Plant Physiol, December 2002, Vol. 130, pp. 2019-2026

Molecular Characterization of an Arabidopsis Acyl-Coenzyme A Synthetase Localized on Glyoxysomal Membranes¹

Department of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan (H.H., L.D.B., Y.H., K.N., A.K., M.H., M.N.); Department of Bioscience, Fukui Prefectural University, Matsuoka, Fukui 910-1195, Japan (H.H.); Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, via Provinciale Lecce-Monteroni, 73100 Lecce, Italy (L.D.B.); Faculty of Science, Niigata University, Niigata 950-2181, Japan (Y.H., A.K.); and Department of Botany, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan (I.H.-N.)

In higher plants, fat-storing seeds utilize storage lipids as a source of energy during germination. To enter the -oxidation pathway, fatty acids need to be activated to acyl-coenzyme As (CoAs) by the enzyme acyl-CoA synthetase (ACS; EC 6.2.1.3). Here, we report the characterization of an Arabidopsis cDNA clone encoding for a glyoxysomal acyl-CoA synthetase designated AtLACS6. The cDNA sequence is 2,106 bp long and it encodes a polypeptide of 701 amino acids with a calculated molecular mass of 76,617 D. Analysis of the amino-terminal sequence indicates that acyl-CoA synthetase is synthesized as a larger precursor containing a cleavable amino-terminal presequence so that the mature polypeptide size is 663 amino acids. The presequence shows high similarity to the typical PTS2 (peroxisomal targeting signal 2). The AtLACS6 also shows high amino acid identity to prokaryotic and eukaryotic fatty acyl-CoA synthetases. Immunocytochemical and cell fractionation analyses indicated that the AtLACS6 is localized on glyoxysomal membranes. AtLACS6 was overexpressed in insect cells and purified to near homogeneity. The purified enzyme is particularly active on long-chain fatty acids (C16:0). Results from immunoblot analysis revealed that the expression of both AtLACS6 and -oxidation enzymes coincide with fatty acid degradation. These data suggested that AtLACS6 might play a regulatory role both in fatty acid import into glyoxysomes by making a complex with other factors, e.g. PMP70, and in fatty acid -oxidation activating the fatty acids.