae
strains were grown aerobically at 30 °C in SC or YP medium, and cells were harvested in logarithmic growth phase (OD600 < 1.3). Mitochondria were isolated by one of two different methods. One method involved differential centrifugation followed by a Nycodenz density gradient (Glick and Pon 1995
), where the progress of mitochondrial purification was controlled by Western blot analysis using organelle-specific marker protein antibodies. In the other method, isolated mitochondria were purified by zone electrophoresis using a ProTeam FFE Free-Flow Electrophoresis apparatus (Tecan, Grödig, Austria) (Zischka et al. 2003
). The anodic and cathodic circuit electrolytes consisted of 100 mM acetic acid and 100 mM triethanolamine acetate (pH 7.4). The electrolyte stabilizer was 280 mM sucrose, 100 mM acetic acid, and 100 mM triethanolamine (pH 7.4). The separation medium was 280 mM sucrose, 10 mM acetic acid, and 10 mM triethanolamine (pH 7.4). The counterflow medium was 280 mM sucrose. Table S1
lists the strains, growth conditions, and purification methods used for each dataset.
Prior to FFE fractionation, the mitochondria sample was equilibrated with separation medium and adjusted to a final protein concentration of 1–2 mg/mL. Electrophoresis was performed in horizontal mode at 5 °C with a total flow rate of 280 mL/h within the separation chamber at a voltage of 750 V. The samples were applied to the separation chamber with a flow rate of 1–2 mL/h via the cathodic inlet. Fractions were collected in 96-well plates, and the distribution of separated particles was monitored at a wavelength of 260 nm with a SynergyHT reader (Bio-Tek, Winooski, Vermont, United States). The peak fraction was isolated, shock-frozen in liquid nitrogen, and used for electron microscopy.
To assess purity, the preparations were analyzed by electron microscopy. The mitochondrial preparations were fixed with 4% formaldehyde, 2% glutaraldehyde, 4% sucrose, 2 mM calcium acetate, and 50 mM sodium cacodylate (pH 7.2) at 4 °C. The fixed samples were dissected with a scalpel, washed for 1 h in cacodylate buffer with 1% osmium tetroxide, and dehydrated with alcohol in increasing concentrations. After embedding in Araldite, the preparations were cut into 50-nm slices by means of an ultramicrotome (LKB-Produkter, Bromma, Sweden) and then analyzed on a Zeiss (Oberkochen, Germany) EM 10 electron microscope.
Fractionation of matrix and membrane proteins
Reagents used for the preparation of peptide samples were purchased from the indicated suppliers. Ammonium bicarbonate and methanol were from Fisher Scientific (Fair Lawn, New Jersey, United States). Sodium carbonate, urea, dithiothreitol, and calcium chloride were obtained from Sigma-Aldrich (St. Louis, Missouri, United States). Thiourea, trifluoroacetic acid, and acetonitrile were from Aldrich Chemical Company (Milwaukee, Wisconsin, United States). Sequencing-grade, modified porcine trypsin was obtained from Promega (Madison, Wisconsin, United States). Ammonium formate was obtained from Fluka (St. Louis, Missouri, United States). CHAPS and bicinchoninic acid (BCA) assay reagents and standards were from Pierce (Rockford, Illinois, United States). Purified water was generated using a Barnstead Nanopure Infinity water purification system (Dubuque, Iowa, United States).
Purified mitochondrial samples were disrupted using a Mini Beadbeater-8 (Biospec Products, Bartlesville, Oklahoma, United States) for 3 min at 4,500 rpm with 0.1 mm zirconia/silica beads (Biospec Products) in a 0.5-mL, sterile siliconized microcentrifuge tube. The lysed mitochondria, containing membrane and matrix proteins, were removed from the beads through a puncture at the bottom of the microcentrifuge tube, by centrifugation at 16,000 xg for 2 min at 4 °C, and the flow-through was collected in a second microcentrifuge tube. The collected lysate was then centrifuged at 356,000 xg for 10 min at 4 °C to pellet the mitochondrial membranes. The soluble supernatant was used for the study of mitochondrial matrix proteins, and the pellet was retained for identifying mitochondrial membrane proteins.
Mitochondrial membrane protein preparation
Using a sonication bath (Branson 1510, Danbury, Connecticut, United States), the membrane pellet was resuspended in 50 mM ammonium bicarbonate (pH 7.8) in an ice bath. The resuspended sample was diluted with ice-cold 100 mM sodium carbonate (pH 11.0) and incubated on ice for 10 min. The membranes were then pelleted by ultracentrifugation at 356,000 xg for 10 min at 4 °C. The pelleted membranes were washed using two aliquots of ice-cold water and pelleted again by centrifugation. The BCA protein assay was performed to determine protein concentration.
