Abstract
The Arabidopsis ABC transporter Comatose (CTS; AtABCD1) is required for uptake into the peroxisome of a wide range of substrates for β-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 pxa2Δ 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 pxa2Δ mutant, and restored β-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.
Highlights
Peroxisomes perform a range of different functions, including -oxidation of fatty acids (FA)2 and synthesis and degradation of bioactive, lipid-derived molecules
CTS Is Targeted to the Peroxisome in S. cerevisiae—To determine the suitability of S. cerevisiae (BSL1-11B) as a heterologous system for the biochemical characterization of CTS, the CTS ORF was expressed from a low copy plasmid under the control of the strong, constitutive glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter [34]
To investigate whether CTS was appropriately targeted in the heterologous host, peroxisomes were purified on Iodixanol gradients from a “light mitochondrial fraction” of oleate-grown cells transformed with CTS/pRS416-GPD or the non-recombinant vector, as described [30]
Summary
Yeast transformants were selected and grown on minimal medium with appropriate supplements. A single colony of yeast was inoculated into 10 ml of WOYD medium (0.67% (w/v) yeast nitrogen base (BD Biosciences, Oxford, UK), 0.3% (w/v) glucose, 0.1% (w/v) yeast extract) and incubated at 28 °C for 18 h. 5 ml of the resultant yeast suspension was used to inoculate a liter of oleate medium (0.5% (w/v) Bacto-peptone, 0.3% (w/v) yeast extract, 0.12% (v/v) oleic acid, 0.2% (v/v) Tween 20, 0.5% (w/v) KH2PO4, pH 6.0) followed by incubation at 28 °C for 18 h (A600 of 1.0 to 1.5) [30]. Construction of a pxa1⌬ Mutant Strain—Gene replacement was used to generate a BSL1-11 B strain lacking the PXA1 gene
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