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. 2004 Jan;15(1):310–322. doi: 10.1091/mbc.E03-07-0508

Figure 3.

Figure 3.

Overproducing Mhr1p enhances concatemer formation in mhr1-1 cells at 34°C. (A and B) One-dimensional gel electrophoretic analysis of mt-pMK2 DNA. The AFS984a-pMK2 (mhr1-1) cells, with the overproducing plasmid harboring the MHR1 gene (pYESTrp1MHR1) or without the ORF (pYESTrp1), were grown at 34°C in SD medium supplemented with all of the required amino acids, except tryptophan (lanes 2–8), from 1.0 × 105 cells/ml to 5.0 × 107 cells/ml. The cells were transferred to raffinose medium containing 2% galactose for the induction of the GAL1 promoter, and aliquots were obtained at the various times indicated in the figure during the incubation at 34°C. Whole cellular DNA was isolated and analyzed by one-dimensional electrophoresis without restriction endonuclease-treatment. (A) Gel stained with ethidium bromide. (B) Signals from the mt-pMK2 DNA, obtained by Southern hybridization by using the [32P]pMK2 DNA probe. CC, closed circular monomer; NC and m (region), nicked circular monomer; c (region), concatemers. Lane 1, 5-kb ladder as a size marker. Lane 9, the isolated plasmid pMK2 DNA. (C) Quantification of the signals in B. Based on the Southern hybridization experiment, the 32P signals of the pMK2 DNA probe from the areas indicated in B (m, circular monomers; c, concatemers) were plotted. ▪, □ (squares), concatemeric mtDNA; ⋄, ⋄ (diamonds), circular monomeric mtDNA. Closed symbols, the mhr1-1 cells containing the MHR1 expression plasmid; open symbols, the mhr1-1 cells containing the vector without MHR1. The net signals for circular monomers were corrected as described in Figure 2E. The experiment was performed twice to confirm that the results were reproducible. A single experimental result is indicated. (D) The proteins were extracted from the sampled cells (∼2 × 105) by the use of a yeast protein kit (Zymo Research) and were subjected to immunoblotting. Both Mhr1p and mhr1-1p in the extracts were detected with the antiserum against Mhr1p. The amounts of mitochondrial proteins are indicated relative to the amount of the mitochondrial outer membrane protein, porin, detected by its antiserum.