Chen et al. 10.1073/pnas.0703078104. |
Table 1. Genotypes and sources of yeast strains used in this study
Strain name | Genotype | Source |
4741/∆cce1 | as BY4741, but cce1∆::kan | Research Genetics |
4741/∆rpo41 | as BY4741, but rpo41∆::kan | Research Genetics |
4741/∆rtg3 | as BY4741, but rtg3∆::kan | Research Genetics |
α MCC109HS40 | MAT α, ade2, ura3, kar1-1, [HS40] | This laboratory |
α MCC109VAR1 | MAT α, ade2, ura3, kar1-1, [VAR1] | This laboratory |
BY4741 | MAT a, met15∆0, his3∆0, leu2∆0, ura3∆0 | Research Genetics |
CS615-7D | MAT α, met15∆0, his3∆0, leu2∆0, ura3∆0, rtg1∆::kan, | This study |
mks1 ∆::kan | ||
CS654/2-6C | MAT a, lys2∆0, his3∆0, leu2∆0, ura3∆0, rtg1∆::kan, | This study |
ade2::ADH1-ACO1-URA3 | ||
CS655/3 | MAT a, met15∆0, ura3∆0, leu2∆0, his3∆0, [HS40] | This study |
CS657/1 | MAT a, lys2∆0, ura3∆0, leu2∆0, his3∆0, | This study |
mks1 ∆::kan, [HS40] | ||
CS686-3C | MAT α, lys2∆0, his3∆0, leu2∆0, ura3∆0, rtg1∆::kan | This study |
CS699/1 | as BY4741, but ade2::ACO1-URA3 | This study |
CS745-2C | MAT a, met15∆0, his3∆0, leu2∆0, ura3∆0, aco1∆::kan, | This study |
ade2::GAL10-ACO1-URA3 | ||
CS776/1 | MAT a, met15∆0, his3∆0, leu2∆0, ura3∆0, aco1∆::kan, | This study |
ade2::GAL10-ACO1-URA3 , [HS40] | ||
CS777/2 | MAT a, met15∆0, his3∆0, leu2∆0, ura3∆0, aco1∆::kan, | This study |
ade2::GAL10-ACO1-URA3 , [VAR1] | ||
CS880-4A | MAT a, met15∆0, ura3∆0, leu2∆0, his3∆0, aco1∆::kan, | This study |
rpo41 ∆::kan | ||
CS844/2 | a cytoductant of RG∆aco1-2B containing the ρ- HS40 | This study |
genome at low copy number | ||
CS910 | as 4741/∆rpo41, but [VAR1] | This study |
CS911 | as CS880-4A, but [VAR1] | This study |
CS914-6D | MAT α, lys2∆0, his3∆0, leu2∆0, ura3∆0, cce1∆::kan, | This study |
rtg1 ∆::URA3 | ||
CS915-1B | MAT α, lys2∆0, his3∆0, leu2∆0, ura3∆0, cce1∆::kan, | This study |
rtg3 ∆::kan | ||
CS954-3B | MAT α, lys2∆0, his3∆0, leu2∆0, ura3∆0, cce1∆::kan, | This study |
mks1 ∆::LEU2 | ||
CS956 | MAT a/α, met15∆0/+, lys2∆0/+, his3∆0/+, leu2∆0/+, | This study |
ura3 ∆0/+, pif1∆::kan/+, mks1∆::LEU2/+ | ||
CS998/1 | as RG∆pif1-5A, but ade2::ADH1-ACO1-URA3 | This study |
CS1304 | MAT a/α, met15∆0/+, lys2∆0/+, his3∆0/+, leu2∆0/+, | This study |
ura3 ∆0/+, aco1∆::kan/+, ade2::aco1R476S-URA3/+ | ||
CS1309 | MAT a/α, met15∆0/+, lys2∆0/+, his3∆0/+, leu2∆0/+, | This study |
ura3 ∆0/+, aco1∆::kan/+, ade2::aco1R668S-URA3/+ | ||
CS1337/2 | as 4741/∆rtg3, but ade2::ADH1-ACO1-URA3 | This study |
CS1338-4C | MAT a, met15∆0, his3∆0, leu2∆0, ura3∆0, rrm3∆::kan | This study |
CS1338-4D | MAT α, lys2∆0, met15∆0, his3∆0, leu2∆0, ura3∆0, | This study |
rrm3 ∆::kan, rtg1∆::URA3 | ||
RG∆aco1p-2B | MAT a, met15∆0, lys2∆0, his3∆0, ura3∆0, leu2∆0, | This study |
aco1 ∆::kan | ||
RG∆mks1-6C | MAT a, lys2∆0, his3∆0, leu2∆0, ura3∆0, mks1∆::kan | This study |
RG∆pif1-5A | MAT a, met15∆0, his3∆0, leu2∆0, ura3∆0, pif1∆::kan | This study |
SI Text
We found that inactivation of NUC1 and DIN7, which encode mitochondrial nucleases (1, 2), failed to suppress the loss of mtDNA in an aco1D mutant, ruling out the possibility that Aco1p protects mtDNA from attack by these endogenous nucleases. Moreover, neither the inactivation of NTG1, OGG1, and APN1, involved in base excision repair of oxidatively damaged mtDNA, nor the disruption of SOD2, encoding the mitochondrial manganese superoxide dismutase, further destabilizes mtDNA in the rtg1D mutant with reduced ACO1 expression. Single mutants disrupted in these four genes have very mild or no detectable phenotype with respect to petite formation (3, 4).
