The 70-kDa peroxisomal membrane protein (PMP70) and the adrenoleukodystrophy protein (ALDP) are half ATP binding cassette (ABC) transporters in the peroxisome membrane. Mutations in the ALD gene encoding ALDP result in the X-linked neurodegenerative disorder adrenoleukodystrophy. Plausible models exist to show a role for ATP hydrolysis in peroxisomal ABC transporter functions. Here, we describe the first measurements of the rate of ATP binding and hydrolysis by purified nucleotide binding fold (NBF) fusion proteins of PMP70 and ALDP. Both proteins act as an ATP specific binding subunit releasing ADP after ATP hydrolysis; they did not exhibit GTPase activity. Mutations in conserved residues of the nucleotidases (PMP70: G478R, S572I; ALDP: G512S, S606L) altered ATPase activity. Furthermore, our results indicate that these mutations do not influence homodimerization or heterodimerization of ALDP or PMP70. The study provides evidence that peroxisomal ABC transporters utilize ATP to become a functional transporter.
The 70-kDa peroxisomal membrane protein (PMP70) and the adrenoleukodystrophy protein (ALDP) are half ATP binding cassette (ABC) transporters in the peroxisome membrane. Mutations in the ALD gene encoding ALDP result in the X-linked neurodegenerative disorder adrenoleukodystrophy. Plausible models exist to show a role for ATP hydrolysis in peroxisomal ABC transporter functions. Here, we describe the first measurements of the rate of ATP binding and hydrolysis by purified nucleotide binding fold (NBF) fusion proteins of PMP70 and ALDP. Both proteins act as an ATP specific binding subunit releasing ADP after ATP hydrolysis; they did not exhibit GTPase activity. Mutations in conserved residues of the nucleotidases (PMP70: G478R, S572I; ALDP: G512S, S606L) altered ATPase activity. Furthermore, our results indicate that these mutations do not influence homodimerization or heterodimerization of ALDP or PMP70. The study provides evidence that peroxisomal ABC transporters utilize ATP to become a functional transporter.
The 70-kDa peroxisomal membrane protein (PMP70) and the adrenoleukodystrophy protein (ALDP) are half ATP binding cassette (ABC) transporters in the peroxisome membrane. Mutations in the ALD gene encoding ALDP result in the X-linked neurodegenerative disorder adrenoleukodystrophy. Plausible models exist to show a role for ATP hydrolysis in peroxisomal ABC transporter functions. Here, we describe the first measurements of the rate of ATP binding and hydrolysis by purified nucleotide binding fold (NBF) fusion proteins of PMP70 and ALDP. Both proteins act as an ATP specific binding subunit releasing ADP after ATP hydrolysis; they did not exhibit GTPase activity. Mutations in conserved residues of the nucleotidases (PMP70: G478R, S572I; ALDP: G512S, S606L) altered ATPase activity. Furthermore, our results indicate that these mutations do not influence homodimerization or heterodimerization of ALDP or PMP70. The study provides evidence that peroxisomal ABC transporters utilize ATP to become a functional transporter.
Adrenoleukodystrophy (ALD) is an X-linked disease affecting 1/20,000 males either as cerebral ALD in childhood or as adrenomyeloneuropathy (AMN) in adults. Childhood ALD is the more severe form, with onset of neurological symptoms between 5-12 years of age. Central nervous system demyelination progresses rapidly and death occurs within a few years. AMN is a milder form of the disease with onset at 15-30 years of age and a more progressive course. Adrenal insufficiency (Addison's disease) may remain the only clinical manifestation of ALD. The principal biochemical abnormality of ALD is the accumulation of very-long-chain fatty acids (VLCFA) because of impaired beta-oxidation in peroxisomes. The normal oxidation of VLCFA-CoA in patients' fibroblasts suggested that the gene coding for the VLCFA-CoA synthetase could be a candidate gene for ALD. Here we use positional cloning to identify a gene partially deleted in 6 of 85 independent patients with ALD. In familial cases, the deletions segregated with the disease. An identical deletion was detected in two brothers presenting with different clinical ALD phenotypes. Candidate exons were identified by computer analysis of genomic sequences and used to isolate complementary DNAs by exon connection and screening of cDNA libraries. The deduced protein sequence shows significant sequence identity to a peroxisomal membrane protein of M(r) 70K that is involved in peroxisome biogenesis and belongs to the 'ATP-binding cassette' superfamily of transporters.
