title: Molecular characterization of an Arabidopsis acyl-coenzyme a synthetase localized on glyoxysomal membranes.
abstract: In higher plants, fat-storing seeds utilize storage lipids as a source of energy during germination. To enter the beta-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 beta-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 beta-oxidation activating the fatty acids.
authors.affiliation: Department of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Hayashi
authors.forename: Luigi
authors.initials: L
authors.lastname: De Bellis
authors.forename: Yasuko
authors.initials: Y
authors.lastname: Hayashi
authors.forename: Kazumasa
authors.initials: K
authors.lastname: Nito
authors.forename: Akira
authors.initials: A
authors.lastname: Kato
authors.forename: Makoto
authors.initials: M
authors.lastname: Hayashi
authors.forename: Ikuko
authors.initials: I
authors.lastname: Hara-Nishimura
authors.forename: Mikio
authors.initials: M
authors.lastname: Nishimura
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D003066:Coenzyme A Ligases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D018525:Germination / Q000235:genetics; D020676:Glyoxysomes / Q000201:enzymology* / Q000648:ultrastructure; D015151:Immunoblotting; D007425:Intracellular Membranes / Q000201:enzymology* / Q000648:ultrastructure; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D012639:Seeds / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D017422:Sequence Analysis, DNA
chemical_list: D029681:Arabidopsis Proteins; D018076:DNA, Complementary; D003066:Coenzyme A Ligases; C470374:LACS6 protein, Arabidopsis