Intraperoxisomal localization of very-long-chain fatty acyl-CoA synthetase: implication in X-adrenoleukodystrophy

X-adrenoleukodystrophy (X-ALD) is a demyelinating disorder characterized by the accumulation of saturated very-long-chain (VLC) fatty acids (>C(22:0)) due to the impaired activity of VLC acyl-CoA synthetase (VLCAS). The gene responsible for X-ALD was found to code for a peroxisomal integral membrane protein (ALDP) that belongs to the ATP binding cassette superfamily of transporters. To understand the function of ALDP and how ALDP and VLCAS interrelate in the peroxisomal beta-oxidation of VLC fatty acids we investigated the peroxisomal topology of VLCAS protein. Antibodies raised against a peptide toward the C-terminus of VLCAS as well as against the N-terminus were used to define the intraperoxisomal localization and orientation of VLCAS in peroxisomes. Indirect immunofluorescent and electron microscopic studies show that peroxisomal VLCAS is localized on the matrix side. This finding was supported by protease protection assays and Western blot analysis of isolated peroxisomes. To further address the membrane topology of VLCAS, Western blot analysis of total membranes or integral membranes prepared from microsomes and peroxisomes indicates that VLCAS is a peripheral membrane-associated protein in peroxisomes, but an integral membrane in microsomes. Moreover, peroxisomes isolated from cultured skin fibroblasts from X-ALD patients with a mutation as well as a deletion in ALDP showed a normal amount of VLCAS. The consequence of VLCAS being localized to the luminal side of peroxisomes suggests that ALDP may be involved in stabilizing VLCAS activity, possibly through protein-protein interactions, and that loss or alterations in these interactions may account for the observed loss of peroxisomal VLCAS activity in X-ALD.     

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.     

The ABC transporter proteins Pat1 and Pat2 are required for import of long-chain fatty acids into peroxisomes of Saccharomyces cerevisiae.

Peroxisomes of Saccharomyces cerevisiae are the exclusive site of fatty acid beta-oxidation. We have found that fatty acids reach the peroxisomal matrix via two independent pathways. The subcellular site of fatty acid activation varies with chain length of the substrate and dictates the pathway of substrate entry into peroxisomes. Medium-chain fatty acids are activated inside peroxisomes hby the acyl-CoA synthetase Faa2p. On the other hand, long-chain fatty acids are imported from the cytosolic pool of activated long-chain fatty acids via Pat1p and Pat2p, peroxisomal membrane proteins belonging to the ATP binding cassette transporter superfamily. Pat1p and Pat2p are the first examples of membrane proteins involved in metabolite transport across the peroxisomal membrane.     

Transport of fatty acids into human and rat peroxisomes. Differential transport of palmitic and lignoceric acids and its implication to X-adrenoleukodystrophy.

The different topology of palmitoyl-CoA ligase (on the cytoplasmic surface) and of lignoceroyl-CoA ligase (on the luminal surface) in peroxisomal membranes suggests that these fatty acids may be transported in different form through the peroxisomal membrane (Lazo, O., Contreras, M., and Singh, I. (1990) Biochemistry 29, 3981-3986), and this differential transport may account for deficient oxidation of lignoceric acid in X-adrenoleukodystrophy (X-ALD) (Singh, I., Moser, A. B., Goldfisher, S., and Moser, H. W. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 4203-4207). To define the transport mechanism for these fatty acids through the peroxisomal membrane and its possible implication to lignoceric acid metabolism in X-ALD, we examined cofactors and energy requirements for the transport of palmitic and lignoceric acids in isolated peroxisomes from rat liver and peroxisomes isolated from X-ALD and control fibroblasts. The similar rates of transport of palmitoyl-CoA (87.6 +/- 6.3 nmol/h/mg protein) and palmitic acid in the fatty acid activating conditions (83.4 +/- 5.1 nmol/h/mg protein) and lack of transport of palmitic acid (4% of palmitoyl-CoA transport) when ATP and/or CoASH were removed or substituted by alpha,beta-methyleneadenosine-5'-triphosphate (AMPCPOP) and/or desulfoCoA-agarose from assay medium clearly demonstrate that transport of palmitic acid requires prior synthesis of palmitoyl-CoA by palmitoyl-CoA ligase on the cytoplasmic surface of peroxisomes. The 10-fold higher rate of transport of lignoceric acid (5.3 +/- 0.6 nmol/h/mg protein) as compared with lignoceroyl-CoA (0.41 +/- 0.11 nmol/h/mg protein) and lack of inhibition of transport of lignoceric acid when ATP and/or CoASH were removed or substituted with AMPCPOP or desulfoCoA-agarose suggest that lignoceric acid is transported through the peroxisomal membrane as such. Moreover, the lack of effect of removal of ATP or substitution with AMPOPCP (a nonhydrolyzable substrate) demonstrates that the translocation of palmitoyl-CoA and lignoceric acid across peroxisomal membrane does not require energy. The transport, activation, and oxidation of palmitic acid are normal in peroxisomes from X-ALD. The deficient lignoceroyl-CoA ligase (13% of control) and oxidation of lignoceric acid (10% of control) as compared with normal transport of lignoceric acid into peroxisomes from X-ALD clearly demonstrates that pathogenomonic accumulation of very long chain fatty acids (greater than C22) in X-ALD is due to the deficiency of peroxisomal lignoceroyl-CoA ligase activity.     

