FIGURE 6.

Polymerization of Yfh1 in response to heat stress. A, mitochondria were isolated under the same conditions used in Fig. 5 (A–C), and the total mitochondrial lysates (T) were extracted with 100 mm Na₂CO₃ or 1% dodecyl maltoside for 30 min on ice prior to separation into supernatant (S) and pellet (P). B, total mitochondrial lysates (4.5 mg of total protein/ml) were left untreated or extracted with 1% dodecyl maltoside for 20 or 60 min on ice. After centrifugation at 10,000 × g for 20 min at 4 °C, aliquots of the supernatants were analyzed by blue native PAGE and Western blotting (WB) with anti-Yfh1 antibody. Purified monomer, trimer, and 24-mer (α, α₃, and α₂₄) were used as molecular mass standards. In another experiment, total mitochondrial lysates were incubated in the presence of low concentrations of SDS followed by centrifugation at 5,000 × g for 20 min. Two identical sets of samples from the resulting supernatants were analyzed by native PAGE (3–12% Bis-Tris) on the same gel. One half of the gel was stained with Prussian blue for iron detection, and the second half was analyzed by Western blotting for Yfh1 detection. C and F, yeast cells were preconditioned in SD and further grown in 1,500 ml of SD in the presence of 100μm FeCl₃ for ∼24 h at 37 °C. The mitochondria were isolated, and a soluble mitochondrial fraction (1.5 mg of total protein) was analyzed by size exclusion chromatography and Western blotting as described in the legend of Fig. 3 (C–F). Because of the very low levels of soluble Yfh1 present in mitochondria under the conditions used in these experiments, long exposures were required for Yfh1 detection resulting in high backgrounds.