以酵母菌雙雜交實驗證明酵母菌 Pxa2p 的COOH端會與Pxa1p、Pxa2p的NBF結合

Use Yeast Two Hybrid experiment to identify Pxa2p COOH-terminus interact with the NBF of Pxa1p and Pxa2p in Saccharomyces cerevisiae

陳詩銘(Shih-Ming Chen)

指導教授 ： 陳凌雲

中山醫學大學/醫學院/生化暨生物科技研究所/碩士(2010年)

https://doi.org/10.6834/CSMU.2010.00023

摘要

酵母菌過氧化小體half ABC transporter：Pxa1p、Pxa2p 是人類ALDP(Adrenoleukodystrophy protein)、PMP70(70-kDa peroxisome membrane protein)的同源蛋白，目前已知ALDP是與VLCFA(Vary long chain fatty acid)的運輸有關，而PMP70的功能目前僅止於推測與LCFA(long chain fatty acid)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，在1999年，Liu等人針對ALDP、ALDRP(Adrenoleukodystrophy-related protein)及PMP70的COOH端透過酵母菌雙雜交實驗及免疫共沈澱發現這些ABC transporter皆有可能形成同源二聚體或彼此之間形成異源二聚體。
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。本篇研究主要針對Pxa1p、Pxa2p的COOH端進行酵母菌雙雜交實驗，結果發現Pxa2p的COOH端對於蛋白交互作用扮演重要的角色，再透過胺基酸片段刪除得知交互作用區域是位於Pxa2p的COOH端第724到773號胺基酸之間，最後利用胺基酸定點突變以及beta-galactosidase活性分析鑑定出幾個可能影響交互作用的胺基酸。

關鍵字

酵母菌雙雜交 ； 過氧化小體 ； 長鏈脂肪酸運輸 ； 蛋白交互作用

並列摘要

The Yeast peroxisomal half ABC transporters, Pxa1p and Pxa2p, are the homologous proteins of human ALDP and PMP70. The function of ALDP and PMP70 are understood limitedly. ALDP is thought to be involved in the uptake of activated VLCFAs into mammalian peroxisomes. PMP70 is speculated to be involved in the transportation of LCFA into mammalian peroxisomes. To our knowledge, the half ABC transporters have to dimerize to be functional. In 1999, Liu and co-workers studied the COOH terminus of the human ALDP, ALDRP and PMP70 by Yeast Two Hybrid and co-immunopricipitation experiments to show that the COOH terminus of the human ALDP, ALDRP and PMP70 will form homodimer or heterodimer.
Pxa1p and Pxa2p, the peroxisomal membrane proteins, are known to form heterodimer to transport LCFA into peroxisomes. There are no reports about the binding domains between Pxa1p and Pxa2p. Using the Yeast Two hybrid system, we show that the interactions occur between NBF_CT(Pxa2p) and NBF(Pxa2p)or NBF(Pxa2p) but not NBF_CT(Pxa1p). Moreover, the interaction area of the Pxa2p COOH terminus is from 724th to 774th amino acids by the deletion mutants. Finally, using site-directed mutagenesis and beta-galactosidase activity assay, we determine several conserved amino acids could affect the protein-protein interaction.

並列關鍵字

Yeast two hybrid ； peroxisome ； Pxa1p ； Pxa2p ； interaction ； lon chain fatty acid ； ATP binding cassette transporter ； beta-galactosidase activity assay

參考文獻

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