Materials and Methods
Strain and Plasmid Construction.
The R476S and R668S mutants of ACO1 were generated by changing the codons 476 and 668 for arginine from AGA, CGT to AGT that codes for serine. The aco1R476S and aco1R668S alleles were cloned into a URA3-based integrative vector, pURA3-ade2, that contains sequences from the ADE2 locus truncated at both the amino and carboxyl termini of its open reading frame. The resulting plasmids were linearized by digestion with HpaI and integrated into the ADE2 locus. This generated two truncated copies of ade2 that can be readily identified by adenine auxotrophy and the associated red color in haploid strains. For ectopic expression of ACO1, the open reading frame was amplified by PCR, placed under the control of the ADH1 or GAL10 promoter. The expression cassettes were cloned into pURA3-ade2 for chromosomal integration. The expression levels of Aco1p were monitored by Western blot using a specific antibody against Aco1p (which was kindly provided by R. Lill).
Cytoduction.
For cytoduction, aMCC109HS40 and aMCC109VAR1 were used as donors for the HS40 and VAR1 r- genomes. All of the recipient strains express the kan gene that confers resistance to G418. Exponentially growing donor and recipient strains were mixed in YPD medium at a ratio of 10:1, mated, and over-grown at 30°C for 15 h. Cells were then diluted in water and plated on YPD plus G418 plates for selecting recipient cells. G418-resistant colonies were examined for auxotrophic markers, and the presence of the r- genomes was confirmed by colony hybridization and Southern blot analysis.
Two-Dimensional Agarose Gel Electrophoresis (2DAGE).
The strains CS655/3 (ACO1, [HS40]) and CS657/1 (mks1D, [HS40]) were grown in YPD overnight, and cells were harvested and grown in fresh YPD for another 8 h before being subjected to DNA extraction. For Aco1p-depletion, the strain CS776/1 (GAL10-ACO1, [HS40]) was grown in 500 ml of YPGal at 30°C for overnight. The cells were harvested and washed with water. Half of the cells were inoculated in 500 ml of fresh YPD (10% glucose) and allowed to grow for another 24 h to repress the expression of GAL10-ACO1. This growth corresponds to about 3-4 doublings, after which cells are depleted of Aco1p but partially retain the HS40 r- genome allowing 2DAGE analysis.
Expression and Purification of Aco1p Variants from E. coli.
For expressing the GST-Aco1 fusion, the ACO1 ORF lacking the first 16 codons was cloned into the pGEX-3X expression vector to generate an in-frame fusion of ACO1 with the N-terminal GST. PCR fragments harboring ACO1, and its aco1R476S, aco1R668S and aco1C448S variants, were cloned into the pRSF-Duet-1 expression vector (Novagen) to create His6 fusions on their N terminus. His6-Aco1DD4 was generated by an in-frame fusion between His6 and Aco117-576 lacking the entire domain 4 of aconitase. The fusion proteins were produced in the E. coli BL21-Codonplus (DE3)-RIL strain upon IPTG induction at 22°C for overnight. The His6-tagged proteins were purified by Ni-NTA agarose (Qiagen), while the GST-fused protein was purified with Glutathione Sepharose 4B (Amersham).
Gel Retardation Assay and Southwestern Analysis.
For electrophoretic mobility-shift assays, oligonucleotide probes were end-labeled with [g-32P]ATP (6,000 Ci/mmol; PerkinElmer) using T4 Polynucleotide kinase (3'-phosphatase-free; Roche). Recombinant proteins were mixed with 12.5 fmol of [g-32P]ATP (6,000 Ci/mmol; PerkinElmer) end-labeled oligonucleotide probes in the binding buffer of 20 mM Tris·HCl (pH 7.4), 50 mM NaCl, 4 mM MgCl2, 1 mM EDTA (pH 8.0), 12% glycerol, 1 mM DTT, 300 mg/ml BSA, and 1 mM PMSF, and incubated at room temperature for 30 min. The samples were then loaded onto a pre-run 4% native acrylamide gel containing 2.5% (vol/vol) glycerol, in 0.5´ TBE buffer at 4°C.
Conditions for Southwestern analysis are modifications of a protocol previously described by Tomaska and Nosek (5). 0.125-2.0 mg of the recombinant proteins were dissociated in SDS/PAGE sample buffer [45 mM Tris·HCl (pH 6.8)/10% glycerol/1% SDS/0.01% bromophenol blue/50 mM DTT] at 42°C for 15 min. The proteins were resolved on a SDS/4-20% PAGE gel in 25 mM Tris·HCl/192 mM glycine/0.1% SDS (pH 8.3) at a constant voltage of 120 V for 1.5-2 h. Separated proteins were then electrotransferred to Protran BA85 nitrocellulose (Schleicher & Schuell) in buffer containing 12 mM Tris, 96 mM glycine, and 20% (vol/vol) methanol at 5-10 V overnight using the Criterion Blotter (Bio-Rad). The membrane was incubated in the DNA-binding buffer [10 mM Tris·HCl (pH 7.4)/1 mM EDTA (pH 7.4)/50 mM NaCl] containing 0.05% Tween 20 for 30 min at 4°C. The membrane then was hybridized at room temperature for 1-2 h in the DNA-binding buffer containing 2% PVP-40 and 0.25 pmol/ml [g-32P]ATP (6,000 Ci/mmol; PerkinElmer) end-labeled oligonucleotide probes. The membrane was washed three times for 30 min each in the DNA-binding buffer including 2% PVP40 before being autoradiographed.
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