The roles of yeast peroxisomal half abc transporter pxa1p and pxa2p carboxyl-terminal region in Pxa1p-Pxa2p heterodimer and Pxa1p-Pxa1p homodimer formation

• Main Authors: CHENG-YI CHUANG, 莊秉毅
• Other Authors: 陳凌雲
• Format: Others
• Language: zh-TW
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/52817405223862435548

博士 === 中山醫學大學 === 生化暨生物科技研究所 === 103 === The peroxisome is a single membrane-bound organelle in eukaryotic cells involved in lipid metabolism, including β-oxidation of fatty acids. The human genetic disorder X-linked adrenoleukodystrophy (X-ALD) is caused by mutations in the ABCD1 gene (encoding ALDP, a peroxisomal half ATP-binding cassette (ABC) transporter). This disease is characterized by defective peroxisomal β-oxidation and a large accumulation of very long-chain fatty acids in brain white matter, adrenal cortex, and testis. ALDP forms a homodimer proposed to be the functional transporter, whereas the peroxisomal transporter in yeast is a heterodimer comprising two half ABC transporters, Pxa1p and Pxa2p, both orthologs of human ALDP. While the carboxyl-terminal domain of ALDP is engaged in dimerization, it remains unknown whether the same region is involved in the interaction between Pxa1p and Pxa2p. Using a yeast two-hybrid assay, we found that the carboxyl-terminal region (CT) of Pxa2p, but not of Pxa1p, is required for their interaction. Further analysis indicated that the central part of the CT (designated CT2) of Pxa2p was indispensable for its interaction with the carboxyl terminally truncated Pxa1_NBD. An interaction between the CT of Pxa2p and Pxa1_NBD was not detected, but could be identified in the presence of Pxa2_NBD-CT1. A single mutation of two conserved residues (aligned with X-ALD-associated mutations at the same positions in ALDP) in the CT2 of the Pxa2p impaired its interaction with Pxa1p, resulting in a mutant protein that exhibited a proteinase K digestion profile different from that of the wild-type protein. Functional analysis of these mutant proteins on oleate plates indicated that they were defective in transporter function. In addition, using a yeast two-hybrid and co-immunoprecipitation assay, we found that Pxa1p could form homodimer and its carboxyl-terminal region was required for this homodimer interaction. By analyzing the CT fragment deletion and CT-point mutations of Pxa1p, we found that a conserved residues E868 was critical in its self-interaction. By oleate plate assay, we showed that the growth of yeast strains was suppressed due to the overexpression of Pxa1p. We also proved that Pxa1p homodimer formed through its carboxyl-terminal region was required for the interference to endogenous Pxa1p/Pxa2p heterodimer function and cell growth, indicating a negative regulation role of Pxa1p in Pxa1p/Pxa2p transporter function. The CT of Pxa2p and Pxa1p is involved in its interaction with Pxa1p and in transporter function. This concept may be applied to human ALDP studies, helping to establish the pathological mechanism for CT-related X-ALD disease.