Results: 4

1.
Figure 3

Figure 3. From: Truncation of the Mrp20 protein reveals new ribosome-assembly subcomplex in mitochondria.

Isolation of the Mrp20ΔC subcomplex. (AD) Mitochondria isolated from WT or mrp20ΔC mutant expressing GAL10-driven Mrp20ΔC or Mrp20ΔCHis proteins were analysed by SDS–PAGE, western blotting and immunedecoration with Mrp20- or His-specific antisera (A), or subjected to detergent solubilization and Ni-NTA purification, western blotting and immunedecoration (B,D) or silver staining (C). Total, 5% of the solubilized mitochondria; Bound, the Ni-NTA eluted material. (EG) Mitochondria were isolated from WT, GAL10-Mrp20ΔC, GAL10-Mrp20ΔCHis strains and GAL10-Mrp20ΔC strains expressing the His-tagged MrpL25 or MrpL27 (E,F) or Prx1His (G) proteins, as indicated. Following detergent solubilization, samples were subjected to Ni-NTA purification and further analysed as described in (B). MW, molecular weight; Prx, peroxiredoxin; SDS–PAGE, SDS–polyacrylamide gel electrophoresis; WT, wild type.

Jasvinder Kaur, et al. EMBO Rep. 2011 September;12(9):950-955.
2.
Figure 4

Figure 4. From: Truncation of the Mrp20 protein reveals new ribosome-assembly subcomplex in mitochondria.

Non-assembled ribosomal proteins are membrane associated. (A) Isolated GAL10-mrp20ΔC or WT/ρ0 mitochondria were sonicated under different salt concentrations, and following the addition of Triton X-100 (1%), as indicated, were subjected to low-speed centrifugation. Membrane pellet (LP) and supernatant (S) fractions were analysed by SDS–PAGE, western blotting and immunedecoration. Note that the exposure times for the WT/ρ0 blots were longer because of the reduced levels of ribosomal proteins in this mutant (see Fig 2B). (B) Mitochondria isolated from GAL10-mrp20ΔC, Δyta10 and wild-type (WT/ρ+) strains were sonicated under low-salt conditions and subjected to membrane floatation assay. The resulting fractions were analysed further as described in (A). AAC, ADP/ATP carrier protein; SDS–PAGE, SDS–polyacrylamide gel electrophoresis; WT, wild type.

Jasvinder Kaur, et al. EMBO Rep. 2011 September;12(9):950-955.
3.
Figure 2

Figure 2. From: Truncation of the Mrp20 protein reveals new ribosome-assembly subcomplex in mitochondria.

The mrp20ΔC mutant has defective ribosome assembly. (A) Steady-state levels of ribosomal proteins in isolated mitochondria (50 μg) of the indicated strains. (B) Detergent-solubilized mitochondria from WT and GAL10-mrp20ΔC cells were subjected to sucrose-density sedimentation analysis. Immunodecoration of the resulting fractions (numbered from top to bottom) for indicated proteins was performed. (C) Haploid (mating type a) strains were crossed to a haploid WT/ρ0 (mating type α) strain, followed by growth on YPD and YPG media (see Fig 1B). The Δoxa1 (YPG+) and the WT/ρ0 (mating type a) strains were used as controls. (D) Detergent-solubilized GAL10-mrp20ΔC mitochondria were subjected to shallow sucrose-density sedimentation analysis using 5–25% sucrose gradients, and the resulting fractions were analysed further as described in A. The fractionation behaviour of control marker protein complexes harvested from a parallel sucrose gradient is indicated above with their mass (kDa). WT, wild type; YPD, YP plates containing glucose; YPG, YP plates containing glycerol.

Jasvinder Kaur, et al. EMBO Rep. 2011 September;12(9):950-955.
4.
Figure 1

Figure 1. From: Truncation of the Mrp20 protein reveals new ribosome-assembly subcomplex in mitochondria.

The C-terminal region of Mrp20 protein is important for mitochondrial translation and oxidative-phosphorylation function. (A) The conserved L23 domain and the C-terminal mitospecific region that are truncated in the Mrp20ΔC derivative are indicated. (B) Serial 10-fold dilutions of WT, mrp20ΔC and GAL10-Mrp20ΔC cells were spotted on YPD, YPG or YPG supplemented with the minimal amount of galactose required to induce the GAL10 promoter (YPG+0.1% Gal) and grown at 30 °C. (C) In organello translation was monitored in WT, mrp20ΔC and GAL10-Mrp20ΔC mitochondria. Cox1, Cox2 and Cox3 of the cytochrome c oxidase; Atp6, Atp8 and Atp9 subunits of the F1F0-ATP synthase. (D) Steady-state levels of the OXPHOS subunits in the mitochondria (50 μg) isolated from the indicated strains. Tim17 was used as a loading control. Cytb, cytochrome b; FeS, Rieske FeS protein; OXPHOS, oxidative phosphorylation; Su e, subunit e of the F1F0-ATP synthase; WT, wild type; YPD, YP plates containing glucose; YPG, YP plates containing glycerol.

Jasvinder Kaur, et al. EMBO Rep. 2011 September;12(9):950-955.

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