以免疫沉澱、膠體過濾層析法及鎳離子親和性管柱層析法探討 Pxa2p 的 COOH 端在Pxa1p 及 Pxa2p 之 NBF 區域的交互作用

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。本實驗室針對 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，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，本篇再透過免疫共沉澱、膠體過濾層析法、鎳離子親和性管柱層析法作進一步分析。免疫共沉澱和鎳離子親和性管柱層析法的實驗結果雖與酵母雙雜交實驗有一致性，但是從膠體過濾層析法的實驗得知，我們所建構的 Pxa1p 和 Pxa2p 在打破細胞後會形成一個龐大的聚合體，這可能說明 Pxa1p 和 Pxa2p 的交互作用無法用生化實驗來探討。

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. This study, using Co-Immunoprecipitation, gelfiltration and Ni-charged affinity column to study the interactions occur between NBF_CT (Pxa2p) and NBF (Pxa2p) or NBF (Pxa2p) .