Mouse very long-chain acyl-CoA synthetase in X-linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disorder characterized by accumulation of very long-chain fatty acids (VLCFA). This accumulation has been attributed to decreased VLCFA beta-oxidation and peroxisomal very long-chain acyl-CoA synthetase (VLCS) activity. The X-ALD gene, ABCD1, encodes a peroxisomal membrane ATP binding cassette transporter, ALDP, that is hypothesized to affect VLCS activity in peroxisomes by direct interaction with the VLCS enzyme. Recently, a VLCS gene that encodes a protein with significant sequence identity to known rat and human peroxisomal VLCS protein has been identified in mice. We find that the mouse VLCS gene (Vlcs) encodes an enzyme (Vlcs) with VLCS activity that localizes to peroxisomes and is expressed in X-ALD target tissues. We show that the expression of Vlcs in the peroxisomes of X-ALD mouse fibroblasts improves VLCFA beta-oxidation in these cells, implying a role for this enzyme in the biochemical abnormality of X-ALD. X-ALD mice, which accumulate VLCFA in tissues, show no change in the expression of Vlcs, the subcellular localization of Vlcs, or general peroxisomal VLCS activity. These observations imply that ALDP is not necessary for the proper expression or localization of Vlcs protein, and the control of VLCFA levels does not depend on the direct interaction of Vlcs and ALDP.     

Adrenoleukodystrophy: impaired oxidation of fatty acids due to peroxisomal lignoceroyl-CoA ligase deficiency

Very long chain fatty acids (lignoceric acid) are oxidized in peroxisomes and pathognomonic amounts of these fatty acids accumulate in X-adrenoleukodystrophy (X-ALD) due to a defect in their oxidation. However, in cellular homogenates from X-ALD cells, lignoceric acid is oxidized at a rate of 38% of control cells. Therefore, to identify the source of this residual activity we raised antibody to palmitoyl-CoA ligase and examined its effect on the activation and oxidation of palmitic and lignoceric acids in isolated peroxisomes from control and X-ALD fibroblasts. The normalization of peroxisomal lignoceric acid oxidation in the presence of exogenously added acyl-CoA ligases and along with the complete inhibition of activation and oxidation of palmitic and lignoceric acids in peroxisomes from X-ALD by antibody to palmitoyl-CoA ligase provides direct evidence that lignoceroyl-CoA ligase is deficient in X-ALD and demonstrates that the residual activity for the oxidation of lignoceric acid was derived from the activation of lignoceric acid by peroxisomal palmitoyl-CoA ligase. This antibody inhibited the activation and oxidation of palmitic acid but had little effect on these activities for lignoceric acid in peroxisomes from control cells. Furthermore, these data provide evidence that peroxisomal palmitoyl-CoA and lignoceroyl-CoA ligases are two different enzymes.     

Molecular organization of peroxisomal enzymes: protein-protein interactions in the membrane and in the matrix

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.     

