Principles of alcohol oxidase assembly in Hansenula polymorpha

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Peroxisomes are important organelles that are present in almost all eukaryotic cells. During the last decade several proteins involved in peroxisomal matrix protein import have been identified. Most peroxisomal proteins are imported as enzymatically active, oligomeric enzymes. Alcohol oxidase (AO) in methylotrophic yeast appears to be an exception on this rule, because this protein is imported as a monomer, whereas oligomerization to the active octamer occurs after import in the peroxisomal matrix. Recently evidence was obtained that the enzyme pyruvate carboxylase (Pyc) is essential for import and assembly of AO. Mutational analysis revealed that Pyc has affinity for FAD and is capable to physically interact with AO protein. This suggests that Pyc plays a dual role in that, besides its well-characterized metabolic function as anapleurotic enzyme, the protein fulfils a specific role in AO sorting and essembly, possibly as a Co-factor binding protein. The research outlined in this proposal aims to elucidate the molecular mechanisms of the function of Pyc in AO import and assembly in detail using a combination of molecular, biochemical and biophysical approaches.