Subcellular distribution of serine acetyltransferase from Pisum sativum and characterization of an Arabidopsis thaliana putative cytosolic isoform.
The intracellular compartmentation of serine acetyltransferase, a key enzyme in the L-cysteine biosynthesis pathway, has been investigated in pea (Pisum sativum) leaves, by isolation of organelles and fractionation of protoplasts. Enzyme activity was mainly located in mitochondria (approximately 76% of total cellular activity). Significant activity was also identified in both the cytosol (14% of total activity) and chloroplasts (10% of total activity). Three enzyme forms were separated by anion-exchange chromatography, and each form was found to be specific for a given intracellular compartment. To obtain cDNA encoding the isoforms, functional complementation experiments were performed using an Arabidopsis thaliana expression library and an Escherichia coli mutant devoid of serine acetyltransferase activity. This strategy allowed isolation of three distinct cDNAs encoding serine acetyltransferase isoforms, as confirmed by enzyme activity measurements, genomic hybridizations, and nucleotide sequencing. The cDNA and related gene for one of the three isoforms have been characterized. The predicted amino acid sequence shows that it encodes a polypeptide of M(r) 34,330 exhibiting 41% amino acid identity with the E. coli serine acetyltransferase. Since none of the general features of transit peptides could be observed in the N-terminal region of this isoform, we assume that it is a cytosolic form.
Ruffet
M L
ML
Unité mixte Centre National de la Recherche Scientifique, Rhône-Poulenc Agrochimie, Lyon, France.
Lebrun
M
M
Droux
M
M
Douce
R
R
D000123:Acetyltransferases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology*; D001483:Base Sequence; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D005816:Genetic Complementation Test; D007527:Isoenzymes / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D018532:Pisum sativum / Q000201:enzymology*; D010957:Plasmids; D017386:Sequence Homology, Amino Acid; D051050:Serine O-Acetyltransferase; D013347:Subcellular Fractions / Q000201:enzymology*
D018076:DNA, Complementary; D007527:Isoenzymes; D000123:Acetyltransferases; D051050:Serine O-Acetyltransferase