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 i n pea (Pisum sativum)leaves, by isolation of organelles and fractionat ion 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 ch loroplasts (10% of total activity). Three enzyme forms were separated by anion-exchange chromatography, and each form was found to be specif ic 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 mut ant devoid of serine acetyltransferase activity. This strategy allowed isolation of three distinct cDNAs encoding serine acetyltransferase i soforms, as confirmed by enzyme activity measurements, genomic hybridi zations, and nucleotide sequencing. The cDNA and related gene for one of the three isoforms have been characterized. The predicted amino aci d sequence shows that it encodes a polypeptide of M(r) 34330 exhibitin g 41% amino acid identity with the E. coli serine acetyltransferase. S ince none of the general features of transit peptides could be observe d in the N-terminal region of this isoform, we assume that it is a cyt osolic form.