Thermogenic flux induced by lignoceric acid in peroxisomes isolated from HepG2 cells and from X-adrenoleukodystrophy and control fibroblasts

This work analyzes the thermogenic flux induced by the very long-chain fatty acid (VLCFA) lignoceric acid (C24:0) in isolated peroxisomes. Specific metabolic alterations of peroxisomes are related to a variety of disorders, the most frequent one being the neurodegenerative inherited disease X-linked adrenoleukodystrophy (X-ALD). A peroxisomal transport protein is mutated in this disorder. Due to reduced catabolism and enhanced fatty acid (FA) elongation, VLCFA accumulates in plasma and in all tissues, contributing to the clinical manifestations of this disorder. During peroxisomal metabolism, heat is produced but it is considered lost. Instead, it is a form of energy that could play a role in molecular mechanisms of this pathology and other neurodegenerative disorders. The thermogenic flux induced by lignoceric acid (C24:0) was estimated by isothermal titration calorimetry in peroxisomes isolated from HepG2 cells and from fibroblasts obtained from patients with X-ALD and healthy subjects. Heat flux induced by lignoceric acid in HepG2 peroxisomes was exothermic, indicating normal peroxisomal metabolism. In X-ALD peroxisomes the heat flux was endothermic, indicating the requirement of heat/energy, possibly for cellular metabolism. In fibroblasts from healthy subjects, the effect was less pronounced than in HepG2, a kind of cell known to have greater FA metabolism than fibroblasts. Our hypothesis is that heat is not lost but it could act as an activator, for example on the heat-sensitive pathway related to TRVP2 receptors. To investigate this hypothesis we focused on peroxisomal metabolism, considering that impaired heat generation could contribute to the development of peroxisomal neurodegenerative disorders.     

Impaired Very Long-chain Acyl-CoA β-Oxidation in Human X-linked Adrenoleukodystrophy Fibroblasts Is a Direct Consequence of ABCD1 Transporter Dysfunction

X-linked adrenoleukodystrophy (X-ALD), an inherited peroxisomal disorder, is caused by mutations in the ABCD1 gene encoding the peroxisomal ATP-binding cassette (ABC) transporter ABCD1 (adrenoleukodystrophy protein, ALDP). Biochemically, X-ALD is characterized by an accumulation of very long-chain fatty acids and partially impaired peroxisomal β-oxidation. In this study, we used primary human fibroblasts from X-ALD and Zellweger syndrome patients to investigate the peroxisomal β-oxidation defect. Our results show that the degradation of C26:0-CoA esters is as severely impaired as degradation of unesterified very long-chain fatty acids in X-ALD and is abolished in Zellweger syndrome. Interestingly, the β-oxidation rates for both C26:0-CoA and C22:0-CoA were similarly affected, although C22:0 does not accumulate in patient fibroblasts. Furthermore, we show that the β-oxidation defect in X-ALD is directly caused by ABCD1 dysfunction as blocking ABCD1 function with a specific antibody reduced β-oxidation to levels observed in X-ALD fibroblasts. By quantification of mRNA and protein levels of the peroxisomal ABC transporters and by blocking with specific antibodies, we found that residual β-oxidation activity toward C26:0-CoA in X-ALD fibroblasts is mediated by ABCD3, although the efficacy of ABCD3 appeared to be much lower than that of ABCD1. Finally, using isolated peroxisomes, we show that β-oxidation of C26:0-CoA is independent of additional CoA but requires a cytosolic factor of >10-kDa molecular mass that is resistant to N-ethylmaleimide and heat inactivation. In conclusion, our findings in human cells suggest that, in contrast to yeast cells, very long-chain acyl-CoA esters are transported into peroxisomes by ABCD1 independently of additional synthetase activity.     

Effect of clofibrate on peroxisomal lignoceroyl-CoA ligase activity

The effect of a 2-week clofibrate (0.5%)-fortified diet on peroxisomal palmitoyl-CoA and lignoceroyl-CoA ligases was studied. The activities of palmitoyl-CoA and lignoceroyl-CoA ligases in peroxisomes isolated from clofibrate-treated animals were 4.4- and 4.0-fold higher than those of the controls. The different degrees of increases in these two enzyme activities support the previous conclusions that in peroxisomes palmitoyl-CoA ligase and lignoceroyl-CoA ligase are different enzymes. Since clofibrate treatment increases both of these peroxisomal acyl-CoA ligase activities and normal palmitoyl-CoA ligase is the source of the partial activity for the oxidation of lignoceric acid in X-ALD, treatment with a hypolipidemic drug, which can increase human peroxisomal enzyme activities, may be helpful in lowering the amount of the pathogen, VLC fatty acids, in X-ALD.     

