The Arabidopsis ALDP protein homologue COMATOSE is instrumental in peroxisomal acetate metabolism

Abstract

The Arabidopsis acn (acetate non-utilizing) mutants were isolated by fluoroacetate-resistant germination and seedling establishment. We report the characterization of the acn2 mutant. Physiological analyses of acn2 showed that it possessed characteristics similar to those of the mutants cts (COMATOSE)-1 and pxa [peroxisomal ABC (ATP-binding-cassette) transporter]1. The acn2 locus was mapped to within 3 cM of the CTS gene on the bottom arm of chromosome IV using CAPS (cleavage amplification polymorphism) and SSLP (simple sequence-length polymorphism) markers. Crossing acn2 and cts-1 failed to restore the fluoroacetate-sensitive phenotype, suggesting that these mutations were allelic. Sequencing of the ACN2 locus revealed a C-->T nonsense mutation in exon 13, which would have resulted in the elimination of the C-terminal hemitransporter domain of the encoded protein. Neither the full-length CTS protein nor the truncated protein was detected on immunoblots using either C-terminal- or N-terminal-specific anti-CTS antibodies respectively, demonstrating the absence of the entire CTS protein in acn2 mutants. Emerged seedlings of both cts-1 and pxa1 alleles displayed increased resistance to FAc (monofluoroacetic acid) compared with the corresponding wild-type seedlings. Complementation studies showed that mutation of the CTS gene was responsible for the FAc-resistant phenotype, as when the wild-type protein was expressed in both the cts-1 and pxa1 mutant lines, the strains became FAc-sensitive. Feeding studies confirmed that both acn2 and cts-1 mutants were compromised in their ability to convert radiolabelled acetate into soluble carbohydrate. These results demonstrate a role for the ABC protein CTS in providing acetate to the glyoxylate cycle in developing seedlings.

The ABC Transporter PXA1 and Peroxisomal β-Oxidation Are Vital for Metabolism in Mature Leaves ofArabidopsisduring Extended Darkness

“…This function is supported by numerous studies in plants (Zolman et al, 2001;Footitt et al, 2002;Hayashi et al, 2002). In Arabidopsis, PXA1 has also been associated with other substrates like acetate (Hooks et al, 2007), IBA, or OPDA, an intermediate of JA biosynthesis (Theodoulou et al, 2005). However, because opr3 and dde2 mutants, impaired specifically in JA biosynthesis, are unaffected by exposure to extended darkness (data not shown), the reported pxa1 and kat2 mutant phenotype is apparently unlinked to OPDA and/ or JA biosynthesis.…”

“…Later, it became evident that PED3 corresponds to the same locus as COMATOSE (Russell et al, 2000;Footitt et al, 2002) and functionally represents the full-size peroxisomal ABC transporter PXA1 (Schwacke et al, 2003;Theodoulou et al, 2006). Recently, PXA1 has also been implicated in import of substrates as diverse as 12-oxo phytodienoic acid (OPDA), an intermediate of jasmonic acid (JA) biosynthesis (Theodoulou et al, 2005) and acetate (Hooks et al, 2007), suggesting a relatively broad substrate spectrum for PXA1. However, in germinating oilseeds, the assumed function of PXA1 is the import of fatty acids into peroxisomes providing substrates for b-oxidation.…”

Peroxisomal Fatty Acid Uptake Mechanism in Saccharomyces cerevisiae

“…18 O]H 2 O, the carboxyl group of the fatty acid will be labeled with 18 O, and this can be used directly to determine whether or not hydrolysis occurs during or prior to uptake of acyl-CoA esters into the peroxisomes. The carboxyl group has two oxygen atoms, which are in a tautomeric equilibrium upon hydrolysis of the acyl-CoA ester into the free fatty acid.…”

“…in wild type and fox3⌬ cells, respectively, are close to the theoretically predicted ratio when the uptake process involves hydrolysis and subsequent re-formation of the acyl-CoA ester. Moreover, the observation of a double 18 O incorporation in wild type cells suggests that the hydrolysis step (and subsequent esterification) is an obligatory step prior to ␤-oxidation. As a control, we incubated WT and fox3⌬ cells, both transformed with CPT2per with octanoic acid (C8:0) (Fig.…”

“…As a control, we incubated WT and fox3⌬ cells, both transformed with CPT2per with octanoic acid (C8:0) (Fig. 4C), and observed no incorporation and single 18 O incorporation into the ␤-oxidation intermediate 3-hydroxyoctanoylcarnitine but no double 18 O incorporation, indicating that C8:0 presumably enters yeast peroxisomes by passive diffusion as a free acid independently of Pxa1p-Pxa2p.…”