Principles of alcohol oxidase assembly in Hansenula polymorpha

过氧化物酶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，已经鉴定了几种参与过氧化物酶体基质蛋白输入的蛋白质。大多数过氧化物酶体蛋白是作为具有酶活性的寡聚酶输入的。甲醇营养型酵母中的醇氧化酶（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，而在过氧化物酶体基质中输入后发生寡聚化为活性八聚体。最近的证据表明，丙酮酸羧化酶（Pyc）是必不可少的进口和组装AO。突变分析显示Pyc对FAD具有亲和力，并且能够与AO蛋白物理相互作用。这表明Pyc发挥双重作用，除了其作为anapleurotic酶的充分表征的代谢功能之外，该蛋白质在AO分选和鉴定中发挥特定作用，可能作为辅因子结合蛋白。本提案中概述的研究旨在使用分子，生物化学和生物物理方法的组合详细阐明Pyc在AO导入和组装中功能的分子机制。

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.