酵母菌過氧化體ABC運輸蛋白Pxa2p可形成同源二聚體

The Yeast Peroxisomal ABC Transporter Pxa2p, a Human ALDP Homolog, Forms a Homodimer

王建程(Chien-Cheng Wang)

指導教授 ： 蔡榮宗

中山醫學大學/醫學院/生化暨生物科技研究所/碩士(2009年)

https://doi.org/10.6834/CSMU.2009.00009

摘要

不論在人類或酵母菌中，過氧化體在β氧化作用中扮演一個重要的角色。過氧化體膜上有特定的ABC運輸蛋白(ATP-binding cassette transporter)已經被鑑定出來，而且和人類的遺傳疾病ALD (Adrenoleukodystrophy;腎上腺腦白質退化症)息息相關。人類過氧化體膜中相關的ABC運輸蛋白，包括ALDP和PMP70，酵母菌( Saccharomyces cerevisiae )中同樣也有跟ALDP及PMP70同功異物種的蛋白：Pxa1p和Pxa2p。ALDP證實會形成同源二聚體或與其它過氧化體膜上蛋白形成異源二聚體，參與β氧化作用的執行。Pxa1p同源於ALDP同樣也被證實會與Pxa2p形成異源二聚體，在酵母菌中負責極長鏈脂肪酸的運送。同源於Pxa2p的PMP70被證實會形成同源二聚體或多聚體，這對於Pxa2p是否也會形成同源二聚體或多聚體的研究，是非常有意義的。實驗中在E.coli內利用IPTG誘導的方式表現His-Pxa2pC1-HA蛋白，進一步以8 M Urea denature透析法、鎳離子親和性管柱層析法純化以及膠體過濾層析法分析His-Pxa2pC1-HA蛋白的分子量，但His-Pxa2pC1-HA蛋白呈現不可溶的形式。接著，以同樣的方式表現His-Pxa2pC2-HA蛋白，並在透析時加入Triton-X 100，再以膠體過濾層析法分析，發現Triton-X 100會干擾膠體過濾層析法的分析。在酵母菌中，用免疫共沉澱的方式分析具不同蛋白標籤的Pxa2p之間的相互作用，證實酵母菌過氧化體膜上的Pxa2p可以形成同源二聚體或多聚體的能力。

關鍵字

腎上腺腦白質退化症 ； ABC運輸蛋白 ； 過氧化體

並列摘要

Peroxisomal beta-oxidation play an important role in mammalian and yeast. The ABC transporter (ATP-binding cassette transporter) of peroxisomal membrane has been identified that is closely related to human ALD (Adrenoleukodystrophy). So far four ABC transporter have been detected in mammalian peroxisomes that contains four ABC transporters named ALDP (Adrenoleukodystrophy protein), ALDRP (ALD-related protein), PMP70 (The 70-kDa peroxisomal membrane protein) and PMP69 (The 69-kDa peroxisomal membrane protein). Saccharomyces cerevisiae contains two peroxisomal half-ABC transporters named Pxa1p (peroxisomal ABC transporter1 protein) and Pxa2p (peroxisomal ABC transporter2 protein). The ALDP and PMP70 are homolog Pxa1p and Pxa2p. ALDP are located in the peroxisome, where function as homo- and/or heterodimers in the regulation of very long chain fatty acid transport. The yeast Pxa1p and Pxa2p dimerize to form a functional transporter involved in very long chain fatty acid oxidation in the peroxisome. The formation of PMP70 assembles as dimeric or oligomeric forms on peroxisomal membranes implies that Pxa2p may form a homodimers. We used IPTG to induce His-Pxa2pC1-HA protein. Then, we purified His-Pxa2pC1-HA proteins sequentially by 8 M urea denaturation, dialysis and nicole’s column purification. We analysed the molecular size of this purified His-Pxa2pC1-HA protein by FPLC. The His-Pxa2pC1-HA proteins are expressed at low levels and insoluble. Then, we prepare His-Pxa2pC2-HA by the same way, but dialyse with Triton-X 100 to increase its solubility and skip nicole’s column purification step. We analysed the molecular size of this His-Pxa2pC2-HA protein by FPLC. However, the molecular size determination was interferenced by Triton-X 100. In yeast, by using coimmunoprecipitation assays of differentially tagged full-length Pxa2p, we demonstrated that Pxa2p can form a homodimer or homo-oligomer.

並列關鍵字

Pxa2 ； Pxa2p ； Peroxisome ； Adrenoleukodystrophy ； ATP-binding cassette transporter

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