Homo- and Heterodimerization of Peroxisomal ATP-binding Cassette Half-transporters

Abstract

The peroxisomal membrane is equipped with four ATP-binding cassette (ABC) 1 transporters, which include the adrenoleukodystrophy protein (ALDP) (1), the adrenoleukodystrophyrelated protein (ALDRP) (2), the 70-kDa peroxisomal membrane protein (PMP70) (3), and the PMP70-related protein (4, 5). Typical mammalian ABC transporters, like the multidrug-resistant P-glycoprotein, are single functional proteins with two related halves comprised of one hydrophobic transmembrane domain and one hydrophilic nucleotide-binding fold (NBF) (6). In contrast, the peroxisomal ABC transporters, as well as TAP1/TAP2 (7) and ABC7 (8), which are respectively located within the endoplasmic reticulum or mitochondria, are half-transporters with only one hydrophobic domain and one NBF.X-linked adrenoleukodystrophy (X-ALD) is the only genetic disease known to result from a peroxisomal ABC transporter gene defect. This neurodegenerative disorder is characterized by progressive demyelination within the central nervous system, adrenal insufficiency, and accumulation of very long-chain fatty acids because of an impaired peroxisomal ␤-oxidation (9 -12). Although it is firmly established that the loss of ALDP function is responsible for the abnormality in VLCFA metabolism, its precise role is unknown. Similarly, no precise function has been assigned to ALDRP, PMP70, or PMP70-related proteins. ALDRP, PMP70, and PMP70-related protein present 66, 38, and 27% amino acid identity with ALDP, respectively, suggesting functional similarity of these four transporters. These proteins display specific but sometimes overlapping patterns of expression in different cell types (13-15). Because it is likely that half-transporters need to dimerize to exert their function (6), this raises the possibility that different types of peroxisomal ABC dimers could allow the import of distinct substrates. Few ABC transporters are known to dimerize. Genetic evidence suggests that the bacterial hemolysin transporter B ABC half-transporter forms homodimers (16), whereas the Drosophila white, brown, and scarlet gene products form heterodimers (17). Heterodimerization of ABC transporters has also been reported for the transporters of antigenic peptides, TAP1 and TAP2 (18), and the two yeast peroxisomal ABC proteins, Pxa1 and Pxa2 (19,20).This study used the yeast two-hybrid assay to show that the carboxyl-terminal half of ALDP, ALDRP, and PMP70 can engage in homo-and heterodimerization. These two processes were confirmed by co-immunoprecipitation methods. We examined the effect of four different ALD patient mutations upon these interactions and attempted to map the carboxyl-terminal subdomains of ALDP allowing dimerization of the protein. EXPERIMENTAL PROCEDURESAntibodies, Yeast Strains, and Cell Lines-Monoclonal antibody 1D6 against human ALDP (hALDP) and polyclonal antibodies 1664 and 7373 raised against mouse ALDP (mALDP) and mouse ALDRP (mALDRP), respectively, have been described previously (13,15,21).

ydaG and ydbA of Lactococcus lactis Encode a Heterodimeric ATP-binding Cassette-type Multidrug Transporter

“…Half-transporters localized in peroxisomes that belong to the ABCD subfamily, namely adrenoleukodystrophy protein, adrenoleukodystrophy-related protein, and peroxisomal protein of 70 kDa, were shown to homoand heterodimerize as suggested by yeast two-hybrid studies. Accordingly, it was proposed that different combinations of such dimers vary in the substrate spectrum (48). Our data suggest that YdaG and YdbA heterodimerize to form an active MDR transporter.…”

Comparative Genomics and Adaptive Selection of the ATP-Binding-Cassette Gene Family in Caenorhabditis Species

“…The ABCD genes are expected to be functional only after formation of homo-or hetrodimers. Homodimerization of ALDPs and heterodimerization of ALDP with ALDR or PMP70 have been demonstrated in vitro (Liu et al 1999;Smith et al 1999). Instead of four members, there are five ABCD subfamily members in C. elegans, two of which result from tandem duplication (Table 1; supplemental Figure 2 at http://www.genetics.org/supplemental/).…”

Substrate Specificity Overlap and Interaction between Adrenoleukodystrophy Protein (ALDP/ABCD1) and Adrenoleukodystrophy-related Protein (ALDRP/ABCD2)

“…Although a mirror expression pattern is often observed between ALDP and ALDRP when specific cell types are analyzed (9), peroxisomal ABC transporters have overlapping expression patterns rendering possible such interactions (5, 10). Coimmunoprecipitation experiments or FRET analysis have demonstrated heterodimerization in cells overexpressing the peroxisomal ABC transporters (11,12). Although the peroxisomal localization of ALDP, ALDRP, and PMP70 is clearly demonstrated, PMP69 has recently been described to be localized in the endoplasmic reticulum and was found to be absent in the peroxisome.…”