The beta-oxidation of fatty acids in peroxisomes produces hydrogen peroxide (H2O2), a toxic metabolite, as a bi-product. Fatty acids beta-oxidation activity is deficient in X-linked adrenoleukodystrophy (X-ALD) because of mutation in ALD-gene resulting in loss of very long chain acyl-CoA synthetase (VLCS) activity. It is also affected in disease with catalase negative peroxisomes as a result of inactivation by H2O2. Therefore, the following studies were undertaken to delineate the molecular interactions between both the ALD-gene product (adrenoleukodystrophy protein, ALDP) and VLCS as well as H2O2 degrading enzyme catalase and proteins of peroxisomal beta-oxidation. Studies using a yeast two hybrid system and surface plasmon resonance techniques indicate that ALDP, a peroxisomal membrane protein, physically interacts with VLCS. Loss of these interactions in X-ALD cells may result in a deficiency in VLCS activity. The yeast two-hybrid system studies also indicated that catalase physically interacts with L-bifunctional enzyme (L-BFE). Interactions between catalase and L-BFE were further supported by affinity purification, using a catalase-linked resin. The affinity bound 74-kDa protein, was identified as L-BFE by Western blot with specific antibodies and by proteomic analysis. Additional support for their interaction comes from immunoprecipitation of L-BFE with antibodies against catalase as a catalase- L-BFE complex. siRNA for L-BFE decreased the specific activity and protein levels of catalase without changing its subcellular distribution. These observations indicate that L-BFE might help in oligomerization and possibly in the localization of catalase at the site of H2O2 production in the peroxisomal beta-oxidation pathway.
J. Biol. Chem. 274, 32738-32743 (1999)[PubMed:10551832]
Mammalian peroxisomal proteins adrenoleukodystrophy protein (ALDP), adrenoleukodystrophy-related protein (ALDRP), and 70-kDa peroxisomal protein (PMP70) belong to the superfamily of ATP-binding cassette (ABC) transporters. Unlike many ABC transporters that are single functional proteins with two related halves, ALDP, ALDRP, and PMP70 have the structure of ABC half-transporters. The dysfunction of ALDP is responsible for X-linked adrenoleukodystrophy (X-ALD), a neurodegenerative disorder in which saturated very long-chain fatty acids accumulate because of their impaired peroxisomal beta-oxidation. No disease has so far been associated with mutations of adrenoleukodystrophy-related or PMP70 genes. It has been proposed that peroxisomal ABC transporters need to dimerize to exert import functions. Using the yeast two-hybrid system, we show that homo- as well as heterodimerization occur between the carboxyl-terminal halves of ALDP, ALDRP, and PMP70. Two X-ALD disease mutations located in the carboxyl-terminal half of ALDP affect both homo- and heterodimerization of ALDP. Co-immunoprecipitation demonstrated the homodimerization of ALDP, the heterodimerization of ALDP with PMP70 or ALDRP, and the heterodimerization of ALDRP with PMP70. These results provide the first evidence of both homo- and heterodimerization of mammalian ABC half-transporters and suggest that the loss of ALDP dimerization plays a role in X-ALD pathogenesis.
Catalysis of the reaction: ATP + H2O + fatty acyl CoA(cis) = ADP + phosphate + fatty acyl CoA(trans). The transport of fatty acyl CoA into and out of peroxisomes.
Evidence
1:
Inferred from Genetic InteractionUniProtKB
Peroxisomes play a major role in human cellular lipid metabolism, including the beta-oxidation of fatty acids. The most frequent peroxisomal disorder is X-linked adrenoleukodystrophy (X-ALD), which is caused by mutations in the ABCD1 gene. The protein involved, called ABCD1, or alternatively ALDP, is a member of the ATP-binding-cassette (ABC) transporter family and is located in the peroxisomal membrane. The biochemical hallmark of X-ALD is the accumulation of very long-chain fatty acids (VLCFAs), due to an impaired peroxisomal beta-oxidation. Although this suggests a role of ALDP in VLCFA import, no experimental evidence is available to substantiate this. In the yeast Saccharomyces cerevisiae, peroxisomes are the exclusive site of fatty acid beta-oxidation. Earlier work has shown that uptake of fatty acids into peroxisomes may occur via two routes, either as free fatty acids thus requiring intraperoxisomal activation into acyl-CoA esters or as long-chain acyl-CoA esters. The latter route involves the two peroxisomal half ABC transporters Pxa1p and Pxa2p that form a heterodimeric complex in the peroxisomal membrane. Using different strategies, including the analysis of intracellular acyl-CoA esters by tandem-MS, we show that the Pxa1p/Pxa2p heterodimer is involved in the transport of a spectrum of acyl-CoA esters. Interestingly, we found that the mutant phenotype of the pxa1/pxa2Delta mutant can be rescued, at least partially, by the sole expression of the human ABCD1 cDNA coding for ALDP, the protein that is defective in the human disease X-linked adrenoleukodystrophy. Our data indicate that ALDP can function as a homodimer and is involved in the transport of acyl-CoA esters across the peroxisomal membrane.
Interacting selectively and non-covalently with any protein or protein complex (a complex of two or more proteins that may include other nonprotein molecules).