The membrane pellet was resuspended in 7 M urea, 2 M thiourea, 1% CHAPS in 50 mM ammonium bicarbonate (pH 7.8), using vortexing and sonication in an ice bath. Dithiothreitol was added to a final concentration of 9.7 mM in the resuspended sample, and the proteins were then treated with thermal denaturation for 45 min at 60 °C. The denatured and reduced protein sample was then diluted 10-fold with 50 mM ammonium bicarbonate (pH 7.8), and calcium chloride was added to a final sample concentration of 1 mM. Tryptic digestion was performed for 5 h at 37 °C using a 1:50 (w/w) trypsin-to-protein ratio. Snap-freezing the sample in liquid nitrogen quenched the digestion. The tryptic peptides were cleaned using a 1-mL strong cation exchange column (Discovery DSC-SCX , Supelco, Bellefonte, Pennsylvania, United States) per the manufacturer's instructions. The eluted peptide sample was concentrated by lyophilization and a BCA assay was performed to determine final peptide concentration. The peptide sample was stored at −80 °C until time for LC/MS/MS analysis.
Mitochondrial matrix protein preparation
The BCA protein assay was performed on the soluble matrix supernatant. The proteins were thermally denatured and reduced using 7 M urea, 2 M thiourea, and 5 mM dithiothreitol and incubating at 60°C for 30 min. The denatured and reduced protein sample was diluted 10-fold with 50 mM ammonium bicarbonate (pH 7.8), and the concentration of calcium chloride was adjusted to a final concentration of 1 mM. The tryptic digestion of the protein sample was performed in the same manner as described above for the membrane protein sample. The tryptic peptides were cleaned using a 1-mL LC-18 SPE column (Reversed Phase Supelclean LC-18 SPE, Supelco) per the manufacturer's instructions. The eluted peptide sample was concentrated by lyophilization, a BCA protein assay was performed, and the sample was stored at −80 °C until time for LC/MS/MS analysis.
Identification of potential mass and time tags by LC/MS/MS
The LC/MS/MS analysis of the tryptically digested peptides was performed as previously reported (Shen et al. 2001
). In brief, the high-resolution reversed phase capillary liquid chromatography (LC) system was composed of a column assembled in-house using a 150-μm id × 360-μm od × 65-cm capillary (Polymicro Technologies, Phoenix, Arizona, United States) fixed with a 2-μm retaining mesh and packed with 3-μm Jupiter C18 stationary phase (Phenomenex, Torrence, California, United States). The column was equilibrated with 100% mobile phase A (0.05% trifluoroacetic acid in water) at 5,000 psi. Ten minutes after injecting a 10-μL sample (~0.5 μg/μL), the exponential gradient began mixing mobile phase A with mobile phase B (0.1% trifluoroacetic acid:90% acetonitrile:9.9% water [vol/vol/vol]) while maintaining constant pressure. Using an in-house-manufactured electrospray ionization source, the capillary LC was interfaced with an LCQ ion trap mass spectrometer (ThermoFinnigan, San Jose, California, United States) with settings of 2.2 kV and 200 o
C for the ESI voltage and heated capillary, respectively. The data-dependent tandem MS analysis was conducted using a series of segmented mass/charge (m/z
) ranges. A collision energy setting of 45% was employed for the collision-induced dissociation of the three most abundant ions detected in each MS scan. Dynamic exclusion was used to discriminate against previously analyzed ions. Peptides were identified by searching the tandem MS spectra against the complete annotated S. cerevisiae
genome database (available at http://www.yeastgenome.org/
) using SEQUEST (ThermoFinnigan) (Eng et al. 1994
). “MudPIT” filtering rules were adopted as the acceptance criteria for peptides generated from the SEQUEST results (Washburn et al. 2001
). Fully tryptic peptides with a 1+ charge state that had a cross-correlation (Xcorr) factor of 1.9 or greater were accepted. Fully or partially tryptic peptides with a 2+ charge state that had an Xcorr of 2.2 or greater were accepted as well. Peptides with a 2+ charge state that had an Xcorr of 3.0 or greater were accepted. Finally, fully or partially tryptic peptides with a 3+ charge state were accepted if an Xcorr of 3.75 or greater was obtained.
Identification of accurate mass and time tags by LC/FTICR
Some of the samples analyzed by LC/MS/MS were further analyzed by LC/FTICR. In LC/FTICR, tryptic peptides are analyzed using the same high-resolution reversed phase capillary LC described in the previous section, coupled to an electrospray ionization interface with a Fourier transform-ion cyclotron resonance mass spectrometer (Smith et al. 2002
). We used both a custom-made 11.5 Tesla FTICR instrument, designed and constructed in house at Pacific Northwest National Laboratory, and a commercial 9.4 Tesla Bruker Apex III FTICR instrument (Bruker Daltonics, Billerica, Massachusetts, United States).
The acquired FTICR spectra (105 resolution) were processed and deconvoluted using ICR-2LS (software written in-house at Pacific Northwest National Laboratory) to obtain peak lists containing the monoisotopic mass, observed charge, and intensity of the major ions in each spectrum. The masses were calibrated using the masses of internal calibrant peaks infused at the beginning and end of each LC/FTICR analysis. The peak lists for each analysis were then matched against the potential mass and time (PMT) tags defined previously (see above; by LC/MS/MS analyses among any of the previous samples) using VIPER (software written in-house at Pacific Northwest National Laboratory). The matching involved finding the groups of ions in the data, computing a median monoisotopic mass for each group, and then comparing the mass and elution time of the group with the mass and normalized elution time of each peptide in the PMT tag database (match tolerance of ± 8 ppm and ± 0.05 normalized elution time), resulting in the generation of an accurate mass and time (AMT) tag. Because the PMT tag database consisted only of the peptide tags produced via the previous LC/MS/MS analyses (a PMT tag database for the whole genome does not exist to date), the LC/FTICR analysis could identify only AMT tags which corresponded to previously identified PMT tags from one of the LC/MS/MS runs.
