Principles of alcohol oxidase assembly in Hansenula polymorpha

过氧化物体是存在于几乎所有真核细胞中的重要细胞器。在过去的十年中，已经确定了几种与过氧化体基质蛋白输入有关的蛋白质。大多数过氧化物体蛋白是作为具有酶活性的寡聚酶进口的。甲醇酵母中的乙醇氧化酶(AO)似乎是这一规则的例外，因为这种蛋白质是作为单体输入的，而活性八聚体的寡聚是在输入过氧体基质后发生的。最近有证据表明，丙酮酸羧基酶(PYC)在AO的输入和组装过程中是必不可少的。突变分析表明，PYC与FAD有亲和力，并能与AO蛋白发生物理作用。这表明，PYC具有双重作用，除了其作为去胸膜蛋白的代谢功能外，它还在AO分选和必需的过程中发挥着特殊的作用，可能是作为辅因子结合蛋白。这项研究旨在结合分子、生化和生物物理方法，详细阐明PYC在AO导入和组装中的作用的分子机制。

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.