title: Live cell FRET microscopy: homo- and heterodimerization of two human peroxisomal ABC transporters, the adrenoleukodystrophy protein (ALDP, ABCD1) and PMP70 (ABCD3).
abstract: The adrenoleukodystrophy protein (ALDP) and the 70-kDa peroxisomal membrane protein (PMP70) are half-ATP-binding cassette (ABC) transporters in the mammalian peroxisome membrane. Mutations in the gene encoding ALDP result in a devastating neurodegenerative disorder, X-linked adrenoleukodystrophy (X-ALD) that is associated with elevated levels of very long chain fatty acids because of impaired peroxisomal beta-oxidation. The interactions of peroxisomal ABC transporters, their role in the peroxisomal membrane, and their functions in disease pathogenesis are poorly understood. Studies on ABC transporters revealed that half-transporters have to dimerize to gain functionality. So far, conflicting observations are described for ALDP. By the use of in vitro methods (yeast two-hybrid and immunoprecipitation assays) on the one hand, it was shown that ALDP can form homodimers as well as heterodimers with PMP70 and ALDR, while on the other hand, it was demonstrated that ALDP and PMP70 exclusively homodimerize. To circumvent the problems of artificial interactions due to biochemical sample preparation in vitro, we investigated protein-protein interaction of ALDP in its physiological environment by FRET microscopy in intact living cells. The statistical relevance of FRET data was determined in two different ways using probability distribution shift analysis and Kolmogorov-Smirnov statistics. We demonstrate in vivo that ALDP and PMP70 form homodimers as well as ALDP/PMP70 heterodimers where ALDP homodimers predominate. Using C-terminal deletion constructs of ALDP, we demonstrate that the last 87 C-terminal amino acids harbor the most important protein domain mediating these interactions, and that the N-terminal transmembrane region of ALDP has an additional stabilization effect on ALDP homodimers. Loss of ALDP homo- or heterodimerization is highly relevant for understanding the disease mechanisms of X-ALD.
authors.affiliation: Department of Pediatrics and Pediatric Neurology, Georg August University, Faculty of Medicine, Robert-Koch-Strasse 40, 37075 Göttingen, Germany.
authors.forename: Merle
authors.initials: M
authors.lastname: Hillebrand
authors.affiliation: Department of Neurobiology, Max Planck Institute of Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
authors.forename: Sophie E
authors.initials: SE
authors.lastname: Verrier
authors.affiliation: Department of Pediatrics and Pediatric Neurology, Georg August University, Faculty of Medicine, Robert-Koch-Strasse 40, 37075 Göttingen, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Ohlenbusch
authors.affiliation: Department of Pediatrics and Pediatric Neurology, Georg August University, Faculty of Medicine, Robert-Koch-Strasse 40, 37075 Göttingen, Germany.
authors.forename: Annika
authors.initials: A
authors.lastname: Schäfer
authors.affiliation: Department of Neurobiology, Max Planck Institute of Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
authors.forename: Hans-Dieter
authors.initials: HD
authors.lastname: Söling
authors.affiliation: Cell Biophysics Group, European Neuroscience Institute, Waldweg 33, 37073 Göttingen, Germany.
authors.forename: Fred S
authors.initials: FS
authors.lastname: Wouters
authors.affiliation: Department of Pediatrics and Pediatric Neurology, Georg August University, Faculty of Medicine, Robert-Koch-Strasse 40, 37075 Göttingen, Germany. Electronic address: gaertnj@med.uni-goettingen.de.
authors.forename: Jutta
authors.initials: J
authors.lastname: Gärtner
mesh_terms: D000074063:ATP Binding Cassette Transporter, Subfamily D, Member 1; D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000502:physiology; D000326:Adrenoleukodystrophy / Q000209:etiology; D001483:Base Sequence; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008853:Microscopy; D008969:Molecular Sequence Data; D013329:Structure-Activity Relationship
chemical_list: C115572:ABCD1 protein, human; C063305:ABCD3 protein, human; D000074063:ATP Binding Cassette Transporter, Subfamily D, Member 1; D018528:ATP-Binding Cassette Transporters