Phosphorylation of tomato 1-aminocyclopropane-1-carboxylic acid synthase, LE-ACS2, at the C-terminal region.
1-aminocyclopropane-1-carboxylic acid synthase is a key enzyme in the ethylene biosynthesis pathway. Recent studies raise the possibility that 1-aminocyclopropane-1-carboxylic acid synthase (ACS) is regulated not only transcriptionally but also post-translationally. To elucidate post-translational ACS regulation, we analyzed the modification of LE-ACS2 protein, a wound-inducible isozyme in the ACS family, in tomato fruit (Lycopersicon esculentum L.) using an anti-LE-ACS2 antibody. We detected phosphorylated LE-ACS2 at 55-kDa using immunoprecipitation from an extract of wounded fruit fed with [32P]inorganic phosphate. Analysis of LE-ACS2 phosphoamino acids indicated that serine residue(s) were phosphorylated. In vitro phosphorylation analyses using site-directed mutagenesis of recombinant LE-ACS2 as a substrate demonstrate that serine 460 located at the C-terminal region of ACS is phosphorylated. During tomato ripening stages, expression of both LE-ACS2 and LE-ACS4 mRNA increased. LE-ACS4, however, was not phosphorylated in vitro. These results suggest that ACS isozymes have different post-translational regulatory mechanisms, such as phosphorylation.
Tatsuki
M
M
Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho Chikusa-ku, Nagoya, Aichi 464-8601, Japan.
Mori
H
H
D000595:Amino Acid Sequence; D000598:Amino Acids, Cyclic / Q000737:chemistry* / Q000378:metabolism; D015153:Blotting, Western; D003066:Coenzyme A Ligases / Q000737:chemistry* / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D018551:Solanum lycopersicum / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D013237:Stereoisomerism; D013997:Time Factors; D014158:Transcription, Genetic
D000598:Amino Acids, Cyclic; D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins; C023863:1-aminocyclopropane-1-carboxylic acid; D012694:Serine; D003066:Coenzyme A Ligases; C403665:ACSL6 protein, human