Isoform-dependent differences in feedback regulation and subcellular localization of serine acetyltransferase involved in cysteine biosynthesis from Arabidopsis thaliana.
Serine acetyltransferase (SATase; EC 2.3.1.30), which catalyzes the formation of O-acetyl-L-serine (OAS) from acetyl-CoA and L-serine, plays a regulatory role in the biosynthesis of cysteine by its property of feedback inhibition by cysteine in bacteria and certain plants. Three cDNA clones encoding SATase isoforms (SAT-c, SAT-p, and SAT-m) have been isolated from Arabidopsis thaliana. However, the significance of the feedback regulation has not yet been clear in these different isoforms of SATase from A. thaliana. We constructed the overexpression vectors for cDNAs encoding three SATase isoforms of A. thaliana and analyzed the inhibition of SATase activity by cysteine using the recombinant SATase proteins. In the case of SAT-c, the activity was feedback-inhibited by a low concentration of cysteine (the concentration that inhibits 50% activity; IC50 = 1.8 microM). By contrast, SAT-p and SAT-m were feedback inhibition-insensitive isozymes. We also determined the subcellular localization of three SATase isozymes by the transient expression of fusion proteins of each SATase N-terminal region with jellyfish green fluorescent protein (GFP) in 4-week-old Arabidopsis leaves. The SAT-c-GFP fusion protein was stayed in cytosol, whereas SAT-p-GFP and SAT-m-GFP fusion proteins were localized in chloroplasts and in mitochondria, respectively. These results suggest that these three SATase isoforms, which are localized in the different organelles, are subjected to different feedback regulation, presumably so as to play the particular roles for the production of OAS and cysteine in Arabidopsis cells. Regulatory circuit of cysteine biosynthesis in the plant cells is discussed.
Noji
M
M
Faculty of Pharmaceutical Sciences, Laboratory of Molecular Biology and Biotechnology in Research Center of Medicinal Resources, Chiba University, Yayoi-cho 1-33, Inage-ku, Chiba 263-8522, Japan.
Inoue
K
K
Kimura
N
N
Gouda
A
A
Saito
K
K
D000123:Acetyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology / Q000378:metabolism*; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D003545:Cysteine / Q000096:biosynthesis*; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D005246:Feedback; D049452:Green Fluorescent Proteins; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D051050:Serine O-Acetyltransferase; D013347:Subcellular Fractions / Q000201:enzymology*
D017931:DNA Primers; D007527:Isoenzymes; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000123:Acetyltransferases; D051050:Serine O-Acetyltransferase; D003545:Cysteine