Evidence
1:
Inferred from Physical InteractionIntAct
J. Biol. Chem. 274, 32738-32743 (1999)[PubMed:10551832]
Mammalian peroxisomal proteins adrenoleukodystrophy protein (ALDP), adrenoleukodystrophy-related protein (ALDRP), and 70-kDa peroxisomal protein (PMP70) belong to the superfamily of ATP-binding cassette (ABC) transporters. Unlike many ABC transporters that are single functional proteins with two related halves, ALDP, ALDRP, and PMP70 have the structure of ABC half-transporters. The dysfunction of ALDP is responsible for X-linked adrenoleukodystrophy (X-ALD), a neurodegenerative disorder in which saturated very long-chain fatty acids accumulate because of their impaired peroxisomal beta-oxidation. No disease has so far been associated with mutations of adrenoleukodystrophy-related or PMP70 genes. It has been proposed that peroxisomal ABC transporters need to dimerize to exert import functions. Using the yeast two-hybrid system, we show that homo- as well as heterodimerization occur between the carboxyl-terminal halves of ALDP, ALDRP, and PMP70. Two X-ALD disease mutations located in the carboxyl-terminal half of ALDP affect both homo- and heterodimerization of ALDP. Co-immunoprecipitation demonstrated the homodimerization of ALDP, the heterodimerization of ALDP with PMP70 or ALDRP, and the heterodimerization of ALDRP with PMP70. These results provide the first evidence of both homo- and heterodimerization of mammalian ABC half-transporters and suggest that the loss of ALDP dimerization plays a role in X-ALD pathogenesis.
Evidence
2:
Inferred from Physical InteractionUniProtKB
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.
Evidence
3:
Inferred from Physical InteractionUniProtKB
Four ABC half transporters (ALDP, ALDRP, PMP70, and PMP69) have been identified in the mammalian peroxisomal membrane but no function has been unambiguously assigned to any of them. To date X-linked adrenoleukodystrophy (X-ALD) is the only human disease known to result from a defect of one of these ABC transporters, ALDP. Using the yeast two-hybrid system and in vitro GST pull-down assays, we identified the peroxin PEX19p as a novel interactor of ALDP, ALDRP, and PMP70. The cytosolic farnesylated protein PEX19p was previously shown to be involved in an early step of the peroxisomal biogenesis. The PEX19p interaction occurs in an internal N-terminal region of ALDP which we verified to be important for proper peroxisomal targeting of this protein. Farnesylated wild-type PEX19p and a farnesylation-deficient mutant PEX19p did not differ in their ability to bind to ALDP. Our data provide evidence that PEX19p is a cytosolic acceptor protein for the peroxisomal ABC transporters ALDP, PMP70, and ALDRP and might be involved in the intracellular sorting and trafficking of these proteins to the peroxisomal membrane.
Evidence
4:
Inferred from Physical InteractionUniProtKB
PEX19 has been shown to play a central role in the early steps of peroxisomal membrane synthesis. Computational database analysis of the PEX19 sequence revealed three different conserved domains: D1 (aa 1--87), D2 (aa 88--272), and D3 (aa 273--299). However, these domains have not yet been linked to specific biological functions. We elected to functionally characterize the proteins derived from two naturally occurring PEX19 splice variants: PEX19DeltaE2 lacking the N-terminal domain D1 and PEX19DeltaE8 lacking the domain D3. Both interact with peroxisomal ABC transporters (ALDP, ALDRP, PMP70) and with full-length PEX3 as shown by in vitro protein interaction studies. PEX19DeltaE8 also interacts with a PEX3 protein lacking the peroxisomal targeting region located at the N-terminus (Delta66aaPEX3), whereas PEX19DeltaE2 does not. Functional complementation studies in PEX19-deficient human fibroblasts revealed that transfection of PEX19DeltaE8-cDNA leads to restoration of both peroxisomal membranes and of functional peroxisomes, whereas transfection of PEX19DeltaE2-cDNA does not restore peroxisomal biogenesis. Human PEX19 is partly farnesylated in vitro and in vivo. The farnesylation consensus motif CLIM is located in the PEX19 domain D3. The finding that the protein derived from the splice variant lacking D3 is able to interact with several peroxisomal membrane proteins and to restore peroxisomal biogenesis challenges the previous assumption that farnesylation of PEX19 is essential for its biological functionality. The data presented demonstrate a considerable functional diversity of the proteins encoded by two PEX19 splice variants and thereby provide first experimental evidence for specific biological functions of the different predicted domains of the PEX19 protein.
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.
