C. W. T. van Roermund

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.

The membrane of peroxisomes in Saccharomyces cerevisiae is impermeable to NAD(H) and acetyl-CoA under in vivo conditions

We investigated how NADH generated during peroxisomal beta‐oxidation is reoxidized to NAD+ and how the end product of beta‐oxidation, acetyl‐CoA, is transported from peroxisomes to mitochondria in Saccharomyces cerevisiae. Disruption of the peroxisomal malate dehydrogenase 3 gene (MDH3) resulted in impaired beta‐oxidation capacity as measured in intact cells, whereas beta‐oxidation was perfectly normal in cell lysates. In addition, mdh3‐disrupted cells were unable to grow on oleate whereas growth on other non‐fermentable carbon sources was normal, suggesting that MDH3 is involved in the reoxidation of NADH generated during fatty acid beta‐oxidation rather than functioning as part of the glyoxylate cycle. To study the transport of acetyl units from peroxisomes, we disrupted the peroxisomal citrate synthase gene (CIT2). The lack of phenotype of the cit2 mutant indicated the presence of an alternative pathway for transport of acetyl units, formed by the carnitine acetyltransferase protein (YCAT). Disruption of both the CIT2 and YCAT gene blocked the beta‐oxidation in intact cells, but not in lysates. Our data strongly suggest that the peroxisomal membrane is impermeable to NAD(H) and acetyl‐CoA in vivo, and predict the existence of metabolite carriers in the peroxisomal membrane to shuttle metabolites from peroxisomes to cytoplasm and vice versa.

Peroxisomal and mitochondrial carnitine acetyltransferases of Saccharomyces cerevisiae are encoded by a single gene.

Carnitine acetyltransferase (CAT) is present in mitochondria and peroxisomes of oleate‐grown Saccharomyces cerevisiae. Both proteins are encoded by the same gene, YCAT, which encodes a protein with a mitochondrial targeting signal (MTS) at the N‐terminus, and a peroxisomal targeting signal type 1 (PTS‐1) at the C‐terminus. Deletion of both motifs revealed the presence of an additional internal targeting sequence. Import of CAT via this internal signal was shown to be dependent on PAS10, a protein which is required for the import of PTS‐1 containing proteins. An interaction of PAS10 with this internal targeting signal was demonstrated using the yeast two‐hybrid technique. Expression of the YCAT gene behind a heterologous promoter resulted in loss of peroxisomal targeting, indicating that differential targeting is controlled at transcriptional or translational level. Determination of the 5′‐ends of YCAT mRNAs revealed that YCAT transcripts initiating after the first AUG were present in oleate‐grown cells. These transcripts were virtually absent in acetate‐ or glycerol‐grown cells. We propose that in response to oleate, shorter transcripts are produced from which the peroxisomal form of CAT is translated, resulting in a CAT protein without a MTS, which can be targeted to peroxisomes.

Direct demonstration that the deficient oxidation of very long chain fatty acids in X-linked adrenoleukodystrophy is due to an impaired ability of peroxisomes to activate very long chain fatty acids

A method was developed to prepare peroxisome-enriched fractions depleted of microsomes and mitochondria from cultured skin fibroblasts. The method consists of differential centrifugation of a postnuclear supernatant followed by density gradient centrifugation on a discontinuous Metrizamide gradient. The activity of hexacosanoyl-CoA synthetase was subsequently measured in postnuclear supernatants and peroxisome-enriched fractions prepared from cultured skin fibroblasts from control subjects and patients with X-linked adrenoleukodystrophy. Whereas the hexacosanoyl-CoA synthetase activity in postnuclear supernatants of X-linked adrenoleukodystrophy fibroblasts was only slightly decreased (77.8 +/- 4.4% of control (n = 15], enzyme activity was found to be much more markedly reduced in peroxisomal fractions isolated from the mutant fibroblasts (19.6 +/- 6.7% of control (n = 5]. This is a direct demonstration that the defect in X-linked adrenoleukodystrophy is at the level of a deficient ability of peroxisomes to activate very long chain fatty acids, as first suggested by Hashmi et al. [Hashmi, M., Stanley, W. and Singh, I. (1986) FEBS Lett. 86, 247-250].

Peroxisomal beta-oxidation of polyunsaturated fatty acids in Saccharomyces cerevisiae: isocitrate dehydrogenase provides NADPH for reduction of double bonds at even positions.

The β‐oxidation of saturated fatty acids in Saccharomyces cerevisiae is confined exclusively to the peroxisomal compartment of the cell. Processing of mono‐ and polyunsaturated fatty acids with the double bond at an even position requires, in addition to the basic β‐oxidation machinery, the contribution of the NADPH‐dependent enzyme 2,4‐dienoyl‐CoA reductase. Here we show by biochemical cell fractionation studies that this enzyme is a typical constituent of peroxisomes. As a consequence, the β‐oxidation of mono‐ and polyunsaturated fatty acids with double bonds at even positions requires stoichiometric amounts of intraperoxisomal NADPH. We suggest that NADP‐dependent isocitrate dehydrogenase isoenzymes function in an NADP redox shuttle across the peroxisomal membrane to keep intraperoxisomal NADP reduced. This is based on the finding of a third NADP‐dependent isocitrate dehydrogenase isoenzyme, Idp3p, next to the already known mitochondrial and cytosolic isoenzymes, which turned out to be present in the peroxisomal matrix. Our proposal is strongly supported by the observation that peroxisomal Idp3p is essential for growth on the unsaturated fatty acids arachidonic, linoleic and petroselinic acid, which require 2,4‐dienoyl‐CoA reductase activity. On the other hand, growth on oleate which does not require 2,4‐dienoyl‐CoA reductase, and NADPH is completely normal in Δidp3 cells.

