Wouter F. Visser

The human peroxisomal ABC half transporter ALDP functions as a homodimer and accepts acyl-CoA esters

Peroxisomes play a major role in human cellular lipid metabolism, including the β‐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 im paired peroxisomal β‐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 β‐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 Pxalp and Pxa2p that form a heterodimeric complex in the perox isomal 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/pxa2Δ mutant can be rescued, at least par tially, 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 ho modimer and is involved in the transport of acyl‐CoA esters across the peroxisomal membrane.— van Roer mund, C. W. T., Visser, W. F., IJlst, L., van Cruchten, A., Boek, M., Kulik, W., Waterham, H. R., Wanders, R. J. A. The human peroxisomal ABC half transporter ALDP functions as a homodimer and accepts acyl–CoA esters. FASEB J. 22, 4201–4208 (2008)

Metabolite transport across the peroxisomal membrane

In recent years, much progress has been made with respect to the unravelling of the functions of peroxisomes in metabolism, and it is now well established that peroxisomes are indispensable organelles, especially in higher eukaryotes. Peroxisomes catalyse a number of essential metabolic functions including fatty acid beta-oxidation, ether phospholipid biosynthesis, fatty acid alpha-oxidation and glyoxylate detoxification. The involvement of peroxisomes in these metabolic pathways necessitates the transport of metabolites in and out of peroxisomes. Recently, considerable progress has been made in the characterization of metabolite transport across the peroxisomal membrane. Peroxisomes posses several specialized transport systems to transport metabolites. This is exemplified by the identification of a specific transporter for adenine nucleotides and several half-ABC (ATP-binding cassette) transporters which may be present as hetero- and homo-dimers. The nature of the substrates handled by the different ABC transporters is less clear. In this review we will describe the current state of knowledge of the permeability properties of the peroxisomal membrane.

The peroxisomal ABC transporter family

This review describes the current state of knowledge about the ABCD family of peroxisomal half adenosine-triphosphate-binding cassette (ABC) transporters. ABCDs are predicted to be present in a variety of eukaryotic organisms, although at present, only ABCDs in the yeast Saccharomyces cerevisiae, the plant Arabidopsis thaliana, and different mammalian species have been identified and characterized to any significant extent. The functional role of none of these ABCDs has been established definitively and awaits successful reconstitution of ABCDs, either as homo- or heterodimers into liposomes, followed by transport studies. Data obtained in S. cerevisiae suggest that the two ABCDs, which have been identified in this organism, form a heterodimer, which actually transports acyl coenzyme A esters across the peroxisomal membrane. In mammals, four ABCDs have been identified, of which one [adrenoleukodystrophy protein (ALDP)] has been implicated in the transport of the coenzyme A esters of very-long-chain fatty acids. Mutations in the gene (ABCD1) encoding ALDP are the cause of a severe X-linked disease, called X-linked adrenoleukodystrophy. The availability of mutant mice in which Abcd1, Abcd2, or Abcd3 have been disrupted will help to resolve the true role of the peroxisomal half-ABC transporters.

Identification of human PMP34 as a peroxisomal ATP transporter.

In recent years much has been learned about the essential role of peroxisomes in cellular metabolism. Much less, however, is known about the permeability properties of peroxisomes although it is well established now that peroxisomes are impermeable to small molecules which implies the existence of transporters in the peroxisomal membrane. In this paper we report the identification of PMP34, a peroxisomal membrane protein belonging to the mitochondrial solute carrier family, as an adenine nucleotide transporter. This is concluded from different experimental findings including rescue of the defect in medium-chain fatty acid oxidation in Saccharomyces cerevisiae cells in which the ANT1 gene coding for Ant1p, the peroxisomal adenine nucleotide carrier, was disrupted. Furthermore, we have purified PMP34, reconstituted the protein in proteoliposomes, and provide direct proof that PMP34 is an adenine nucleotide transporter.

Differential substrate specificities of human ABCD1 and ABCD2 in peroxisomal fatty acid β-oxidation

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.

