title: Structure-function analysis of peroxisomal ATP-binding cassette transporters using chimeric dimers.
abstract: ABCD1 and ABCD2 are two closely related ATP-binding cassette half-transporters predicted to homodimerize and form peroxisomal importers for fatty acyl-CoAs. Available evidence has shown that ABCD1 and ABCD2 display a distinct but overlapping substrate specificity, although much remains to be learned in this respect as well as in their capability to form functional heterodimers. Using a cell model expressing an ABCD2-EGFP fusion protein, we first demonstrated by proximity ligation assay and co-immunoprecipitation assay that ABCD1 interacts with ABCD2. Next, we tested in the pxa1/pxa2Δ yeast mutant the functionality of ABCD1/ABCD2 dimers by expressing chimeric proteins mimicking homo- or heterodimers. For further structure-function analysis of ABCD1/ABCD2 dimers, we expressed chimeric dimers fused to enhanced GFP in human skin fibroblasts of X-linked adrenoleukodystrophy patients. These cells are devoid of ABCD1 and accumulate very long-chain fatty acids (C26:0 and C26:1). We checked that the chimeric proteins were correctly expressed and targeted to the peroxisomes. Very long-chain fatty acid levels were partially restored in transfected X-linked adrenoleukodystrophy fibroblasts regardless of the chimeric construct used, thus demonstrating functionality of both homo- and heterodimers. Interestingly, the level of C24:6 n-3, the immediate precursor of docosahexaenoic acid, was decreased in cells expressing chimeric proteins containing at least one ABCD2 moiety. Our data demonstrate for the first time that both homo- and heterodimers of ABCD1 and ABCD2 are functionally active. Interestingly, the role of ABCD2 (in homo- and heterodimeric forms) in the metabolism of polyunsaturated fatty acids is clearly evidenced, and the chimeric dimers provide a novel tool to study substrate specificity of peroxisomal ATP-binding cassette transporters.
authors.affiliation: From the Laboratoire Bio-PeroxIL, EA7270 University of Bourgogne, 6 Bd. Gabriel, 21000 Dijon, France.
authors.forename: Flore
authors.initials: F
authors.lastname: Geillon
authors.affiliation: From the Laboratoire Bio-PeroxIL, EA7270 University of Bourgogne, 6 Bd. Gabriel, 21000 Dijon, France.
authors.forename: Catherine
authors.initials: C
authors.lastname: Gondcaille
authors.affiliation: From the Laboratoire Bio-PeroxIL, EA7270 University of Bourgogne, 6 Bd. Gabriel, 21000 Dijon, France.
authors.forename: Soëli
authors.initials: S
authors.lastname: Charbonnier
authors.affiliation: the Laboratory of Genetic Metabolic Diseases, Room F0-226, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ, Amsterdam, The Netherlands.
authors.forename: Carlo W
authors.initials: CW
authors.lastname: Van Roermund
authors.affiliation: From the Laboratoire Bio-PeroxIL, EA7270 University of Bourgogne, 6 Bd. Gabriel, 21000 Dijon, France.
authors.forename: Tatiana E
authors.initials: TE
authors.lastname: Lopez
authors.affiliation: From the Laboratoire Bio-PeroxIL, EA7270 University of Bourgogne, 6 Bd. Gabriel, 21000 Dijon, France.
authors.forename: Alexandre M M
authors.initials: AM
authors.lastname: Dias
authors.affiliation: Lipidomic Analytical Platform-INSERM U866, University of Bourgogne, 21079 Dijon, France, and.
authors.forename: Jean-Paul
authors.initials: JP
authors.lastname: Pais de Barros
authors.affiliation: INRA, UMR1347 Agroécologie, ERL CNRS6300, Plateforme DImaCell, Centre de Microscopie INRA/Université de Bourgogne, BP86510, F-21000 Dijon, France.
authors.forename: Christine
authors.initials: C
authors.lastname: Arnould
authors.affiliation: the Laboratory of Genetic Metabolic Diseases, Room F0-226, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ, Amsterdam, The Netherlands.
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Wanders
authors.affiliation: From the Laboratoire Bio-PeroxIL, EA7270 University of Bourgogne, 6 Bd. Gabriel, 21000 Dijon, France.
authors.forename: Doriane
authors.initials: D
authors.lastname: Trompier
authors.affiliation: From the Laboratoire Bio-PeroxIL, EA7270 University of Bourgogne, 6 Bd. Gabriel, 21000 Dijon, France, stsavary@u-bourgogne.fr.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Savary
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D019281:Dimerization; D006801:Humans; D051379:Mice; D020675:Peroxisomes / Q000378:metabolism*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D051381:Rats; D013329:Structure-Activity Relationship
chemical_list: D018528:ATP-Binding Cassette Transporters; D017931:DNA Primers
keywords: ABC Transporter; Fatty Acid; Peroxisome; Protein Chimera; Protein-Protein Interaction