The Arabidopsis peroxisomal ABC transporter, comatose, complements the Saccharomyces cerevisiae pxa1 pxa2Delta mutant for metabolism of long-chain fatty acids and exhibits fatty acyl-CoA-stimulated ATPase activity.
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.
Nyathi
Yvonne
Y
Faculty of Biological Sciences, University of Leeds, Centre for Plant Sciences, Leeds LS2 9JT, United Kingdom.
De Marcos Lousa
Carine
C
van Roermund
Carlo W
CW
Wanders
Ronald J A
RJ
Johnson
Barbara
B
Baldwin
Stephen A
SA
Theodoulou
Frederica L
FL
Baker
Alison
A
D018528:ATP-Binding Cassette Transporters; D000251:Adenosine Triphosphatases; D020816:Amino Acid Motifs; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins; D005227:Fatty Acids / Q000378:metabolism*; D005816:Genetic Complementation Test; D010084:Oxidation-Reduction; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity
D018528:ATP-Binding Cassette Transporters; D029681:Arabidopsis Proteins; D005227:Fatty Acids; C098338:PXA1 protein, S cerevisiae; C103561:PXA2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D000251:Adenosine Triphosphatases; C456843:At4g39850 protein, Arabidopsis