Figure 2.
Oxa1 associates specifically with newly synthesized Atp9. (A) In organello translation was performed in the presence of [35S]methionine for 30 min at 25°C in mitochondria isolated from the wild-type (WT) yeast strain, a strain overexpressing Oxa1 (GAL-Oxa1) and from a strain expressing histidine-tagged Oxa1 (GAL-Oxa1His). Incorporation of [35S]methionine was stopped by addition of excess unlabeled methionine, and the mitochondria were solubilized in SDS buffer and subjected to SDS-PAGE. The bands marked with an asterisk (*) indicated the complex formed by Oxa1 and a translation product. The positions of the molecular mass standards and mitochondria-encoded proteins, Var1, Cox1, Cox2, cytochrome b (Cytb), Cox3, Atp6, Atp8, and Atp9, are indicated. (B) In organello translation was performed in mitochondrial harboring overexpressed Oxa1 or Oxa1His as described in A. The complex formed between Oxa1His and the radiolabeled translation product(s) were purified by Ni-NTA chromatography and either loaded directly on the gel (−TCA) or after TCA precipitation (+TCA). The mobility of the SDS-resistant Oxa1His-Atp9 complex and the monomeric Atp9 protein after release by TCA precipitation are indicated.