Yeast strains and growth media
All strains used in this study (Table S1
) were isogenic to either the wild-type strain YPH499 (Sikorski and Hieter, 1989
) or W303. The strains carrying the intronless or the cob::ARG8m
mitochondrial genome were contributed by A. Tzagoloff (Columbia University, New York, NY), and the strain carrying the cox2::ARG8m
mitochondrial genome was contributed by N. Bonnefoy (Centre National de la Recherche Scientifique, Gif-sur-Yvette, France). His7- or ProteinAHis7-tagged variants of Mrpl4, Cbp3, Cbp6, Cbs1, and Cbs2 were generated by replacing the stop codons of the endogenous ORFs by a sequence encoding a heptahistidine or ProteinA-heptahistidine tag using a HIS3 selection cassette (Lafontaine and Tollervey, 1996
, and CBP1
were disrupted with a Kanamycin resistance cassette. Yeast cultures were grown at 30°C in lactate medium and YP (1% yeast extract and 2% peptone) medium supplemented with 2% dextrose, 2% galactose, or 2% glycerol.
Isolation of mitochondria
Yeast cells were grown to midexponential phase (OD595nm = 1.3), harvested by centrifugation (3,000 g for 5 min), washed once with distilled water, resuspended (2 ml/g of cell wet weight) in MP1 buffer (0.1 M Tris base and 10 mM dithiothreitol), and incubated for 10 min at 30°C. Cells were then washed once with 1.2 M sorbitol, resuspendend (6.7 ml/g of cell wet weight) in MP2 buffer (20 mM KPi, pH 7.4, 0.6 M sorbitol, and 3 mg/g of cell wet weight zymolyase 20T [Seikagaku Biobusiness]), and incubated, shaking for 1 h at 30°C to digest the cell wall (spheroplastation). Spheroplasts were harvested (3,000 g for 5 min at 4°C) and resuspended (13.4 ml/g of cell wet weight) in homogenization buffer (10 mM Tris, pH 7.4, 0.6 M sorbitol, 1 mM EDTA, 1 mM PMSF, and 0.2% Albumin bovine Fraction V, fatty acid free [Serva]). All subsequent steps were performed on ice; centrifugations were performed at 4°C. The spheroplast suspension was homogenized in two portions by 10–15 strokes of a Teflon plunger in a tight-fitting homogenizer (Sartorius Stedim Biotech S.A.). The homogenate was centrifuged at 3,000 g for 5 min, and the supernatants were combined. Centrifugation was repeated until no residual cell debris was visible. Then mitochondria were harvested by centrifugation at 17,000 g for 12 min. The pellet was resuspended in isotonic buffer (0.6 M sorbitol and 20 mM Hepes, pH 7.4) to give a final concentration of 10 mg/ml.
Construction of a mitochondrial genome with the COB coding sequence replaced by ARG8m
mitochondrial genome was constructed by A. Tzagoloff using PCR amplification of the 5′ and 3′ UTR of COB
using the primer pairs 5′-GGCGAATTCGATATCATTAATATTAATATAATCGTC-3′/5′-GGCGGATCCTGATTTTCTAAATGCCATATTATTATT-3′ and 5′-GGCGGATCCCGGTAGAGTTAATAAATAATATAT-3′/5′-GGGTCTAGAGATTCTATAATAATTATGCTTTATG-3′. The two fragments were amplified from mitochondrial DNA of the respiratory-competent haploid strain MR6 (Rak et al., 2007
) as the template. The PCR products were digested with EcoRI–BamHI and XbaI–BamHI, respectively, and ligated to the EcoRI and XbaI sites of pJM2 (Steele et al., 1996
). The resultant plasmid was termed pCOB/ST3. ARG8m
was amplified using the primer pair 5′-GGCGGATCCTTCAAAAGATATTTATCATCAAC-3′/5′-GGCGGATCCTTAAGCATATACAGC-3′ and cloned into the BamHI site of pCOB/ST3. The resultant plasmid pCOB/ST5 was introduced into the kar1-1
by biolistic transformation (Bonnefoy and Fox, 2007
) with the PDS-1000/He particle delivery system (Bio-Rad Laboratories). Transformants were selected for their ability to rescue the cox2
mutation of M9-94/A3 (Tzagoloff et al., 1975
). A transformant verified to have acquired the modified cob::ARG8m
gene (αDFS160/COB/ST5) was crossed to the ARG8
-deficient mutant MRSI0
, a derivative of MR6 and SDC22 (Duvezin-Caubet et al., 2003
; Rak et al., 2007
) that had previously been cytoduced with an intronless mitochondrial genome from strain MCC109 (a gift from P. Perlman, University of Texas Southwestern Medical Center, Dallas, TX). Cytoductants in which the coding sequence of COB
had been replaced by that of ARG8m
were identified by their ability to grow on media lacking arginine and by lack of respiratory growth.