Peroxisomes play a major role in human cellular lipid metabolism, including the beta-oxidation of fatty acids. The most frequent peroxisomal disorder is X-linked adrenoleukodystrophy (X-ALD), which is caused by mutations in the ABCD1 gene. The protein involved, called ABCD1, or alternatively ALDP, is a member of the ATP-binding-cassette (ABC) transporter family and is located in the peroxisomal membrane. The biochemical hallmark of X-ALD is the accumulation of very long-chain fatty acids (VLCFAs), due to an impaired peroxisomal beta-oxidation. Although this suggests a role of ALDP in VLCFA import, no experimental evidence is available to substantiate this. In the yeast Saccharomyces cerevisiae, peroxisomes are the exclusive site of fatty acid beta-oxidation. Earlier work has shown that uptake of fatty acids into peroxisomes may occur via two routes, either as free fatty acids thus requiring intraperoxisomal activation into acyl-CoA esters or as long-chain acyl-CoA esters. The latter route involves the two peroxisomal half ABC transporters Pxa1p and Pxa2p that form a heterodimeric complex in the peroxisomal membrane. Using different strategies, including the analysis of intracellular acyl-CoA esters by tandem-MS, we show that the Pxa1p/Pxa2p heterodimer is involved in the transport of a spectrum of acyl-CoA esters. Interestingly, we found that the mutant phenotype of the pxa1/pxa2Delta mutant can be rescued, at least partially, by the sole expression of the human ABCD1 cDNA coding for ALDP, the protein that is defective in the human disease X-linked adrenoleukodystrophy. Our data indicate that ALDP can function as a homodimer and is involved in the transport of acyl-CoA esters across the peroxisomal membrane.
The gene mutated in X-linked adrenoleukodystrophy (X-ALD) codes for the HsABCD1 protein, also named ALDP, which is a member of the superfamily of ATP-binding cassette (ABC) transporters and required for fatty acid transport across the peroxisomal membrane. Although a defective HsABCD1 results in the accumulation of very long-chain fatty acids in plasma of X-ALD patients, there is still no direct biochemical evidence that HsABCD1 actually transports very long-chain fatty acids. We used the yeast Saccharomyces cerevisiae to study the transport of fatty acids across the peroxisomal membrane. Our earlier work showed that in yeast the uptake of fatty acids into peroxisomes may occur via two routes, either as (1.) free fatty acid or as (2.) acyl-CoA ester. The latter route involves the two peroxisomal half-ABC transporters, Pxa1p and Pxa2p, which form a heterodimeric complex in the peroxisomal membrane. We here report that the phenotype of the pxa1/pxa2Δ yeast mutant, i.e. impaired growth on oleate containing medium and deficient oxidation of oleic acid, cannot only be partially rescued by human ABCD1, but also by human ABCD2 (ALDRP), which indicates that HsABCD1 and HsABCD2 can both function as homodimers. Fatty acid oxidation studies in the pxa1/pxa2Δ mutant transformed with either HsABCD1 or HsABCD2 revealed clear differences suggesting that HsABCD1 and HsABCD2 have distinct substrate specificities. Indeed, full rescue of beta-oxidation activity in cells expressing human ABCD2 was observed with C22:0 and different unsaturated very long-chain fatty acids including C24:6 and especially C22:6 whereas in cells expressing HsABCD1 rescue of beta-oxidation activity was best with C24:0 and C26:0 as substrates.
Adrenoleukodystrophy (ALD) is an X-linked disease affecting 1/20,000 males either as cerebral ALD in childhood or as adrenomyeloneuropathy (AMN) in adults. Childhood ALD is the more severe form, with onset of neurological symptoms between 5-12 years of age. Central nervous system demyelination progresses rapidly and death occurs within a few years. AMN is a milder form of the disease with onset at 15-30 years of age and a more progressive course. Adrenal insufficiency (Addison's disease) may remain the only clinical manifestation of ALD. The principal biochemical abnormality of ALD is the accumulation of very-long-chain fatty acids (VLCFA) because of impaired beta-oxidation in peroxisomes. The normal oxidation of VLCFA-CoA in patients' fibroblasts suggested that the gene coding for the VLCFA-CoA synthetase could be a candidate gene for ALD. Here we use positional cloning to identify a gene partially deleted in 6 of 85 independent patients with ALD. In familial cases, the deletions segregated with the disease. An identical deletion was detected in two brothers presenting with different clinical ALD phenotypes. Candidate exons were identified by computer analysis of genomic sequences and used to isolate complementary DNAs by exon connection and screening of cDNA libraries. The deduced protein sequence shows significant sequence identity to a peroxisomal membrane protein of M(r) 70K that is involved in peroxisome biogenesis and belongs to the 'ATP-binding cassette' superfamily of transporters.
The chemical reactions and pathways resulting in the breakdown of ATP, adenosine 5'-triphosphate, a universally important coenzyme and enzyme regulator.
