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Biochim Biophys Acta. 1998 May 27;1403(1):72-84.

Characterization of a chloroplast isoform of serine acetyltransferase from the thermo-acidiphilic red alga Cyanidioschyzon merolae.

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1
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

Abstract

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.

PMID:
9622597
DOI:
10.1016/s0167-4889(98)00031-7
[Indexed for MEDLINE]
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