Figure 6.
Oxa1 interacts with Atp9 before its assembly into an F1Fo-ATP synthase complex. In organello translation in the presence of [35S]methionine was performed in mitochondria bearing overexpressed Oxa1 or Oxa1His (Oxa1 or Oxa1His) or in mitochondria harboring a histidine-tagged Atpβ subunit (AtpβHis), as indicated. After a chase reaction as described in Figure 2, the mitochondria were reisolated and lysed in Triton X-100 containing buffer and subjected to Ni-NTA chromatography, as described in Figure 5D. The Ni-NTA bound material (indicated by B) and a sample corresponding to 10% of the total material applied to the Ni-NTA beads were analyzed by SDS-PAGE, Western blotting, and autoradiography (right panels, indicated by 35S), followed by immunedecoration with Atp9 and Atp6 specific antisera (α-Atp9 and α-Atp6, respectively; left panel). The electrophoretic positions of Atp6, Atp8, and monomeric Atp9 are indicated. The position of the 72-kDa Oxa1-Atp9 SDS-resistant oligomer is indicated by an asterisk (*).