(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

• Main Authors: Meng-Shian, 陳孟賢
• Other Authors: Rong-Tzong Tsai
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/68962324504137507009

碩士 === 中山醫學大學 === 生化暨生物科技研究所 === 99 === 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.