Yeast Pxa1 COOH-terminal region interact with Pxa2 NBD domain by yeast two-hybrid analysis

• Main Authors: Fu-Sheng Yang, 楊富昇
• Other Authors: Ling-Yun Chen
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/72292299316128364392

碩士 === 中山醫學大學 === 生化暨生物科技研究所 === 102 === The yeast peroxisome half ABC transporters "Pxa1p and Pxa2p" are homologous protein with ALDP (Adrenoleukodystrophy protein) and PMP70 (70-kDa peroxisome membrane protein). It is currently known that ALD protein and PMP70 protein function was related with very long chain fatty acid and long chain fatty acid transport. In addition, this type of half ABC transporter must form a dimer to have correct function. In 1999, Liu et al studied in COOH terminus of ALDP, PMP70 and ALDRP. They were found that the ALDP COOH terminus will participate in protein interaction. The Pxa1p and Pxa2p mainly exist in the heterodimer form and they will stay on the peroxisome membranes. There are no studies about the detailed mechanisms of protein interactions them. In our study, we simulate Liu et al study in 1999. We use yeast two-hybrid to observe the COOH terminus of Pxa1p and Pxa2p and found that Pxa1p COOH terminus will interact with Pxa2p NBD but Pxa2p COOH terminus cannot interact with Pxa1p NBD. The Pxa1 COOH terminus seems has a special meaning in this kind of protein interaction. In order to determine the existence of such interactions, we conducted experiments of amino acid fragment deleted. We found that the interaction region was located in Pxa1 COOH 775-785. Then we also referred the mutant database of human ALD disease and produce two mutant proteins which are Pxa1_CT A778T and Pxa1_CT E784K. Finally, we analyzed the protein interaction by yeast two-hybrid system and found that Pxa1_CT E784K and Pxa2_NBD did not have protein interactions, but Pxa1_CT A778T and Pxa2_NBD had protein interactions.