The 70-kDa peroxisomal membrane protein (PMP70) and the adrenoleukodystrophy protein (ALDP) are half ATP binding cassette (ABC) transporters in the peroxisome membrane. Mutations in the ALD gene encoding ALDP result in the X-linked neurodegenerative disorder adrenoleukodystrophy. Plausible models exist to show a role for ATP hydrolysis in peroxisomal ABC transporter functions. Here, we describe the first measurements of the rate of ATP binding and hydrolysis by purified nucleotide binding fold (NBF) fusion proteins of PMP70 and ALDP. Both proteins act as an ATP specific binding subunit releasing ADP after ATP hydrolysis; they did not exhibit GTPase activity. Mutations in conserved residues of the nucleotidases (PMP70: G478R, S572I; ALDP: G512S, S606L) altered ATPase activity. Furthermore, our results indicate that these mutations do not influence homodimerization or heterodimerization of ALDP or PMP70. The study provides evidence that peroxisomal ABC transporters utilize ATP to become a functional transporter.
A fatty acid oxidation process that results in the complete oxidation of a long-chain fatty acid. Fatty acid beta-oxidation begins with the addition of coenzyme A to a fatty acid, and occurs by successive cycles of reactions during each of which the fatty acid is shortened by a two-carbon fragment removed as acetyl coenzyme A; the cycle continues until only two or three carbons remain (as acetyl-CoA or propionyl-CoA respectively).
X-Linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disorder characterized by reduced peroxisomal very long chain fatty acid (VLCFA) beta-oxidation. The X - ALD gene product (ALDP) is a peroxisomal transmembrane protein with an ATP binding cassette (ABC). ALDP and three other ABC proteins (PMP70, ALDR, P70R) localize to the peroxisomal membrane. The function of this family of peroxisomal membrane proteins is unknown. We used complementation studies to begin analysis of their role in VLCFA beta-oxidation and on the peroxisomal membrane. Expression of either ALDP or PMP70 restores VLCFA beta-oxidation in X-ALD fibroblasts, indicating overlapping functions. Their expression also restores peroxisome biogenesis in cells that are deficient in the peroxisomal membrane protein Pex2p. Thus it is likely that complex protein interactions are involved in the function and biogenesis of peroxisomal membranes that may contribute to disease heterogeneity.
Evidence
2:
Inferred from Genetic InteractionUniProtKB
The gene mutated in X-linked adrenoleukodystrophy (X-ALD) codes for the HsABCD1 protein, also named ALDP, which is a member of the superfamily of ATP-binding cassette (ABC) transporters and required for fatty acid transport across the peroxisomal membrane. Although a defective HsABCD1 results in the accumulation of very long-chain fatty acids in plasma of X-ALD patients, there is still no direct biochemical evidence that HsABCD1 actually transports very long-chain fatty acids. We used the yeast Saccharomyces cerevisiae to study the transport of fatty acids across the peroxisomal membrane. Our earlier work showed that in yeast the uptake of fatty acids into peroxisomes may occur via two routes, either as (1.) free fatty acid or as (2.) acyl-CoA ester. The latter route involves the two peroxisomal half-ABC transporters, Pxa1p and Pxa2p, which form a heterodimeric complex in the peroxisomal membrane. We here report that the phenotype of the pxa1/pxa2Δ yeast mutant, i.e. impaired growth on oleate containing medium and deficient oxidation of oleic acid, cannot only be partially rescued by human ABCD1, but also by human ABCD2 (ALDRP), which indicates that HsABCD1 and HsABCD2 can both function as homodimers. Fatty acid oxidation studies in the pxa1/pxa2Δ mutant transformed with either HsABCD1 or HsABCD2 revealed clear differences suggesting that HsABCD1 and HsABCD2 have distinct substrate specificities. Indeed, full rescue of beta-oxidation activity in cells expressing human ABCD2 was observed with C22:0 and different unsaturated very long-chain fatty acids including C24:6 and especially C22:6 whereas in cells expressing HsABCD1 rescue of beta-oxidation activity was best with C24:0 and C26:0 as substrates.