A novel bile acid biosynthesis defect due to a deficiency of peroxisomal ABCD3

ABCD3 is one of three ATP-binding cassette (ABC) transporters present in the peroxisomal membrane catalyzing ATP-dependent transport of substrates for metabolic pathways localized in peroxisomes. So far, the precise function of ABCD3 is not known. Here, we report the identification of the first patient with a defect of ABCD3. The patient presented with hepatosplenomegaly and severe liver disease and showed a striking accumulation of peroxisomal C27-bile acid intermediates in plasma. Investigation of peroxisomal parameters in skin fibroblasts revealed a reduced number of enlarged import-competent peroxisomes. Peroxisomal beta-oxidation of C26:0 was normal, but beta-oxidation of pristanic acid was reduced. Genetic analysis revealed a homozygous deletion at the DNA level of 1758bp, predicted to result in a truncated ABCD3 protein lacking the C-terminal 24 amino acids (p.Y635NfsX1). Liver disease progressed and the patient required liver transplantation at 4 years of age but expired shortly after transplantation. To corroborate our findings in the patient, we studied a previously generated Abcd3 knockout mouse model. Abcd3-/- mice accumulated the branched chain fatty acid phytanic acid after phytol loading. In addition, analysis of bile acids revealed a reduction of C24 bile acids, whereas C27-bile acid intermediates were significantly increased in liver, bile and intestine of Abcd3-/- mice. Thus, both in the patient and in Abcd3-/- mice, there was evidence of a bile acid biosynthesis defect. In conclusion, our studies show that ABCD3 is involved in transport of branched-chain fatty acids and C27 bile acids into the peroxisome and that this is a crucial step in bile acid biosynthesis.

Carnitine-dependent transport of acetyl coenzyme A in Candida albicans is essential for growth on nonfermentable carbon sources and contributes to biofilm formation.

ABSTRACT In eukaryotes, acetyl coenzyme A (acetyl-CoA) produced during peroxisomal fatty acid β-oxidation needs to be transported to mitochondria for further metabolism. Two parallel pathways for acetyl-CoA transport have been identified in Saccharomyces cerevisiae; one is dependent on peroxisomal citrate synthase (Cit), while the other requires peroxisomal and mitochondrial carnitine acetyltransferase (Cat) activities. Here we show that the human fungal pathogen Candida albicans lacks peroxisomal Cit, relying exclusively on Cat activity for transport of acetyl units. Deletion of the CAT2 gene encoding the major Cat enzyme in C. albicans resulted in a strain that had lost both peroxisomal and mitochondrion-associated Cat activities, could not grow on fatty acids or C2 carbon sources (acetate or ethanol), accumulated intracellular acetyl-CoA, and showed greatly reduced fatty acid β-oxidation activity. The cat2 null mutant was, however, not attenuated in virulence in a mouse model of systemic candidiasis. These observations support our previous results showing that peroxisomal fatty acid β-oxidation activity is not essential for C. albicans virulence. Biofilm formation by the cat2 mutant on glucose was slightly reduced compared to that by the wild type, although both strains grew at the same rate on this carbon source. Our data show that C. albicans has diverged considerably from S. cerevisiae with respect to the mechanism of intracellular acetyl-CoA transport and imply that carnitine dependence may be an important trait of this human fungal pathogen.

Demonstration and characterization of phosphate transport in mammalian peroxisomes.

It is now well established that the peroxisomal membrane is not freely permeable to small molecules in vivo, which implies the existence of metabolite transporters in the peroxisomal membrane. A few putative peroxisomal metabolite transporters have indeed been identified, but the function of these proteins has remained largely unresolved so far. The only peroxisomal transporter characterized to a significant extent is the adenine nucleotide transporter, which is presumably required to sustain the activity of the intraperoxisomal very-long-chain-acyl-CoA synthetase. In addition to AMP, this acyl-CoA synthetase also produces pyrophosphate, which must be exported from the peroxisome. In the present study, we demonstrate that the peroxisomal membrane contains a transporter activity that facilitates the passage of phosphate and possibly pyrophosphate across the peroxisomal membrane. By reconstitution of peroxisomal membrane proteins in proteoliposomes, some kinetic parameters of the transporter could be established in vitro. The transporter can be distinguished from the mitochondrial phosphate transporter by its different sensitivity to inhibitors.

Peroxisomal Fatty Acid Alpha-and Beta-Oxidation in Health and Disease: New insights

In humans, peroxisomes play a number of essential metabolic functions, of which most have to do with lipid metabolism including fatty acid alpha-and beta-oxidation. The importance of the peroxisomal alpha-and alpha-and systems in humans is stressed by the existence of a number of peroxisomal disorders, in which one or both of these pathways are disturbed. X-linked adrenoleukodystrophy is the most well known among the disorders of peroxisomal beta-oxidation whereas Refsum disease is the prototype of the group of alpha-oxidation defects. In this paper we will describe the basic aspects of the peroxisomal alpha-and beta-oxidation systems with particular emphasis on recently acquired knowledge.