ATP sulfurylase (
ATP:
sulfate adenylyl transferase, EC 2.7.7.4), the first
enzyme of the
sulfate assimilation pathway, is present in the chloroplast and cytosol of plants. In Arabidopsis thaliana
cDNA cloning revealed the existence of three
ATP sulfurylase isoforms (APS1, -2, and -3) all of which appear to be localized in plastids. In the present study the cytosolic
isoform was sought by searching the expressed sequence tag (EST) database and by screening A. thaliana genomic libraries. A fourth
isoform, APS4, was identified, but it also encodes a plastid-localized
isoform. The APS genes all contain four introns. The introns are located at identical positions within the coding sequence of each of the APS genes. A putative TATA box was identified in the promoter of the APS3 and APS4 genes, but no regions of sequence similarity were found among the other promoters. Combined analysis of an APS4
cDNA and genomic clone revealed that the deduced
protein is 469
amino acids and is most homologous to the A. thaliana APS1 subclass. The APS4
cDNA was able to functionally
complement a yeast
ATP sulfurylase (met3) mutant and the recombinant
enzyme displayed
ATP sulfurylase activity. The APS4
protein exhibits a plastid targeting
peptide at its amino terminus that, when fused to
green fluorescent protein, was able to target the reporter to chloroplasts. APS4
mRNA was detected at a similar steady-state level in roots and leaves, and its expression was not induced by
sulfur starvation or by
O-acetylserine treatment. Having identified a fourth plastid-localized
ATP sulfurylase, the origin of cytosolic
isoform in A. thaliana remains unclear. Based on sequence analysis, it is hypothesized that APS2 may encode the cytosolic
ATP sulfurylase.