Mutation in the X-chromosomal adrenoleukodystrophy gene (ALD; ABCD1) leads to X-linked adrenoleukodystrophy (X-ALD), a severe neurodegenerative disorder. The encoded adrenoleukodystrophy protein (ALDP/ABCD1) is a half-size peroxisomal ATP-binding cassette protein of 745 amino acids in humans. In this study, we chose nine arbitrary mutant human ALDP forms (R104C, G116R, Y174C, S342P, Q544R, S606P, S606L, R617H, and H667D) with naturally occurring missense mutations and examined the intracellular behavior. When expressed in X-ALD fibroblasts lacking ALDP, the expression level of mutant His-ALDPs (S606L, R617H, and H667D) was lower than that of wild type and other mutant ALDPs. Furthermore, mutant ALDP-green fluorescence proteins (S606L and H667D) stably expressed in CHO cells were not detected due to rapid degradation. Interestingly, the wild type ALDP co-expressed in these cells also disappeared. In the case of X-ALD fibroblasts from an ALD patient (R617H), the mutant ALDP was not detected in the cells, but appeared upon incubation with a proteasome inhibitor. When CHO cells expressing mutant ALDP-green fluorescence protein (H667D) were cultured in the presence of a proteasome inhibitor, both the mutant and wild type ALDP reappeared. In addition, mutant His-ALDP (Y174C), which has a mutation between transmembrane domain 2 and 3, did not exhibit peroxisomal localization by immunofluorescense study. These results suggest that mutant ALDPs, which have a mutation in the COOH-terminal half of ALDP, including S606L, R617H, and H667D, were degraded by proteasomes after dimerization. Further, the region between transmembrane domain 2 and 3 is important for the targeting of ALDP to the peroxisome.
Evidence
4:
Inferred from Genetic InteractionUniProtKB
Peroxisomes play a major role in human cellular lipid metabolism, including the beta-oxidation of fatty acids. The most frequent peroxisomal disorder is X-linked adrenoleukodystrophy (X-ALD), which is caused by mutations in the ABCD1 gene. The protein involved, called ABCD1, or alternatively ALDP, is a member of the ATP-binding-cassette (ABC) transporter family and is located in the peroxisomal membrane. The biochemical hallmark of X-ALD is the accumulation of very long-chain fatty acids (VLCFAs), due to an impaired peroxisomal beta-oxidation. Although this suggests a role of ALDP in VLCFA import, no experimental evidence is available to substantiate this. In the yeast Saccharomyces cerevisiae, peroxisomes are the exclusive site of fatty acid beta-oxidation. Earlier work has shown that uptake of fatty acids into peroxisomes may occur via two routes, either as free fatty acids thus requiring intraperoxisomal activation into acyl-CoA esters or as long-chain acyl-CoA esters. The latter route involves the two peroxisomal half ABC transporters Pxa1p and Pxa2p that form a heterodimeric complex in the peroxisomal membrane. Using different strategies, including the analysis of intracellular acyl-CoA esters by tandem-MS, we show that the Pxa1p/Pxa2p heterodimer is involved in the transport of a spectrum of acyl-CoA esters. Interestingly, we found that the mutant phenotype of the pxa1/pxa2Delta mutant can be rescued, at least partially, by the sole expression of the human ABCD1 cDNA coding for ALDP, the protein that is defective in the human disease X-linked adrenoleukodystrophy. Our data indicate that ALDP can function as a homodimer and is involved in the transport of acyl-CoA esters across the peroxisomal membrane.
Peroxisomes play a major role in human cellular lipid metabolism, including the beta-oxidation of fatty acids. The most frequent peroxisomal disorder is X-linked adrenoleukodystrophy (X-ALD), which is caused by mutations in the ABCD1 gene. The protein involved, called ABCD1, or alternatively ALDP, is a member of the ATP-binding-cassette (ABC) transporter family and is located in the peroxisomal membrane. The biochemical hallmark of X-ALD is the accumulation of very long-chain fatty acids (VLCFAs), due to an impaired peroxisomal beta-oxidation. Although this suggests a role of ALDP in VLCFA import, no experimental evidence is available to substantiate this. In the yeast Saccharomyces cerevisiae, peroxisomes are the exclusive site of fatty acid beta-oxidation. Earlier work has shown that uptake of fatty acids into peroxisomes may occur via two routes, either as free fatty acids thus requiring intraperoxisomal activation into acyl-CoA esters or as long-chain acyl-CoA esters. The latter route involves the two peroxisomal half ABC transporters Pxa1p and Pxa2p that form a heterodimeric complex in the peroxisomal membrane. Using different strategies, including the analysis of intracellular acyl-CoA esters by tandem-MS, we show that the Pxa1p/Pxa2p heterodimer is involved in the transport of a spectrum of acyl-CoA esters. Interestingly, we found that the mutant phenotype of the pxa1/pxa2Delta mutant can be rescued, at least partially, by the sole expression of the human ABCD1 cDNA coding for ALDP, the protein that is defective in the human disease X-linked adrenoleukodystrophy. Our data indicate that ALDP can function as a homodimer and is involved in the transport of acyl-CoA esters across the peroxisomal membrane.