Cross-linking and denaturing purification
Mitochondria from yeast strains expressing His7-tagged proteins were incubated in isotonic buffer (0.6 M sorbitol and 20 mM Hepes, pH 7.4). The membrane-permeable, noncleavable cross-linker MBS was dissolved in DMSO and used at a final concentration of 200 µM. DMSO without cross-linker served as a vehicle control. Cross-linking was performed at 25°C for 45 min. For analysis of cross-linking to newly synthesized proteins encoded in the mitochondrial genome, in organello translation was performed at 30°C as described in the Labeling of mitochondrial translation products in organello section. The membrane-permeable, cleavable cross-linker DSP was added 10 min after addition of [35S]methionine, and labeling was allowed to go on for another 30 min. Cross-linking was stopped by addition of 100 mM Tris/HCl, pH 8.0, and 100 mM β-mercaptoethanol and incubating the samples for 10 min at 25°C. Mitochondria were then reisolated by centrifugation at 25,000 g for 10 min at 4°C and for purification of His7-tagged proteins on nickel–nitrilotriacetic acid (Ni-NTA) beads lysed in 50 µl of 1% SDS and briefly heated at 98°C. Next, the sample was adjusted to 1 ml with binding buffer (1% Triton X-100, 300 mM NaCl, 20 mM imidazole, pH 7.4, and 20 mM KPi, pH 7.4), and the His-tagged proteins and their cross-linking partners were purified on Ni-NTA beads.
Ni-NTA purification was performed by incubating the respective samples with 10 µl Ni-NTA beads for 2 h at 4°C. The beads were then washed three times with binding buffer and eluted with binding buffer containing 500 mM imidazole, pH 7.4. In the case of DSP, the cross-linker was cleaved by 100 mM DTT for 30 min at 37°C. The samples were then separated via SDS-PAGE and analyzed by Western blotting and immunodetection or autoradiography.
Native purification of Cbs1, Cbs2, Cbp6, or Cbp3
Mitochondria from the wild type or mutants expressing ProteinAHis7-tagged Cbs1 or Cbs2 or His7-tagged Cbp6 or Cbp3 (1 mg each) were lysed for 30 min in 1 ml of buffer containing 1% Triton X-100 or 1% digitonin, 150 mM KCl, 20 mM Hepes/KOH, pH 7.4, 1 mM PMSF, 1× Complete Protease Inhibitor mix, 20 mM imidazole, pH 7.4, and 20% glycerol. Tagged Cbs1, Cbs2, Cbp6, or Cbp3 and complexed proteins were purified by incubating the respective samples with 10 µl Ni-NTA beads tumbling for 3 h at 4°C. The beads were then washed three times with lysis buffer containing 0.1% detergent and were eluted with lysis buffer containing 0.1% detergent and 500 mM imidazole, pH 7.4. Alternatively, Cbp3 was purified from mitochondrial digitonin lysates by immunoprecipitation using serum against Cbp3 or preimmune serum (as a negative control) and Protein A–Sepharose beads. When interactions with newly synthesized mitochondrially encoded proteins were analyzed, in organello translation was performed at 30°C as described in the Labeling of mitochondrial translation products in organello section, radioactive labeling was stopped by adding 8 mM of unlabeled methionine, mitochondria were reisolated, and immunoprecipitation of Cbp3 was performed. Beads were washed as previously described and an additional time with 20 mM Hepes/KOH, pH 7.4. Bound proteins were eluted with sample buffer. Samples were separated via SDS-PAGE and analyzed by Western blotting and/or autoradiography.
