Fig 1.
Scheme of mitochondrial isolation from yeast using magnetic beads.
Yeast cells expressing Tom70-6xHis are converted to spheroplasts by incubation with Zymolyase, and disrupted using a Dounce homogenizer. The resulting whole-cell lysate is subjected to low-speed centrifugation to remove intact cells, cellular debris and nuclei, followed by high-speed centrifugation of the supernatant to concentrate mitochondria. We refer to the resuspended pellet from the high-speed centrifugation as the mitochondria-enriched fraction. The mitochondria-enriched fraction is the starting point for further purification of mitochondria. For affinity purification, the mitochondria-enriched fraction is incubated with HisPur Ni-NTA magnetic beads. Bead-bound mitochondria are separated from debris and other organelles in a magnetic field and eluted from beads with 500 mM imidazole.
Fig 2.
Cellular and mitochondrial fitness are not affected by tagging Tom70 with 6xHis.
(A) The growth rate of yeast expressing untagged or 6xHis-tagged Tom70 was monitored by measuring the optical density of the culture in YPD medium at 600 nm (OD600) every 20 min for 3 days. (B) Maximum growth rate was calculated from the maximum slope of the growth curve in mid-log phase. There was no significant difference in growth rate of yeast expressing untagged or 6xHis-tagged Tom70 (two-tailed t-test). (C) The respiration-driven growth of yeast expressing untagged or 6xHis-tagged Tom70. Yeast were spotted in 10-fold serial dilutions to YPG plates and incubated at 30°C for 5–7 days. (D) Representative images of mito-roGFP1 in wild-type (WT), Tom70-6xHis cells, and cells treated with 5 mM H2O2 or DTT. The ratio of the reduced to oxidized roGFP signals is shown in heat maps superimposed on a transmitted-light image. Warmer colors represent more reducing enviroments and cooler colors represent more oxidizing environments. Scale bar = 5 μm. (E) Quantification of mitochondrial redox state using mito-roGFP. H2O2 and DTT treatments generate highly oxidized and reduced mito-roGFP1, respectively, and illustrate the dynamic range of the sensor. No significant difference was detected between wild-type and Tom70-6xHis cells (Kruskal-Wallis test with Dunn’s multiple comparisons test). Number of cells analyzed: WT = 160; Tom70-6xHis = 139; H2O2 = 173; DTT = 160. (F) Quantification of mitochondrial redox asymmetry between bud and mother cells of wild type and Tom70-6xHis cells. Buds have a more reduced mitochondrial redox state compared to mother cells in both wild-type and Tom70-6xHis cells (Wilcoxon matched-pairs signed rank test, ****p < 0.0001). Number of cells analyzed: WT = 154; Tom70-6xHis = 130.
Fig 3.
Magnetic bead-purified mitochondria have less contamination compared to crude mitochondria.
(A) Tom70, Porin, cytochrome b2 (Cyb2), ketoglutarate dehydrogenase (Kgd1), hexokinase (Hxk), Sec61, and Nyv1 were probed using Western blot analysis of whole-cell lysate, post-mitochondrial supernatant, mitochondria-enriched fraction, crude mitochondria, and magnetic bead-purified mitochondria. Tom70, Porin, Cyb2, and Kgd1 are markers for mitochondrial outer membrane, intermembrane space, and matrix, respectively. Hxk, Sec61 and Nyv1 are markers for cytosol, ER, and vacuole, respectively. Total protein load was assessed using TCE. (B—H) Quantification of fold enrichment of marker proteins shown in (A). Mitochondrial proteins are enriched in bead-purified mitochondria compared to cell lysate, and ER, cytosol, and vacuole contaminants are reduced compared to crude mitochondria (one-way ANOVA with Sidak’s multiple comparisons test). Results shown are representative of 6 trials.
Fig 4.
Cryo-electron tomographic analysis of mitochondria reveals preserved ultrastructure.
(A and B) Crude and (C and D) magnetic bead-purified mitochondrial preparations were analyzed by cryo-electron tomography. Representative mitochondria are shown. Two-dimensional slices were taken through the middle of the tomographic volumes (left), and three-dimensional models (right) were generated by manual segmentation. Key mitochondrial structural features are indicated, including mitochondrial outer membrane (MOM), crista, matrix, and mitochondrial inner membrane (MIM). Scale bars = 200 nm.
Fig 5.
Magnetic bead-purified mitochondria are intact and have robust COX and mitochondrial protein import activity.
(A) Crude or bead-purified mitochondria were incubated in the presence or absence of proteinase K (PK, 100 μg/ml) for 30 min at 4°C. Levels of mitochondrial outer membrane proteins (Tom70 and Porin) and an intermembrane space protein (Cyb2) were determined using Western blot analysis. A representative blot is shown. (B) Quantification of the experiment shown in (A). For each purification method, the amount of the indicated protein remaining after proteinase K treatment is shown, normalized to the level in the untreated sample (one-way ANOVA with Sidak’s multiple comparisons test). (C) Import of 35S radiolabeled Su9-DHFR into crude and bead-purified mitochondra was carried out as described in Materials and Methods. TCE staining of 10% of the crude and bead-purified mitochondria is shown as a loading control. PK, proteinase K; Val, valinomycin; p, precursor protein; m, mature protein; CM, crude mitochondria; BPM, bead-purified mitochondria. (D) COX activity was measured as the decrease in absorbance at 550 nm resulting from the oxidation of reduced cytochrom c and expressed in micromoles of cytochrome c oxidized/minute/milligram of mitochondrial protein (two-tailed t-test).