Peroxisomes play a major role in human cellular lipid metabolism, including the beta-oxidation of fatty acids. The most frequent peroxisomal disorder is X-linked adrenoleukodystrophy (X-ALD), which is caused by mutations in the ABCD1 gene. The protein involved, called ABCD1, or alternatively ALDP, is a member of the ATP-binding-cassette (ABC) transporter family and is located in the peroxisomal membrane. The biochemical hallmark of X-ALD is the accumulation of very long-chain fatty acids (VLCFAs), due to an impaired peroxisomal beta-oxidation. Although this suggests a role of ALDP in VLCFA import, no experimental evidence is available to substantiate this. In the yeast Saccharomyces cerevisiae, peroxisomes are the exclusive site of fatty acid beta-oxidation. Earlier work has shown that uptake of fatty acids into peroxisomes may occur via two routes, either as free fatty acids thus requiring intraperoxisomal activation into acyl-CoA esters or as long-chain acyl-CoA esters. The latter route involves the two peroxisomal half ABC transporters Pxa1p and Pxa2p that form a heterodimeric complex in the peroxisomal membrane. Using different strategies, including the analysis of intracellular acyl-CoA esters by tandem-MS, we show that the Pxa1p/Pxa2p heterodimer is involved in the transport of a spectrum of acyl-CoA esters. Interestingly, we found that the mutant phenotype of the pxa1/pxa2Delta mutant can be rescued, at least partially, by the sole expression of the human ABCD1 cDNA coding for ALDP, the protein that is defective in the human disease X-linked adrenoleukodystrophy. Our data indicate that ALDP can function as a homodimer and is involved in the transport of acyl-CoA esters across the peroxisomal membrane.
Adrenoleukodystrophy (ALD) is an X-linked disease affecting 1/20,000 males either as cerebral ALD in childhood or as adrenomyeloneuropathy (AMN) in adults. Childhood ALD is the more severe form, with onset of neurological symptoms between 5-12 years of age. Central nervous system demyelination progresses rapidly and death occurs within a few years. AMN is a milder form of the disease with onset at 15-30 years of age and a more progressive course. Adrenal insufficiency (Addison's disease) may remain the only clinical manifestation of ALD. The principal biochemical abnormality of ALD is the accumulation of very-long-chain fatty acids (VLCFA) because of impaired beta-oxidation in peroxisomes. The normal oxidation of VLCFA-CoA in patients' fibroblasts suggested that the gene coding for the VLCFA-CoA synthetase could be a candidate gene for ALD. Here we use positional cloning to identify a gene partially deleted in 6 of 85 independent patients with ALD. In familial cases, the deletions segregated with the disease. An identical deletion was detected in two brothers presenting with different clinical ALD phenotypes. Candidate exons were identified by computer analysis of genomic sequences and used to isolate complementary DNAs by exon connection and screening of cDNA libraries. The deduced protein sequence shows significant sequence identity to a peroxisomal membrane protein of M(r) 70K that is involved in peroxisome biogenesis and belongs to the 'ATP-binding cassette' superfamily of transporters.
A process that is carried out at the cellular level which results in the assembly, arrangement of constituent parts, or disassembly of a peroxisome. A peroxisome is a small, membrane-bounded organelle that uses dioxygen (O2) to oxidize organic molecules.
Adrenoleukodystrophy (ALD) is an X-linked disease affecting 1/20,000 males either as cerebral ALD in childhood or as adrenomyeloneuropathy (AMN) in adults. Childhood ALD is the more severe form, with onset of neurological symptoms between 5-12 years of age. Central nervous system demyelination progresses rapidly and death occurs within a few years. AMN is a milder form of the disease with onset at 15-30 years of age and a more progressive course. Adrenal insufficiency (Addison's disease) may remain the only clinical manifestation of ALD. The principal biochemical abnormality of ALD is the accumulation of very-long-chain fatty acids (VLCFA) because of impaired beta-oxidation in peroxisomes. The normal oxidation of VLCFA-CoA in patients' fibroblasts suggested that the gene coding for the VLCFA-CoA synthetase could be a candidate gene for ALD. Here we use positional cloning to identify a gene partially deleted in 6 of 85 independent patients with ALD. In familial cases, the deletions segregated with the disease. An identical deletion was detected in two brothers presenting with different clinical ALD phenotypes. Candidate exons were identified by computer analysis of genomic sequences and used to isolate complementary DNAs by exon connection and screening of cDNA libraries. The deduced protein sequence shows significant sequence identity to a peroxisomal membrane protein of M(r) 70K that is involved in peroxisome biogenesis and belongs to the 'ATP-binding cassette' superfamily of transporters.
X-Linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disorder characterized by reduced peroxisomal very long chain fatty acid (VLCFA) beta-oxidation. The X - ALD gene product (ALDP) is a peroxisomal transmembrane protein with an ATP binding cassette (ABC). ALDP and three other ABC proteins (PMP70, ALDR, P70R) localize to the peroxisomal membrane. The function of this family of peroxisomal membrane proteins is unknown. We used complementation studies to begin analysis of their role in VLCFA beta-oxidation and on the peroxisomal membrane. Expression of either ALDP or PMP70 restores VLCFA beta-oxidation in X-ALD fibroblasts, indicating overlapping functions. Their expression also restores peroxisome biogenesis in cells that are deficient in the peroxisomal membrane protein Pex2p. Thus it is likely that complex protein interactions are involved in the function and biogenesis of peroxisomal membranes that may contribute to disease heterogeneity.
