Molecular cloning and expression analyses of mitochondrial and plastidic isoforms of cysteine synthase (O-acetylserine(thiol)lyase) from Arabidopsis thaliana

Cysteine synthase, the key enzyme for fixation of inorganic sulfide, cataly ses the formation of cysteine from O-acetylserine and inorganic sulfide. He re we report the cloning of cDNAs encoding cysteine synthase isoforms from Arabidopsis thaliana. The isolated cDNA clones encode for a mitochondrial a nd a plastidic isoform of cysteine synthase (O-acetylserine (thiol)-lyase, EC 4.2.99.8), designated cysteine synthase C (AtCS-C, CSase C) and B (AtCS- B; CSase B), respectively AtCS-C and AtCS-B, having lengths of 1569-bp and 1421-bp, respectively, encode polypeptides of 430 amino acids (similar to 4 5.8 kD) and of 392 amino acids (similar to 41.8 kD), respectively. The dedu ced amino acid sequences of the mitochondrial and plastidic isoforms exhibi t high homology even with respect to the presequences. The predicted preseq uence of AtCS-C has a N-terminal extension of 33 amino acids when compared to the plastidic isoform. Northern blot analysis showed that AtCS-C is high er expressed in roots than in leaves whereas the expression of AtCS-B is st ronger in leaves. Furthermore, gene expression of both genes was enhanced b y sulfur limitation which in turn led to an increase in enzyme activity in crude extracts of plants. Expression of the AtCS-B gene is regulated by lig ht. The mitochondrial, plastidic and cytosolic (Hesse and Altmann, 1995) is oforms of cysteine synthase of Arabidopsis are able to complement a cystein e synthase-deficient mutant of Escherichia coli unable to grow on minimal m edium without cysteine, indicating synthesis of functional plant proteins i n the bacterium. Two lines of evidence proved that AtCS-C encodes a mitocho ndrial form of cysteine synthase; first, import of in vitro translation pro ducts derived from AtCS-C in isolated intact mitochondria and second, Weste rn blot analysis of mitochondria isolated from transgenic tobacco plants ex pressing AtC-SC cDNA/c-myc DNA fusion protein.