Peroxisomes are organelles that perform diverse metabolic functions in different organisms, but a common function is β-oxidation of a variety of long chain aliphatic, branched, and aromatic carboxylic acids. Import of substrates into peroxisomes for β-oxidation is mediated by ATP binding cassette (ABC) transporter proteins of subfamily D, which includes the human adrenoleukodystropy protein (ALDP) defective in X-linked adrenoleukodystrophy (X-ALD). Whether substrates are transported as CoA esters or free acids has been a matter of debate. Using COMATOSE (CTS), a plant representative of the ABCD family, we demonstrate that there is a functional and physical interaction between the ABC transporter and the peroxisomal long chain acyl-CoA synthetases (LACS)6 and -7. We expressed recombinant CTS in insect cells and showed that membranes from infected cells possess fatty acyl-CoA thioesterase activity, which is stimulated by ATP. A mutant, in which Serine 810 is replaced by asparagine (S810N) is defective in fatty acid degradation in vivo, retains ATPase activity but has strongly reduced thioesterase activity, providing strong evidence for the biological relevance of this activity. Thus, CTS, and most likely the other ABCD family members, represent rare examples of polytopic membrane proteins with an intrinsic additional enzymatic function that may regulate the entry of substrates into the β-oxidation pathway. The cleavage of CoA raises questions about the side of the membrane where this occurs and this is discussed in the context of the peroxisomal coenzyme A (CoA) budget. T he peroxisome is the sole site of β-oxidation of fatty acids and related molecules in plants and fungi and is essential in metabolism of very long chain fatty acids and bioactive lipid molecules in mammals. ATP binding cassette (ABC) proteins of subfamily D are required for the transport of these substrates across the peroxisome membrane (1). These are peroxisomal ABC transporter 1 and 2 (Pxa1p/Pxa2p) in yeast, adrenoleukodystrophy protein (ALDP/ABCD1), adrenoleukodystrophy related protein (ALDPR/ ABCD2), and the 70 kDa peroxisome membrane protein (PMP70/ ABCD3) in mammals, and Comatose (CTS; also known as PED3, PXA1, and AtABCD1) in plants. Defects in ALDP result in Xlinked adrenoleukodystrophy, a neurological disorder in which very long chain fatty acids accumulate (2). Similarly, cts mutants are defective in germination and mobilization of stored triacylglycerol (3).Activation by formation of a CoA thioester is a prerequisite for entry of substrates into β-oxidation (4) but whether ABCD proteins accept free fatty acids or acyl-CoAs has been contentious. Arabidopsis mutants lacking CTS accumulate acyl-CoAs (5) and the basal ATPase activity of the protein is stimulated by acyl-CoAs rather than free fatty acids (6). Studies with yeast cells and trypanosomes suggest that Pxa1p/Pxa2p and the trypanosome ABCD protein GAT1 transport acyl-CoAs (7, 8) and human ALDP, ALDR, and Arabidopsis CTS are all able to complement the Saccharomyces cerevisiae pxa1...
The Arabidopsis ABC transporter Comatose (CTS; AtABCD1) is required for uptake into the peroxisome of a wide range of substrates for -oxidation, but it is uncertain whether CTS itself is the transporter or if the transported substrates are free acids or CoA esters. To establish a system for its biochemical analysis, CTS was expressed in Saccharomyces cerevisiae. The plant protein was correctly targeted to yeast peroxisomes, was assembled into the membrane with its nucleotide binding domains in the cytosol, and exhibited basal ATPase activity that was sensitive to aluminum fluoride and abrogated by mutation of a conserved Walker A motif lysine residue. The yeast pxa1 pxa2⌬ mutant lacks the homologous peroxisomal ABC transporter and is unable to grow on oleic acid. Consistent with its exhibiting a function in yeast akin to that in the plant, CTS rescued the oleate growth phenotype of the pxa1 pxa2⌬ mutant, and restored -oxidation of fatty acids with a range of chain lengths and varying degrees of desaturation. When expressed in yeast peroxisomal membranes, the basal ATPase activity of CTS could be stimulated by fatty acyl-CoAs but not by fatty acids. The implications of these findings for the function and substrate specificity of CTS are discussed.Peroxisomes perform a range of different functions, including -oxidation of fatty acids (FA) 2 and synthesis and degradation of bioactive, lipid-derived molecules. Import of substrates for peroxisomal metabolism is mediated by ATP binding cassette (ABC) transporters belonging to subclass D (1, 2). ABC transporters are composed of a minimum of four functional domains: two transmembrane domains, involved in substrate binding and translocation, and two nucleotide binding domains (NBDs) that bind and hydrolyze ATP, providing energy for transport (3, 4). The domains may be fused into a single polypeptide, but are frequently expressed as half-size transporters composed of a transmembrane domain fused to an NBD, which hetero-or homodimerize to form a functional transporter. Bakers' yeast (Saccharomyces cerevisiae) contains two ABCD genes that encode half-size ABC proteins: Pxa1p (peroxisomal ABC-transporter 1), and Pxa2p (5-7). The single pxa1⌬ and pxa2⌬ deletion mutants are unable to grow on oleate (C18:1) as the sole carbon source and exhibit reduced -oxidation of this long-chain FA. It has been proposed that Pxa1p and Pxa2p operate as a heterodimer to form a functional transporter (6,8,9), which has been shown by indirect evidence to be required for the peroxisomal transport of the C18:1-CoA, a long-chain acyl-CoA ester, but not for import of C8:0-CoA (10). In contrast, medium-chain FAs enter yeast peroxisomes as free acids independently of Pxa1p/Pxa2p, and are activated by peroxisomal acyl-CoA synthetase, Faa2p, prior to -oxidation (6).The human ABCD transporter subfamily comprises four half-size members: adrenoleukodystrophy protein (ALDP), ALD-related protein, the 70-kDa peroxisomal membrane protein (PMP70), and PMP70-related protein (PMP70R/PMP69) (1, 2). Although...
The mitochondrial adenine nucleotide carrier, or Ancp, plays a key role in the maintenance of the energetic fluxes in eukaryotic cells. Human disorders have been found associated to unusual human ANC gene (HANC) expression but also to direct inactivation of the protein, either by autoantibody binding or by mutation. However, the individual biochemical properties of the three HAncp isoforms have not yet been deciphered. To do so, the three HANC ORF were expressed in yeast under the control of the regulatory sequences of ScANC2. Each of the three HANC was able to restore growth on a nonfermentable carbon source of a yeast mutant strain lacking its three endogenous ANC. Their ADP/ATP exchange properties could then be measured for the first time in isolated mitochondria. HANC3 was the most efficient to restore yeast growth, and HAnc3p presented the highest V(M) (80 nmol ADP min(-1) mg protein(-1)) and K(ADP)(M)(8.4 microM). HAnc1p and HAnc2p presented similar kinetic constants (V(M) approximately 30-40 nmol ADP min(-(1) mg protein(-1) and K(ADP)(M) approximately 2.5-3.7 microM), whose values were consistent with HANC1's and HANC2's lower capacity to restore yeast growth. However, the HANC genes restored growth at a lower level than ScANC2, indicating that HAncp amount may be limiting in vivo. To optimize the HAncp production, we investigated their biogenesis into mitochondria by mutagenesis of two charged amino acids in the N-terminus of HAnc1p. Severe effects were observed with the D3A and D3K mutations that precluded yeast growth. On the contrary, the K10A mutation increased yeast growth complementation and nucleotide exchange rate as compared to the wild type. These results point to the importance of the N-terminal region of HAnc1p for its biogenesis and transport activity in yeast mitochondria.
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