Figure 8.

Sedimentation of Atp23p-HA in a sucrose gradient. Mitochondria prepared from W303ΔATP23/ST13 at a protein concentration of 10 mg/ml were adjusted to 1% potassium deoxycholate and 1 M KCl. After centrifugation at 105,000 × g_av for 15 min, the clear supernatant (0.25 ml) was diluted with an equal volume of 10 mM Tris-Cl, pH 7.5, containing 1.5 mg hemoglobin and 100 μg of bovine l-lactate dehydrogenase. The mixture was layered on top of 5 ml of a 7–25% linear sucrose gradient prepared in 10 mM Tris-Cl, pH 7.5, and 0.1% Triton X-100. The gradient was centrifuged in a Beckman SW65 rotor (Fullerton, CA) at 65,000 rpm for 5 h and fractionated into 15 equal-size fractions. The fractions were assayed for hemoglobin at 410 nm (○—○) and for lactate dehydrogenase by pyruvate-dependent oxidation of NADH at 340 nm (•—•). The distribution of Atp23p-HA was determined by Western analysis of the fractions with the mouse mAb against the hemagglutinin tag as in Figure 8. The size of Atp23p was estimated from the positions of the peak relative to those of the markers (Martin and Ames, 1961).