Labeling of mitochondrial translation products in vivo
Cells were grown on minimal medium containing 2% galactose, 20 µg/ml arginine, methionine, threonine, and tyrosine, 30 µg/ml isoleucine, 50 µg/ml phenylalanine, 100 µg/ml valine, and 0.1% glucose (Prestele et al., 2009
). A cell amount corresponding to an optical density (OD595nm
) of 0.5 was collected, washed twice with minimal medium containing 2% galactose, and incubated at 30°C for 10 min in the same buffer supplemented with 0.12 mg/ml of all amino acids except methionine. After inhibiting cytosolic protein synthesis with 0.15 mg/ml cycloheximide, labeling of mitochondrial proteins was started by addition of 6 µCi [35
S]methionine. Pulse labeling was stopped after the indicated time points by addition of lysis buffer (1.85 M NaOH and 1.1 M β-mercaptoethanol) containing 8 mM of unlabeled methionine. After incubation for 10 min on ice, proteins were precipitated with 14% TCA. The stability of mitochondrial translation products was chased by stopping a 15-min labeling reaction by the addition of 8 mM of unlabeled methionine and by taking samples after the indicated time points, which were lysed and TCA precipitated as previously described. Pellets were resolved in sample buffer, subjected to SDS-PAGE on 16/0.2% acrylamide/bisacrylamide gels, and analyzed by autoradiography and Western blotting.
Labeling of mitochondrial translation products in organello
Mitochondria were resuspended at a final concentration of 1 mg/ml in translation buffer (0.6 M sorbitol, 150 mM KCl, 15 mM KPi, pH 7.4, 20 mM Hepes, pH 7.4, 12.67 mM MgCl2, 4 mM ATP, 0.5 mM GTP, 5 mM phosphoenolpyruvate, 5 mM α-ketoglutarate, 12.13 µg/ml alanine, arginine, aspartic acid, asparagine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophane, tyrosine, and valine, 66.67 µM cysteine, and 10 µg/ml pyruvate kinase). After 5 min of incubation at 30°C, 4 µCi [35S]methionine/100 µg mitochondria was added, and incubation was pursued for 20 min. Labeling of mitochondrial translation products was stopped by the addition of unlabeled methionine to a final concentration of 8 mM, and mitochondria were reisolated by centrifugation (10,000 g at 4°C for 10 min).
Fractionation of mitochondrial lysates
300 µg of isolated mitochondria was lysed for 30 min on ice in lysis buffer containing 1% Triton X-100 or 1% digitonin, 50 or 150 mM KCl, 1× Complete Protease Inhibitor mix, 0.5 mM MgCl2, and 20 mM Hepes/KOH, pH 7.4. After a clarifying spin for 10 min at 25,000 g at 4°C, one half of the extract was precipitated with 12% TCA (100% total), and the rest was underlayered with 50 µl sucrose solution (1.2 M sucrose and 20 mM Hepes/KOH, pH 7.4) and centrifuged for 105 min at 200,000 g in a TLA100 rotor (Beckman Coulter) at 4°C. The supernatant was then collected, and the ribosome-containing pellet was resuspended in lysis buffer. Proteins of both fractions were precipitated with 12% TCA. The resulting pellets were dissolved in sample buffer, separated by SDS-PAGE, and analyzed by Western blotting.
