The Arabidopsis Peroxisomal ABC Transporter, Comatose, Complements the Saccharomyces cerevisiae pxa1 pxa2Δ Mutant for Metabolism of Long-chain Fatty Acids and Exhibits Fatty Acyl-CoA-stimulated ATPase Activity

Abstract

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 α/β Hydrolase CGI-58 and Peroxisomal Transport Protein PXA1 Coregulate Lipid Homeostasis and Signaling in Arabidopsis

“…On the other hand, the observation that CGI-58 interacts with the C-terminal region of PXA1, which is oriented toward the cytosol in the context of the full-length protein ( Figure 2A) (Nyathi et al, 2010), implies that CGI-58 might localize also to peroxisomes. Indeed, as shown in Figure 3 (top row), transient expression of GFP-CGI-58 in tobacco (Nicotiana tabacum) leaves using Agrobacterium tumefaciens infiltration revealed that at early time points after infection (i.e., 2 d) the protein localized primarily to the cytosol but also was associated with peroxisomes that were labeled with the coexpressed peroxisomal matrix marker Cherry-PTS1 (Ching et al, 2012).…”

Plant Peroxisomes: Biogenesis and Function

“…The S. cerevisiae equivalent transporter Pxa1p/Pxa2p transports acyl-CoAs (Verleur et al, 1997). The Arabidopsis CTS/PXA1/PED3 protein can complement the yeast pxa1 pxa2 double mutant and support the metabolism of a wide range of fatty acid substrates that differ in chain length and degree of unsaturation (Nyathi et al, 2010). Furthermore, the ATPase activity of CTS/PXA1/PED3 is stimulated by acyl-CoAs but not appreciably by free fatty acids, which also supports the notion of acyl-CoAs as substrates (Nyathi et al, 2010).…”

“…The Arabidopsis CTS/PXA1/PED3 protein can complement the yeast pxa1 pxa2 double mutant and support the metabolism of a wide range of fatty acid substrates that differ in chain length and degree of unsaturation (Nyathi et al, 2010). Furthermore, the ATPase activity of CTS/PXA1/PED3 is stimulated by acyl-CoAs but not appreciably by free fatty acids, which also supports the notion of acyl-CoAs as substrates (Nyathi et al, 2010). As proposed by Fulda et al (2004), one possible explanation of this discrepancy is that acyl-CoAs are the substrate, but the CoA is removed during transport and acyl-CoA is resynthesized in the peroxisome by LACS6 and/or LACS7.…”

Structure-Function Analysis of Peroxisomal ATP-binding Cassette Transporters Using Chimeric Dimers

“…Heterologous expression of the full-length CTS protein was shown to complement the pxa1/ pxa2⌬ yeast mutant for ␤-oxidation of fatty acids (35). To prove the functionality of the rat full-length ABCD transporters, we expressed the chimeric dimers in pxa1/pxa2⌬ yeast cells.…”