Immunocytochemical demonstration of the peroxisomal ATPase of yeasts

The presence of an ATPase on yeast peroxisomal membranes was studied by immunological methods. Western blot analysis of purified peroxisomal membranes from several yeasts revealed distinct cross-reaction with specific antibodies against the F1-part or the beta-subunit of the mitochondrial ATPase of Saccharomyces cerevisiae. This was not due to mitochondrial contamination as was demonstrated by analytical sucrose gradient centrifugation. Protein A-gold labelling carried out on Lowicryl-embedded methanol-grown Hansenula polymorpha using these antibodies did not result in significant staining. However, when organelles isolated from this yeast were successively incubated with antibodies and protein A-gold prior to embedding, specific labelling was observed on both the peroxisomal membrane and the membrane of damaged mitochondria but not on intact mitochondria. Specific labelling of the peroxisomal membrane was confirmed by freeze-fracture immunocytochemistry. In addition to the peroxisomal membrane, the mitochondrial membrane was also labelled in these experiments. Freeze-fracture immunocytochemistry was also successful for the localization of peroxisomal matrix proteins, e.g. alcohol oxidase and dihydroxyacetone synthase, and of mitochondrial membrane proteins, e.g. cytochrome c oxidase.