Abstract
The acyl-CoA ligases convert free fatty acids to acyl-CoA derivatives, and these enzymes have been shown to be present in mitochondria, peroxisomes, and endoplasmic reticulum. Because their activity is obligatory for fatty acid metabolism, it is important to identify their substrate specificities and subcellular distributions to further understand the cellular regulation of these pathways. To define the role of the enzymes and organelles involved in the metabolism of very long chain (VLC) fatty acids, we studied human genetic cell mutants impaired for the metabolism of these molecules. Fibroblast cell lines were derived from patients with X-linked adrenoleukodystrophy (X-ALD) and Zellweger's cerebro-hepato-renal syndrome (CHRS). While peroxisomes are present and morphologically normal in X-ALD, they are either greatly reduced in number or absent in CHRS. Palmitoyl-CoA ligase is known to be present in mitochondria, peroxisomes, and endoplasmic reticulum (microsomes). We found enzyme-dependent formation of lignoceroyl-CoA in these same organelles (specific activities were 0.32 +/- 0.12, 0.86 +/- 0.12, and 0.78 +/- 0.07 nmol/h per mg protein, respectively). However, lignoceroyl-CoA synthesis was inhibited by an antibody to palmitoyl-CoA ligase in isolated mitochondria while it was not inhibited in peroxisomes or endoplasmic reticulum (ER). This suggests that palmitoyl-CoA ligase and lignoceroyl-CoA are different enzymes and that mitochondria lack lignoceroyl-CoA ligase. This conclusion is further supported by data showing that oxidation of lignoceric acid was found almost exclusively in peroxisomes (0.17 nmol/h per mg protein) but was largely absent from mitochondria and the finding that monolayers of CHRS fibroblasts lacking peroxisomes showed a pronounced deficiency in lignoceric acid oxidation in situ (1.8% of control). In spite of the observation that lignoceroyl-CoA ligase activity is present on the cytoplasmic surface of ER, our data indicate that lignoceroyl-CoA synthesized by ER is not available for oxidation in mitochondria. This organelle plays no physiological role in the beta-oxidation of VLC fatty acids. Furthermore, the normal peroxisomal oxidation of lignoceroyl-CoA but deficient oxidation of lignoceric acid in X-ALD cells indicates that cellular VLC fatty acid oxidation is dependent on peroxisomal lignoceroyl-CoA ligase. These studies allow us to propose a model for the subcellular localization of various acyl-CoA ligases and to describe how these enzymes control cellular fatty acid metabolism.
Highlights
The acyl-CoA ligases convert free fatty acids to acylCoA derivatives, and these enzymes have been shown to be present in mitochondria, peroxisomes, and endoplasmic reticulum
I n this study, we utilized cell cultures derived from X-linked adrenoleukodystrophy (X-ALD) and cerebro-hepato-renal syndrome (CHRS) patients for both in vitro and in situ experiments to elucidate the physiological mechanisms for the cellular metabolism of very long chain (VLC) fatty acids
Specific activity of oxidation and activation of fatty acids in fibroblast homogenates The specific activities of marker enzymes for different organelles were similar in homogenates of control, X-linked adrenoleukodystrophy (XALD), and CHRS fibroblasts (Table 1)
Summary
The acyl-CoA ligases convert free fatty acids to acylCoA derivatives, and these enzymes have been shown to be present in mitochondria, peroxisomes, and endoplasmic reticulum. While studying the oxidation of different derivatives of lignoceric acid, we surprisingly found in homogenates of CHRS cultured skin fibroblasts that lignoceroyl-CoA was oxidized at a n efficient rate of 55 7' 0of normal activity in contrast to the deficient oxidation of lignoceric acid (14.3% of the normal activity) [23] This suggests that the activated fatty acid, lignoceroyl-CoA, may be oxidized in an organelle other than the peroxisomes [23] because functional peroxisomes are absent in CHRS [8, 22,23,24,25], it does not explain the pathognomonic accumulation of VLC fatty acids in X-ALD and CHRS. I n this study, we utilized cell cultures derived from X-ALD and CHRS patients for both in vitro and in situ experiments to elucidate the physiological mechanisms for the cellular metabolism of VLC fatty acids
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