Characterization of a chloroplast isoform of serine acetyltransferase from the thermo-acidiphilic red alga Cyanidioschyzon merolae.
We isolated a gene for serine acetyltransferase (SAT), a key enzyme in sulfate assimilation, from the primitive red alga Cyanidioschyzon merolae, an inhabitant of sulfurous hot springs, and designated this gene cmSAT. The N-terminal region of the cmSAT protein has characteristics of a chloroplast targeting peptide. cmSAT protein fused with a 6x histidine tag complemented a SAT deficient Escherichia coli mutant. The protein was purified with its SAT activity, which was inhibited by cysteine, using the high affinity of the histidine tag in an Ni-NTA column. The Km values for acetyl-CoA and l-serine were 0.3 and 0.1 mM, respectively. Southern blotting indicated the existence of other SAT isoforms in C. merolae. A 2.4 kb transcript was always detected when growth was synchronized under a 12-h light/dark cycle. Under these conditions, a 31-kDa protein was always detected on immunoblots, indicating processing of the cmSAT protein and constitutive expression of cmSAT. A 45-kDa protein, thought to be the unprocessed cmSAT protein, was detected in the dark period, from M phase to early G1 phase. No significant change in the level of protein expression was detected under continuous darkness or in a sulfate-deficient medium. Using immunoelectron microscopy, the cmSAT protein was primarily detected in the stroma and a few were detected in the cytoplasm, which indicate that cmSAT protein is transported to and functions in a chloroplast.
Toda
K
K
Department of Biological Sciences, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan. ss67232@hongo.ecc.u-tokyo.ac.jp
Takano
H
H
Miyagishima
S
S
Kuroiwa
H
H
Kuroiwa
T
T
D000123:Acetyltransferases / Q000032:analysis / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002736:Chloroplasts / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D018744:DNA, Plant / Q000737:chemistry / Q000235:genetics; D017343:Genes, Plant / Q000235:genetics; D005816:Genetic Complementation Test; D018745:Genome, Plant; D015151:Immunoblotting; D007527:Isoenzymes / Q000032:analysis / Q000235:genetics*; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D000461:Rhodophyta / Q000201:enzymology* / Q000235:genetics / Q000648:ultrastructure; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D051050:Serine O-Acetyltransferase
D018744:DNA, Plant; D007527:Isoenzymes; D000123:Acetyltransferases; D051050:Serine O-Acetyltransferase