Identification of a new fatty acid synthesis-transport machinery at the peroxisomal membrane

The neurodegenerative disease X-linked adrenoleukodystrophy (X-ALD) is characterized by the abnormal accumulation of very long chain fatty acids. Mutations in the gene encoding the peroxisomal ATP-binding cassette half-transporter, adrenoleukodystrophy protein (ALDP), are the primary cause of X-ALD. To gain a better understanding of ALDP dysfunction, we searched for interaction partners of ALDP and identified binary interactions to proteins with functions in fatty acid synthesis (ACLY, FASN, and ACC) and activation (FATP4), constituting a thus far unknown fatty acid synthesis-transport machinery at the cytoplasmic side of the peroxisomal membrane. This machinery adds to the knowledge of the complex mechanisms of peroxisomal fatty acid metabolism at a molecular level and elucidates potential epigenetic mechanisms as regulatory processes in the pathogenesis and thus the clinical course of X-ALD.     

Peroxisomal ABC transporters: Structure, function and role in disease

ATP-binding cassette (ABC) transporters belong to one of the largest families of membrane proteins, and are present in almost all living organisms from eubacteria to mammals. They exist on plasma membranes and intracellular compartments such as the mitochondria, peroxisomes, endoplasmic reticulum, Golgi apparatus and lysosomes, and mediate the active transport of a wide variety of substrates in a variety of different cellular processes. These include the transport of amino acids, polysaccharides, peptides, lipids and xenobiotics, including drugs and toxins. Three ABC transporters belonging to subfamily D have been identified in mammalian peroxisomes. The ABC transporters are half-size and assemble mostly as a homodimer after posttranslational transport to peroxisomal membranes. ABCD1/ALDP and ABCD2/ALDRP are suggested to be involved in the transport of very long chain acyl-CoA with differences in substrate specificity, and ABCD3/PMP70 is involved in the transport of long and branched chain acyl-CoA. ABCD1 is known to be responsible for X-linked adrenoleukodystrophy (X-ALD), an inborn error of peroxisomal β-oxidation of very long chain fatty acids. Here, we summarize recent advances and important points in our advancing understanding of how these ABC transporters target and assemble to peroxisomal membranes and perform their functions in physiological and pathological processes, including the neurodegenerative disease, X-ALD. This article is part of a Special Issue entitled: Metabolic Functions and Biogenesis of Peroxisomes in Health and Disease.     

Plant peroxisomal solute transporter proteins

Plant peroxisomes are unique subcellular organelles which play an indispensable role in several key metabolic pathways, including fatty acid b-oxidation,photorespiration, and degradation of reactive oxygen species. The compartmentalization of metabolic pathways into peroxisomes is a strategy for organizing the metabolic network and improving pathway efficiency. An important prerequisite, however, is the exchange of metabolites between peroxisomes and other cell compartments. Since the first studies in the 1970s scientists contributed to understanding how solutes enter or leave this organelle.This review gives an overview about our current knowledge of the solute permeability of peroxisomal membranes described in plants, yeast, mammals and other eukaryotes. In general, peroxisomes contain in their bilayer membrane specific transporters for hydrophobic fatty acids(ABC transporter) and large cofactor molecules(carrier for ATP, NAD and CoA). Smaller solutes with molecular masses below 300–400 Da, like the organic acids malate, oxaloacetate, and 2-oxoglutarate, are shuttled via non-selective channels across the peroxisomal membrane.In comparison to yeast, human, mammals and other eukaryotes, the function of these known peroxisomal transporters and channels in plants are discussed in this review.     

Conservation of PEX19-binding motifs required for protein targeting to mammalian peroxisomal and trypanosome glycosomal membranes

Glycosomes are divergent peroxisomes found in trypanosomatid protozoa, including those that cause severe human diseases throughout much of the world. While peroxisomes are dispensable for both yeast (Saccharomyces cerevisiae and others) and mammalian cells in vitro, glycosomes are essential for trypanosomes and hence are viewed as a potential drug target. The import of proteins into the matrix of peroxisomes utilizes multiple peroxisomal membrane proteins which require the peroxin PEX19 for insertion into the peroxisomal membrane. In this report, we show that the specificity of peroxisomal membrane protein binding for Trypanosoma brucei PEX19 is very similar to those previously identified for human and yeast PEX19. Our studies show that trafficking is conserved across these distant phyla and that both a PEX19 binding site and a transmembrane domain are required for the insertion of two test proteins into the glycosomal membrane. However, in contrast to T. brucei PEX10 and PEX12, T. brucei PEX14 does not traffic to human peroxisomes, indicating that it is not recognized by the human PEX14 import mechanism.     

