FIG. 7.

Yhm2p is associated with mitochondrial nucleoids. (A) Sucrose gradient centrifugation of mitochondrial nucleoids. Mitochondria from wild-type yeast cells were lysed in 0.5% NP-40 and centrifuged. The pellet was loaded onto a sucrose gradient and centrifuged. The peak fraction containing mtDNA was reloaded onto a sucrose gradient and centrifuged as described in Materials and Methods. The gradient fractions were analyzed for mtDNA and protein by Southern blotting and the Bradford assay, respectively. (B) The protein profiles of the fractions from second sucrose gradient centrifugation were analyzed by SDS-PAGE (15% polyacrylamide). SM, size markers; the numbers indicate fraction numbers. The presence of Yhm2p and CoxIIIp in the purified mitochondrial nucleoids was determined by Western blot analysis. The anti-CoxIIIp antibody was used as a negative control. As shown in Fig. 4, the anti-CoxIIIp antibody used in this study was active and detected CoxIIIp in the inner mitochondrial membrane fraction.