Measurement of peroxisomal fatty acid β-oxidation in cultured human skin fibroblasts

SummaryOne of the main functions of mammalian peroxisomes is the β-oxidation of a variety of fatty acids and fatty acid derivatives, including very long-chain fatty acids. Oxidation of these fatty acids is deficient in a number of different peroxisomal disorders, including the disorders of peroxisome biogenesis (Zellweger syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease), X-linked adrenoleukodystrophy and a number of other disorders of peroxisomal β-oxidation of known and unknown aetiology. Accurate measurement of peroxisomal fatty acid oxidation is of utmost importance for correct postnatal and prenatal diagnosis of these disorders. In this paper we describe a straightforward and accurate assay method to measure the β-oxidation of palmitic acid (C16:0), hexacosanoic acid (C26:0) and pristanic acid in intact fibroblasts.

Fatty acid metabolism in Saccharomyces cerevisiae

Peroxisomes are essential subcellular organelles involved in a variety of metabolic processes. Their importance is underlined by the identification of a large group of inherited diseases in humans in which one or more of the peroxisomal functions are impaired. The yeast Saccharomyces cerevisiae has been used as a model organism to study the functions of peroxisomes. Efficient oxidation of fatty acids does not only require the participation of peroxisomal enzymes but also the active involvement of other gene products. One group of important gene products in this respect includes peroxisomal membrane proteins involved in metabolite transport. This overview discusses the various aspects of fatty acid β-oxidation in S. cerevisiae. Addressed are the various enzymes and their particular functions as well as the various transport mechanisms to take up fatty acids into peroxisomes or to export the β-oxidation products out of the peroxisome to mitochondria for full oxidation to CO2 and H2O.

Peroxisomal fatty acid beta-oxidation in relation to the accumulation of very long chain fatty acids in cultured skin fibroblasts from patients with Zellweger syndrome and other peroxisomal disorders.

The peroxisomal oxidation of the long chain fatty acid palmitate (C16:0) and the very long chain fatty acids lignocerate (C24:0) and cerotate (C26:0) was studied in freshly prepared homogenates of cultured skin fibroblasts from control individuals and patients with peroxisomal disorders. The peroxisomal oxidation of the fatty acids is almost completely dependent on the addition of ATP, coenzyme A (CoA), Mg2+ and NAD+. However, the dependency of the oxidation of palmitate on the concentration of the cofactors differs markedly from that of the oxidation of lignocerate and cerotate. The peroxisomal oxidation of all three fatty acid substrates is markedly deficient in fibroblasts from patients with the Zellweger syndrome, the neonatal form of adrenoleukodystrophy and the infantile form of Refsum disease, in accordance with the deficiency of peroxisomes in these patients. In fibroblasts from patients with X-linked adrenoleukodystrophy the peroxisomal oxidation of lignocerate and cerotate is impaired, but not that of palmitate. Competition experiments indicate that in fibroblasts, as in rat liver, distinct enzyme systems are responsible for the oxidation of palmitate on the one hand and lignocerate and cerotate on the other hand. Fractionation studies indicate that in rat liver activation of cerotate and lignocerate to cerotoyl-CoA and lignoceroyl-CoA, respectively, occurs in two subcellular fractions, the endoplasmic reticulum and the peroxisomes but not in the mitochondria. In homogenates of fibroblasts from patients lacking peroxisomes there is a small (25%) but significant deficiency of the ability to activate very long chain fatty acids. This deficient activity of very long chain fatty acyl-CoA synthetase is also observed in fibroblast homogenates from patients with X-linked adrenoleukodystrophy. We conclude that X-linked adrenoleukodystrophy is caused by a deficiency of peroxisomal very long chain fatty acyl-CoA synthetase.

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.

THE INBORN-ERRORS OF PEROXISOMAL BETA-OXIDATION - A REVIEW

SummaryIn recent years a growing number of inherited diseases in man have been recognized in which there is an impairment in peroxisomal β-oxidation. In some diseases this is due to the (virtual) absence of peroxisomes leading to a generalized loss of peroxisomal functions including peroxisomal β-oxidation. In most inborn errors of peroxisomal β-oxidation, however, peroxisomes are normally present and the impairment in peroxisomal β-oxidation is due to the single or multiple loss of peroxisomal β-oxidation enzyme activities. In all these disorders there is accumulation of very-long-chain fatty acids in plasma, which allows biochemical diagnosis of patients affected by an inborn error of peroxisomal β-oxidation to be done via gas-chromatographic analysis of plasma very-long-chain fatty acids. Subsequent enzymic and immunological investigations are required to identify the precise enzymic defects in these patients. In all inborn errors of peroxisomal β-oxidation known today there are multiple abnormalities, especially neurological with death usually occurring in the first decade of life. Prenatal diagnosis of these disorders has recently become possible.