Disruption of a yeast very-long-chain acyl-CoA synthetase gene simulates the cellular phenotype of X-linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is characterized biochemically by elevated levels of saturated very long-chain fatty acids (VLCFAs) in plasma and tissues. In X-ALD, peroxisomal very-long-chain acyl-CoA synthetase (VLCS) fails to activate VLCFAs, preventing their degradation via β-oxidation. However, the product of the defective XALD gene (ALDP) is not a VLCS, but rather a peroxisomal membrane protein (PMP). Disruption of either or both of two yeast PMP genes related to the XALD gene did not produce a biochemical phenotype resembling that found in X-ALD fibroblasts. The authors identified a candidate yeast VLCS gene (the FAT1 locus) by its homology to rat liver VLCS. Disruption of this gene decreased VLCS activity, but had no effect on long-chain acyl-CoA synthetase activity. In FAT1-disruption strains, VLCS activity was reduced to 30–40% of wild-type in both a microsome-rich 27,000g supernatant fraction and a peroxisome- and mitochondria-rich pellet fraction of yeast spheroplast homogenates. Separation of the latter organelles by density gradient centrifugation revealed that VLCS activity was peroxisomal and not mitochondrial. VLCS gene-disruption strains had increased cellular VLCFA levels, compared to wild-type yeast. The extent of both the decrease in peroxisomal VLCS activity and the VLCFA accumulation in this yeast model resembles that observed in cells from X-ALD patients. Characterization of the gene(s) responsible for the residual peroxisomal VLCS activity may suggest new therapeutic approaches in X-ALD.     

Role of ALDP (ABCD1) and mitochondria in X-linked adrenoleukodystrophy

Peroxisomal disorders have been associated with malfunction of peroxisomal metabolic pathways, but the pathogenesis of these disorders is largely unknown. X-linked adrenoleukodystrophy (X-ALD) is associated with elevated levels of very-long-chain fatty acids (VLCFA; C(>22:0)) that have been attributed to reduced peroxisomal VLCFA beta-oxidation activity. Previously, our laboratory and others have reported elevated VLCFA levels and reduced peroxisomal VLCFA beta-oxidation in human and mouse X-ALD fibroblasts. In this study, we found normal levels of peroxisomal VLCFA beta-oxidation in tissues from ALD mice with elevated VLCFA levels. Treatment of ALD mice with pharmacological agents resulted in decreased VLCFA levels without a change in VLCFA beta-oxidation activity. These data indicate that ALDP does not determine the rate of VLCFA beta-oxidation and that VLCFA levels are not determined by the rate of VLCFA beta-oxidation. The rate of peroxisomal VLCFA beta-oxidation in human and mouse fibroblasts in vitro is affected by the rate of mitochondrial long-chain fatty acid beta-oxidation. We hypothesize that ALDP facilitates the interaction between peroxisomes and mitochondria, resulting, when ALDP is deficient in X-ALD, in increased VLCFA accumulation despite normal peroxisomal VLCFA beta-oxidation in ALD mouse tissues. In support of this hypothesis, mitochondrial structural abnormalities were observed in adrenal cortical cells of ALD mice.     

Lipid composition of peroxisomes from the yeast Pichia pastoris grown on different carbon sources

Highly purified peroxisomes from the yeast Pichia pastoris grown on methanol or oleic acid, respectively, were used to characterize the lipid composition of this organelle. For this purpose, an isolation procedure had to be adapted which yielded highly purified P. pastoris peroxisomes. When peroxisome proliferation was induced by growth on methanol, alcohol oxidase was the predominant peroxisomal protein. Cultivation of P. pastoris on oleic acid led to induction of a family of peroxisomal enzymes catalyzing fatty acid beta-oxidation, whose most prominent members were identified by mass spectrometry. On either carbon source, phosphatidylcholine and phosphatidylethanolamine were the major peroxisomal phospholipids, and cardiolipin was present in peroxisomal membranes at a substantial amount, indicating that this phospholipid is a true peroxisomal component. Ergosterol was the most abundant sterol of P. pastoris peroxisomal membranes irrespective of the culture conditions. The fatty acid composition of whole cells and peroxisomes was highly affected by cultivation of P. pastoris on oleic acid. Under these conditions, oleic acid became the predominant fatty acid in phospholipids from total cell and peroxisomal extracts. Thus, oleic acid was not only utilized as an appropriate carbon source but also as a building block for complex membrane lipids. In summary, our data provide first insight into biochemical properties of P. pastoris peroxisomal membranes, which may become important for the biotechnological use of this yeast.     

