title: Involvement of the carboxyl-terminal region of the yeast peroxisomal half ABC transporter Pxa2p in its interaction with Pxa1p and in transporter function.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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 Pxa2_NBD-CT protein impaired its interaction with Pxa1_NBD or Pxa1_NBD-CT, 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.
  
  
  CONCLUSIONS
  The CT of Pxa2p 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.
authors.affiliation: Institute of Biochemistry and Biotechnology, College of Medicine, Chung Shan Medical University, Taichung, Taiwan.
authors.forename: Cheng-Yi
authors.initials: CY
authors.lastname: Chuang
authors.affiliation: Department of Biochemistry, School of Medicine, Chung Shan Medical University, Taichung, Taiwan.
authors.forename: Ling-Yun
authors.initials: LY
authors.lastname: Chen
authors.affiliation: Graduate Institute of Immunology, China Medical University, Taichung, Taiwan.
authors.forename: Ru-Huei
authors.initials: RH
authors.lastname: Fu
authors.affiliation: Institute of Biochemistry and Biotechnology, College of Medicine, Chung Shan Medical University, Taichung, Taiwan.
authors.forename: Shih-Ming
authors.initials: SM
authors.lastname: Chen
authors.affiliation: Institute of Biochemistry and Biotechnology, College of Medicine, Chung Shan Medical University, Taichung, Taiwan.
authors.forename: Ming-Hua
authors.initials: MH
authors.lastname: Ho
authors.affiliation: Institute of Biochemistry and Biotechnology, College of Medicine, Chung Shan Medical University, Taichung, Taiwan.
authors.forename: Jie-Mau
authors.initials: JM
authors.lastname: Huang
authors.affiliation: Institute of Biochemistry and Biotechnology, College of Medicine, Chung Shan Medical University, Taichung, Taiwan.
authors.forename: Chia-Chi
authors.initials: CC
authors.lastname: Hsu
authors.affiliation: Institute of Biochemistry and Biotechnology, College of Medicine, Chung Shan Medical University, Taichung, Taiwan.
authors.forename: Chien-Cheng
authors.initials: CC
authors.lastname: Wang
authors.affiliation: Institute of Biochemistry and Biotechnology, College of Medicine, Chung Shan Medical University, Taichung, Taiwan.
authors.forename: Meng-Shian
authors.initials: MS
authors.lastname: Chen
authors.affiliation: Institute of Biochemistry and Biotechnology, College of Medicine, Chung Shan Medical University, Taichung, Taiwan; Department of Biochemistry, School of Medicine, Chung Shan Medical University, Taichung, Taiwan.
authors.forename: Rong-Tzong
authors.initials: RT
authors.lastname: Tsai
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000378:metabolism*; D000326:Adrenoleukodystrophy / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D019281:Dimerization; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020675:Peroxisomes / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
chemical_list: D018528:ATP-Binding Cassette Transporters; C098338:PXA1 protein, S cerevisiae; C103561:PXA2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins