2026-11-092026-11-09https://scholars.csmu.edu.tw/handle/123456789/38689ATP-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, PMP70 and ALDRP, individually. In yeast, there are two orthologs of ALDP and PMP70 with the name of PXA1 and PXA2, respectively. The results from biochemical and genetic studies have suggest that the hetero-dimers of PXA1/PXA2 are involved in β-oxidation of the LCFA. Nevertheless, most (if not all) evidences for the dimers formation of these ABC tranporters in yeast and human came from co-immunoprecipitation, pull down assay, genetic interaction and protein stability dependence, and the results of above assay can’t fully prove that these proteins form a dimer, due to inability to precisely determine the molecular weights of the protein complex. Additionally, there have no evidences, indicating that PXA1 and PXA2 can’t form a homo-dimers. From our preliminary data in previous research project funded by NSC, a recombinant protein, containing C-terminal region of PXA1, form a homo-complex, consisted of about 2~6 monomers as determined by gel filtration assay. So, based on the preliminary result, I propose a new three-year research project to explore the structural and functional properties of protein complex formed by PXA1 and PXA2. First year, I will purify the native homo- and hetero-complexes of PXA1 and PXA2 directly from yeast, determine the molecular weights of protein complexes and estimate the number of monomers in a complex. Whether PXA1 and PXA2 have a functional redundancy with each other will be detect by genetic complementation test. The second year, I will determine the ratio of homo- and hetero-complexes existed in physiological condition in yeast by immuno-depletion, and conclude the importance of two different kinds of complex. The putative ATPase activity of PXA1 and PXA2 in forms of homo- and hetero-complex will be proved by ATPase functional assay using recombinant PXA1 and PXA2. Whether the homo- and hetero-complex are stable or transient structures in the transport process will be determine by incubation of the complex with ATP, ADP and non-hydrolysable analog, following gel filtration analysis. The third year, because C-terminal region (contain 169 amino acids) of PXA1 is required for homo-interaction, I will divide this region into four sub-domains, and determine which one is required for homo- and hetero-interaction by yeast two-hybrid and GST pull down assay. From the results of above project, we will unequivocally reveal the structural and functional relationships of homo- and hetero-complexes formed by PXA1 and PXA2, and the new knowledge from our researches will be applicable to other peroxisomal ABC transporter by other scientists.ABC 運輸蛋白(ATP-binding cassette transporter)是屬於一個很大的蛋白 家族，此蛋白家族成員通常為膜蛋白，一般的功能在於協助離子、小分子或 蛋白質等物質穿越膜的運輸過程。目前已知人類過氧化體膜上有四個ABC 運輸蛋白(ATP-binding cassette transporter)，其中的ALDP 參與極長鏈脂肪酸 之b氧化作用，當此蛋白基因發生變異時，會導致腎上腺腦白質退化症(簡稱 為ALD)。目前的證據指出，ALDP 與另外兩個過氧化體ABC 蛋白(ALDRP 及PMP70)會形成同源及異源二聚體。酵母菌中也有ALDP 及PMP70 的同 源性蛋白，分別為PXA1 及PXA2。生化及遺傳的證據指出，PXA1 及PXA2 是以異源二聚體參與長鏈脂肪酸的b氧化作用。然而，來自人類及酵母菌的 研究中，有關同源及異源二聚體的證據不外乎是：免疫共沈澱分析、pull down 分析、酵母菌雙雜交實驗、單獨突變及雙重突變的組合分析、蛋白質穩定的 相關性，這些方法無法測定分子量而不能完全證明是否為二聚體。此外，在 酵母菌中並無證據指出PXA1 及PXA2 不會形成同源二聚體。 我們在上一個針對PXA1 及PXA2 的計畫中所獲得的初步結果指出，只 包含PXA1 蛋白C 端之重組蛋白，經膠體過濾層析法證實可形成由2~6 個 單體蛋白所組成的同源複合體。因此，我依據這個結果為出發點，提出了以 下的三年計畫，繼續來探討PXA1 及PXA2 所形成的複合體之結構及功能的 關係。 第一年、(1)從酵母菌中直接純化由PXA1 及PXA2 所組成的同源及異 源複合體，並測定複合體的分子量，推算複合體所包含的分子數。(2)以互 補測試的實驗，證明PXA1 及PXA2 是否為功能重複的蛋白。 第二年、(1)以免疫耗盡試驗(immuno-depletion)評估同源及異源複合體 所佔的比率，以此評估同源及異源複合體的重要性。(2)利用純化的重組蛋 白來測試其ATPase 的活性，證明PXA1 及PXA2 是否具有典型ATPase 的 活性。(3)測試複合體是一種平時就存在且穩定的結構，還是受ATP 及被運 輸物質控制的非穩定性結構。 第三年、以(1)酵母菌雙雜交實驗及(2)GST 融合蛋白pull down 分析法定 出PXA1 蛋白C 端169 個氨基酸中是哪一個區域參與同源及異源蛋白間的 相互作用。 本研究完成後，將是第一次清楚鑑定酵母菌過氧化體ABC 運輸蛋白之 複合體的大小、分子組成及相互作用區域，對於人類或其他生物的過氧化體 ABC 運輸蛋白的特性研究，也有很高的參考價值。過氧化體ABC 運輸蛋白蛋白複合體PXA1PXA2peroxisomal ABC transporterprotein complexPXA1PXA2Project