The Yeast Peroxisomal ABC Transporter Pxa2p, a Human ALDP Homolog, Forms a Homodimer

• Main Authors: Chien-Cheng, 王建程
• Other Authors: 蔡榮宗
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/50019880119076341200

碩士 === 中山醫學大學 === 生化暨生物科技研究所 === 97 === Peroxisomal beta-oxidation play an important role in mammalian and yeast. The ABC transporter (ATP-binding cassette transporter) of peroxisomal membrane has been identified that is closely related to human ALD (Adrenoleukodystrophy). So far four ABC transporter have been detected in mammalian peroxisomes that contains four ABC transporters named ALDP (Adrenoleukodystrophy protein), ALDRP (ALD-related protein), PMP70 (The 70-kDa peroxisomal membrane protein) and PMP69 (The 69-kDa peroxisomal membrane protein). Saccharomyces cerevisiae contains two peroxisomal half-ABC transporters named Pxa1p (peroxisomal ABC transporter1 protein) and Pxa2p (peroxisomal ABC transporter2 protein). The ALDP and PMP70 are homolog Pxa1p and Pxa2p. ALDP are located in the peroxisome, where function as homo- and/or heterodimers in the regulation of very long chain fatty acid transport. The yeast Pxa1p and Pxa2p dimerize to form a functional transporter involved in very long chain fatty acid oxidation in the peroxisome. The formation of PMP70 assembles as dimeric or oligomeric forms on peroxisomal membranes implies that Pxa2p may form a homodimers. We used IPTG to induce His-Pxa2pC1-HA protein. Then, we purified His-Pxa2pC1-HA proteins sequentially by 8 M urea denaturation, dialysis and nicole’s column purification. We analysed the molecular size of this purified His-Pxa2pC1-HA protein by FPLC. The His-Pxa2pC1-HA proteins are expressed at low levels and insoluble. Then, we prepare His-Pxa2pC2-HA by the same way, but dialyse with Triton-X 100 to increase its solubility and skip nicole’s column purification step. We analysed the molecular size of this His-Pxa2pC2-HA protein by FPLC. However, the molecular size determination was interferenced by Triton-X 100. In yeast, by using coimmunoprecipitation assays of differentially tagged full-length Pxa2p, we demonstrated that Pxa2p can form a homodimer or homo-oligomer.