- 學位論文
酵母菌過氧化體ABC運輸蛋白Pxa1p的結構特性分析
Structural characterizations of peroxisomal ABC transporter Pxa1p in Saccharomyces cerevisiae
摘要
ABC運輸蛋白(ATP-binding cassette transporter)是一類膜蛋白家族，其功用在於幫助分子運送穿過生物膜。在人類過氧化體的膜上有四個ABC輸送蛋白已經被發現，其中之一稱為ALDP，當它發生突變時，會導致ALD (Adrenoleukodystrophy;腎上腺腦白質退化症)的遺傳性的疾病，其病因與過氧化體對極長鏈脂肪酸的beta氧化能力異常有關。這些輸送蛋白已經被證實可經由蛋白C端的相互作用形成同源及異源二聚體。酵母菌中也有兩個過氧化體的ABC輸送蛋白，稱為Pxa1p及Pxa2p，同樣是參與脂肪酸的β氧化作用，是ALDP的異物種同源同功蛋白。這兩個蛋白已經被證實會形成異源二聚體，然而，並無證據指明不會形成同源二聚體，因此，我們想要研究Pxa1p及Pxa2p是否具有形成同源二聚體的特性，先針對與ALDP相似性較高的Pxa1p進行研究。我們製備包含Pxa1p蛋白C端的重組蛋白Pxa1pC-HA，經鎳離子親合性管柱純化後，使用膠體過濾層析法來鑑定Pxa1pC-HA的特性，發現Pxa1pC-HA重組蛋白會形成由2~4單體所形成的複合體。根據這個研究結果，我們預測全長的Pxa1p可能也會形成相似的複合體。
並列摘要
ATP-binding cassette (ABC) transporters are members of a superfamily of membrane proteins involved in the transport of a variety of molecules across biological membranes. So far, four ABC transporters have been detected in mammalian peroxisomes, one of which, named ALDP, is specifically involved in the β-oxidation of the very long chain fatty acid. The marked genetic disorder ALD (adrenoleukodystrophy) was due to the mutation of the ALDP gene. It have been demonstrated that ALDP forms the homo-complex by itself and hetero-complex with other two peroxisomal ABC transporters via its C-terminal domain. In yeast, there are two orthologs of ALDP and PMP70 with the name of Pxa1p and Pxa2p, respectively. The results from biochemical and genetic studies have suggest that Pxa1p/Pxa2p can form hetero-dimers. However, the possibilities of the homo-dimer formation by Pxa1p/Pxa2p have not been excluded with certainty. So, we prepare the recombinant Pxa1pC-HA protein, containing C-terminal domain of Pxa1p, and use gel-filtration to determine its molecular weights. From the calculations of the molecular weights, we found that Pxa1pC-HA can form homo-complex composed of 2~4 monomers. Based on this study, we speculate full-length Pxalp may form homo-complex as Pxa1pC-HA.