Analysis of mitochondrial transcripts by Northern blotting
For RNA isolation, mitochondria were resuspended in AE buffer (50 mM sodium acetate, pH 5.3, and 10 mM EDTA, pH 8) and lysed by addition of 1% SDS. After addition of an equal volume of phenol, pH 4.5–5, the mixture was briefly heated (65°C), rapidly chilled on ice, and stored at −80°C until the phenol was frozen. Next, the aqueous and phenolic phase was separated by centrifugation (13,000 g for 5 min), and the aqueous phase was collected, mixed with an equal amount of phenol/chloroform/isoamylalcohol solution (25:24:1), and centrifuged again. The RNA was precipitated from the aqueous phase in the presence of 0.3 M sodium acetate, pH 5.3, and isopropanol (2 vol) by incubation at −20°C for at least 20 min and centrifugation at 16,100 g for 5 min. The RNA pellet was washed once with 80% ethanol, dried, and resuspended in diethylpyrocarbonate-H2O. For Northern blotting analysis, 2 µg mitochondrial RNA of each strain was separated on a denaturing 1.2% formaldehyde-agarose gel, washed with diethylpyrocarbonate-H2O and TBE (45 mM Tris, 45 mM borate, and 1 mM EDTA), and subsequently transferred to a nylon membrane. After blotting, the membrane was briefly washed in 2× SSC (0.3 M NaCl and 30 mM Na3-citrate, pH 7.0) and dried overnight. Before hybridization, RNA was UV cross-linked and prehybridized with Church buffer (1 mM EDTA, 7% SDS, 1% BSA, and 0.25 M NaPi, pH 7.2). RNA was hybridized with gene-specific 32P-labeled probes overnight and analyzed by autoradiography.
Analysis of the topology of Cbp3, Cbp6, and Cbp4
Alkaline extraction of proteins was performed from mitochondria resuspended in 0.1 M Na2CO3 and incubated for 30 min, shaking at 4°C. Membranes and associated proteins were separated by centrifugation at 30,000 g for 45 min. Proteins from the resulting supernatant were precipitated with 12% TCA. Membrane pellets were directly resuspended in sample buffer. Fractions were analyzed by SDS-PAGE followed by Western blotting.
Alternatively, membrane-spanning and -associated proteins were fractionated from soluble proteins by salt extraction. Mitochondria were resuspended in buffer containing 200 mM KCl and 20 mM Hepes/KOH, pH 7.4, and subjected to three cycles of freeze thawing in liquid nitrogen and 37°C. Membranes were collected by centrifugation, and the fractions were further processed as described for the alkaline extraction procedure.
To analyze the submitochondrial localization of proteins, mitochondria were either resuspended in isoosmotic (600 mM sorbitol and 20 mM Hepes/KOH, pH 7.4) or hypoosmotic (20 mM Hepes/KOH, pH 7.4) buffer to generate mitoplasts. Mitochondria or mitoplasts were then incubated with 100 µg/ml proteinase K for 30 min on ice or left untreated. The protease was inhibited by the addition of 1 mM PMSF, and mitochondria (or mitoplasts) were reisolated, washed three times with a buffer containing 600 mM sorbitol, 80 mM KCl, 20 mM Hepes/KOH, pH 7.4, and 1 mM PMSF, resuspended in sample buffer, and subjected to SDS-PAGE and Western blotting.
The antibody against the His7 tag was purchased from QIAGEN, and the antibody against the ProteinA tag was purchased from Sigma-Aldrich. Antibodies against Cbp3, Cbp4, and Cbp6 were obtained by immunizing rabbits with purified mature Cbp3His6, Cbp4(61–171), and MBP-Cbp6, respectively. The Arg8 antibody was a gift from T. Fox (Cornell University, Ithaca, NY).
Online supplemental material