Peroxisomes play a major role in human cellular lipid metabolism, including the beta-oxidation of fatty acids. The most frequent peroxisomal disorder is X-linked adrenoleukodystrophy (X-ALD), which is caused by mutations in the ABCD1 gene. The protein involved, called ABCD1, or alternatively ALDP, is a member of the ATP-binding-cassette (ABC) transporter family and is located in the peroxisomal membrane. The biochemical hallmark of X-ALD is the accumulation of very long-chain fatty acids (VLCFAs), due to an impaired peroxisomal beta-oxidation. Although this suggests a role of ALDP in VLCFA import, no experimental evidence is available to substantiate this. In the yeast Saccharomyces cerevisiae, peroxisomes are the exclusive site of fatty acid beta-oxidation. Earlier work has shown that uptake of fatty acids into peroxisomes may occur via two routes, either as free fatty acids thus requiring intraperoxisomal activation into acyl-CoA esters or as long-chain acyl-CoA esters. The latter route involves the two peroxisomal half ABC transporters Pxa1p and Pxa2p that form a heterodimeric complex in the peroxisomal membrane. Using different strategies, including the analysis of intracellular acyl-CoA esters by tandem-MS, we show that the Pxa1p/Pxa2p heterodimer is involved in the transport of a spectrum of acyl-CoA esters. Interestingly, we found that the mutant phenotype of the pxa1/pxa2Delta mutant can be rescued, at least partially, by the sole expression of the human ABCD1 cDNA coding for ALDP, the protein that is defective in the human disease X-linked adrenoleukodystrophy. Our data indicate that ALDP can function as a homodimer and is involved in the transport of acyl-CoA esters across the peroxisomal membrane.
X-Linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disorder characterized by reduced peroxisomal very long chain fatty acid (VLCFA) beta-oxidation. The X - ALD gene product (ALDP) is a peroxisomal transmembrane protein with an ATP binding cassette (ABC). ALDP and three other ABC proteins (PMP70, ALDR, P70R) localize to the peroxisomal membrane. The function of this family of peroxisomal membrane proteins is unknown. We used complementation studies to begin analysis of their role in VLCFA beta-oxidation and on the peroxisomal membrane. Expression of either ALDP or PMP70 restores VLCFA beta-oxidation in X-ALD fibroblasts, indicating overlapping functions. Their expression also restores peroxisome biogenesis in cells that are deficient in the peroxisomal membrane protein Pex2p. Thus it is likely that complex protein interactions are involved in the function and biogenesis of peroxisomal membranes that may contribute to disease heterogeneity.
Evidence
3:
Inferred from Genetic InteractionUniProtKB
The gene mutated in X-linked adrenoleukodystrophy (X-ALD) codes for the HsABCD1 protein, also named ALDP, which is a member of the superfamily of ATP-binding cassette (ABC) transporters and required for fatty acid transport across the peroxisomal membrane. Although a defective HsABCD1 results in the accumulation of very long-chain fatty acids in plasma of X-ALD patients, there is still no direct biochemical evidence that HsABCD1 actually transports very long-chain fatty acids. We used the yeast Saccharomyces cerevisiae to study the transport of fatty acids across the peroxisomal membrane. Our earlier work showed that in yeast the uptake of fatty acids into peroxisomes may occur via two routes, either as (1.) free fatty acid or as (2.) acyl-CoA ester. The latter route involves the two peroxisomal half-ABC transporters, Pxa1p and Pxa2p, which form a heterodimeric complex in the peroxisomal membrane. We here report that the phenotype of the pxa1/pxa2Δ yeast mutant, i.e. impaired growth on oleate containing medium and deficient oxidation of oleic acid, cannot only be partially rescued by human ABCD1, but also by human ABCD2 (ALDRP), which indicates that HsABCD1 and HsABCD2 can both function as homodimers. Fatty acid oxidation studies in the pxa1/pxa2Δ mutant transformed with either HsABCD1 or HsABCD2 revealed clear differences suggesting that HsABCD1 and HsABCD2 have distinct substrate specificities. Indeed, full rescue of beta-oxidation activity in cells expressing human ABCD2 was observed with C22:0 and different unsaturated very long-chain fatty acids including C24:6 and especially C22:6 whereas in cells expressing HsABCD1 rescue of beta-oxidation activity was best with C24:0 and C26:0 as substrates.
Protein involved in the transport of a molecule (metabolite, protein, etc), a ion or an electron across cell membranes, inside the cell or in a tissue fluid.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
My favorites 
My labels 
Login