Identification of proteins
For the purpose of deriving a final list of proteins identified by MS, we included only proteins that had been detected by at least two tags in any single experimental dataset. As such we adapted the rules that are standard for minimizing false positives from MS and defining the detected proteins (Wu et al. 2003
Gene expression profiling
Each sample was done in duplicate. Log phase cultures were grown overnight to an O.D. of 1 in 100 mL of YPD, YPL, SCD, or SCL medium. Total RNA was isolated using a hot phenol glass beads protocol. PolyA+ mRNA was purified using Qiagen's Oligotex kit (Qiagen, Valencia, California, United States). Then 4.5 μg of polyA+ mRNA were reverse transcribed to generate single stranded cDNA. Product was fragmented to approximately 50 bp using DNase digestion, biotin end labeled, and hybridized to Affymetrix S98 arrays as described in the Affymetrix user handbook (Affymetrix, Santa Clara, California, United States). Hybridizations were normalized and duplicate samples integrated to arrive at an estimate of absolute transcript abundance using the dChip computational package (Wong Lab, Harvard University). For genes with multiple probe sets on the array, only the probe set with the highest signal was used. For every gene, we calculated the fold difference between fermentable and nonfermentable growth conditions and considered significant only genes with a 1.2-fold or greater difference (either increased or decreased expression). In the final list we included only genes that showed a consistent direction of expression difference (increase or decrease) in both rich and synthetic media conditions.
Comparative genomic analysis between yeast and other organisms
All-against-all comparison of genes belonging to human, yeast, R. prowazekii
, and Encephalitozoon cuniculi
genomes has been conducted using the PSI-BLAST algorithm (Altschul et al. 1997
). For each PSI-BLAST match, the following information has been stored in the MitoP2 database: the identification numbers of two matching proteins, the BLAST E-value of the match, the coverage of the BLAST alignment (defined as the fraction of amino acids of the shorter protein covered by the alignment), and whether the match is a bidirectional best hit (ortholog). A compendium of the yeast–human bidirectional blast hits for all yeast proteins with a MitoP2 score greater than 90 is given in Table S3
Prediction of mitochondrial targeting sequences
Psort was downloaded locally as a perl5 script (from E-mail: nakai/at/imcb.osaka-u.ac.jp)
. MitoProt was run in the same way as in Scharfe et al. (2000)
. Predotar analysis was performed as described by Small et al. (2004)
. The protein lists are available in the MitoP2 database.
Integration of published datasets and calculation of MitoP2 score
To calculate the MitoP2 score, the percentage R of known mitochondrial proteins (reference set of 477 proteins) identified in each single genome-wide experiment (specificity) or in the overlap of all possible combinations of datasets (specificity of the combination of several methods) was calculated. Most proteins belonged to more than one combination, and for those proteins multiple R values were calculated. For example, proteins identified by two approaches received three R values: the specificity of the first approach alone, the specificity of the second approach alone, and the specificity of the overlap of both approaches. The MitoP2 value represented the highest R value calculated for a protein. The relevancy was checked according to the binomial law. The value gives a lower limit of the specificity of a defined combination because the mitochondrial reference dataset is not complete. For more detailed description, please see the MitoP2 database.
Protein import into isolated mitochondria
For T7 polymerase–driven synthesis of preproteins in vitro, the ORFs were amplified from ATG to STOP-codon by PCR, including the T7 RNA polymerase promoter and transcription initiation site within the 5′ primer. Using reticulocyte lysate (Promega), the resulting PCR products were utilized for coupled in vitro transcription/translation reactions to synthesize preproteins in the presence of 35S-radiolabeled methionine. Mitochondria were isolated by differential centrifugation from yeast strain W334 grown on lactate medium and resuspended at 25 °C in import buffer (0.3 mg/mL fatty-acid-free BSA, 0.6 M sorbitol, 80 mM KCl, 10 mM magnesium acetate, 2 mM KH2PO4, 2.5 mM EDTA, 2.5 mM MnCl2, 2 mM ATP, 5 mM NADH, and 50 mM HEPES/KOH [pH 7.2]). Import was initiated by adding 1% to 4% (vol/vol) of reticulocyte lysate containing radiolabelled preprotein. After 15 min, samples were placed on ice and subsequently treated with proteinase K (50 μg/mL) or not for 15 min to remove nonimported proteins. Protease was inhibited by the addition of 2 mM PMSF. Mitochondria were reisolated and analyzed by SDS-PAGE and autoradiography. Control experiments were performed in the absence of membrane potential in the presence of 1 μM valinomycin and 20 μM oligomycin.