(1)建立過氧化體生合成因子Pex19p功能性分析方法，(2)鑑定過氧化體ABC運輸蛋白Pxa1p中調控Pxa1p與Pxa2p相互作用的重要區域

(1)The developments of the function assays for the yeast peroxisome biogenesis factor Pex19p, (2) the identification of the important regions in Pxa1p (a peroxisomal ABC transporter ) that can regulate the interaction of Pxa1p and Pxa2p

陳孟賢(Meng-Shian Chen)

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(2011年)

https://doi.org/10.6834/CSMU.2011.00031

摘要

Part 1
Pex19p在過氧化體生合成中扮演著極為重要的角色。在Pex19p刪除的酵母菌中，過氧化體的形成受到嚴重影響導致在顯微鏡下觀察不到過氧化體的蹤跡。Pex19p是一種極為親水性的酸性蛋白，已知Pex19p在細胞質中被核醣體轉譯出來後，會與許多過氧化體膜蛋白有互相結合的能力，作為過氧化體膜蛋白的受體蛋白，將其運送至過氧化體膜上。我們更想進一步去了解過氧化體生合成基因Pex19p的功能性。所以在本篇研究，我們建立了利用酵母菌代謝油酸去觀察過氧化體功能性的分析方法；另外我們也建立了利用綠色螢光蛋白與過氧化體基質蛋白訊號胜肽的融合蛋白，可以觀察過氧化體的生合成是否正常的分析方法。除了蛋白質一級結構上胺基酸功能探討，我們想進一步去了解 Pex19p的蛋白質的三級結構，因為蛋白質在一級結構上的重要性是無法完全解釋結構和功能之間的關聯性，所以在本研究中，我們將Pex19p的C端重組蛋白純化後，進行養晶，並進行X-射線結晶繞射，目前正在解讀其三級結構，此方法的建立將使我們實驗室能更深入探討Pex19p的結構和功能之間的關聯性。
Part 2
酵母菌過氧化體half ABC transporter：Pxa1p、Pxa2p 是人類ALDP、PMP70的同源蛋白，目前已知ALDP是與VLCFA的運輸有關，而PMP70的功能目前僅止於推測與LCFA的運輸有關。另外，這些half ABC transporter需要形成二聚體的形式才有功能。 Pxa1p、Pxa2p的功能與運輸LCFA有關，並且是以異源二聚體的方式存在於過氧化小體的膜上，目前並沒有相關文獻研究Pxa1p、Pxa2p之間的交互作用區域。在本篇研究，Pxa1p的多重序列比對下，意外發現在Pxa1p第474個到第531個胺基酸有一段高度保留的胺基酸序列，而這段高度保留的胺基酸序列目前尚未被研究定義出有何蛋白修飾或是蛋白功能。在本篇研究針對Pxa1p這段高度保留的胺基酸序列進行一系列的刪除胺基酸以及定位點突變，利用酵母菌雙雜交法去觀察這樣的胺基酸刪除或是定位點突變是否會影響Pxa2p的蛋白質交互作用。另外我們也想探討Pxa1p這樣的胺基酸刪除或是胺基酸的定位點突變是否會影響過氧化體運輸油酸的能力。

關鍵字

pxa1 ； pxa2 ； pex19

並列摘要

Part 1
Pex19p are play important role in peroxisome biogenesis. The peroxisome can’t be observed by the microscope technology in the pex19-knockout strain in yeast. Pex19p is hydrophilic and acidic protein. It has known that Pex19p is translated by free ribosome in the cytoplasm, and function as the targeting receptor for peroxisomal membrane proteins. The peroxisome membrane proteins are first binding to Pex19p in cytoplasm, and then transported to peroxisomes. Because we wanted to understand the function of Pex19p in the peroxisome biogenesis in detail, so we established the functional assay for the peroxisome functionality by using oleic acid as a sole carbon source. The only site of the oleic acid metabolism in yeast is in the peroxisome, so the viability of the yeast strains in oleic acid functional assay plate will represent the ability of peroxisome biogenesis, the process requiring the functions of Pex19p or its derivatives absolutely. We also established the fluorescence microscope assay for examing the peroxisome directly. The C-terminal of green fluorescence proteins was fused to peroxisome target sequences 1 (PTS1). This fusion protein could be transported to the matrix of peroxisomes as long as peroxisomes are existed in the cell. The peroxisomes were look like small green light dots in the cell under the fluorescence microscope observation. Additionally, we were interested in understanding the the tertiary structure of Pex19p, because the importance of Pex19p primary sequences can’t fully explain the structure-function relationship. So, we produced recombinant proteins of the Pex19p C-terminal region with N-terminal His tag (His-∆N-Pex19p). The crystals of His-∆N-Pex19p had been obtained, and the process of 3-D structure determination of His-∆N-Pex19p is ongoing. The tertiary structure of His-∆N-Pex19p will provide a new way to study structure-function relationship of Pex19p for our laboratory.
Part 2
Pxa1p and Pxa2p are half ABC transporter of peroxisomes in yeast. Pxa1p and Pxa2p are homology with ALDP and PMP70 in human. ALDP can transport VLCFA has known, and PMP70 have doubts that it should can transport LCFA. These half ABC transporter are that they have function of proteins by hetrodimer on membrane of peroxisomes. Presently, Pxa1p and Pxa2p how interact is not discussion. In this study, we did multiple sequence alignment, we found that the 474th to 531th of Pxa1p have conserved sequence. This conserved sequence is not defined about the function or motif of domain. We deleted a series deletion of Pxa1p, and we did the point mutation of Pxa1p. We observed that deletion of Pxa1p and point mutation of Pxa1p interacted with Pxa2p, and these mutant type effected the metabolism of oleic acid function of peroxisome in yeast or not.

並列關鍵字

pxa1 ； pxa2 ； pex19

參考文獻

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