Cholesterol-deprivation increases mono-unsaturated very long-chain fatty acids in skin fibroblasts from patients with X-linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is the most common peroxisomal disorder and is characterized by a striking and unpredictable variation in phenotypic expression. It ranges from a rapidly progressive and fatal cerebral demyelinating disease in childhood (CCALD), to the milder slowly progressive form in adulthood (AMN). X-ALD is caused by mutations in the ABCD1 gene that encodes a peroxisomal membrane located ABC half-transporter named ALDP. Mutations in ALDP result in reduced beta-oxidation of very long-chain fatty acids (VLCFA, >22 carbon atoms) in peroxisomes and elevated levels of VLCFA in plasma and tissues. Previously, it has been shown that culturing skin fibroblasts from X-ALD patients in lipoprotein-deficient medium results in reduced VLCFA levels and increased expression of the functionally redundant ALD-related protein (ALDRP). The aim of this study was to further resolve the interaction between cholesterol and VLCFA metabolism in X-ALD. Our data show that the reduction in 26:0 in X-ALD fibroblasts grown in lipoprotein-deficient culture medium (free of cholesterol) is offset by a significant increase in both the level and synthesis of 26:1. We also demonstrate that cholesterol-deprivation results in increased expression of stearoyl-CoA-desaturase (SCD) and increased desaturation of 18:0 to 18:1. Finally, there was no increase in [1-(14)C]-26:0 beta-oxidation. Taken together, we conclude that cholesterol-deprivation reduces saturated VLCFA, but increases mono-unsaturated VLCFA. These data may have implications for treatment of X-ALD patients with lovastatin.     

X-Linked Adrenoleukodystrophy: Genes,Mutations, and Phenotypes

X-linked adrenoleukodystrophy (X-ALD) is a complex and perplexing neurodegenerative disorder. The metabolic abnormality, elevated levels of very long-chain fatty acids in tissues and plasma, and the biochemical defect, reduced peroxisomal very long-chain acyl-CoA synthetase (VLCS) activity, are ubiquitous features of the disease. However, clinical manifestations are highly variable with regard to time of onset, site of initial pathology and rate of progression. In addition, the abnormal gene in X-ALD is not the gene for VLCS. Rather, it encodes a peroxisomal membrane protein with homology to the ATP-binding cassette (ABC) transmembrane transporter superfamily of proteins. The X-ALD protein (ALDP) is closely related to three other peroxisomal membrane ABC proteins. In this report we summarize all known X-ALD mutations and establish the lack of an X-ALD genotype/phenotype correlation. We compare the evolutionary relationships among peroxisomal ABC proteins, demonstrate that ALDP forms homodimers with itself and heterodimers with other peroxisomal ABC proteins and present cDNA complementation studies suggesting that the peroxisomal ABC proteins have overlapping functions. We also establish that there are at least two peroxisomal VLCS activities, one that is ALDP dependent and one that is ALDP independent. Finally, we discuss variable expression of the peroxisomal ABC proteins and ALDP independent VLCS in relation to the variable clinical presentations of X-ALD.     

Identification and functional reconstitution of the yeast peroxisomal adenine nucleotide transporter

The requirement for small molecule transport systems across the peroxisomal membrane has previously been postulated, but not directly proven. Here we report the identification and functional reconstitution of Ant1p (Ypr128cp), a peroxisomal transporter in the yeast Saccharomyces cerevisiae, which has the characteristic sequence features of the mitochondrial carrier family. Ant1p was found to be an integral protein of the peroxisomal membrane and expression of ANT1 was oleic acid inducible. Targeting of Ant1p to peroxisomes was dependent on Pex3p and Pex19p, two peroxins specifically required for peroxisomal membrane protein insertion. Ant1p was essential for growth on medium-chain fatty acids as the sole carbon source. Upon reconstitution of the overexpressed and purified protein into liposomes, specific transport of adenine nucleotides could be demonstrated. Remarkably, both the substrate and inhibitor specificity differed from those of the mitochondrial ADP/ATP transporter. The physiological role of Ant1p in S.cerevisiae is probably to transport cytoplasmic ATP into the peroxisomal lumen in exchange for AMP generated in the activation of fatty acids.