Functional characterization of a gene encoding a fourth ATP sulfurylase isoform from Arabidopsis thaliana.
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.
Hatzfeld
Y
Y
Chiba University, Faculty of Pharmaceutical Sciences, Laboratory of Molecular Biology and Biotechnology, Yayoi-cho 1-33, Inage-ku, Japan.
Lee
S
S
Lee
M
M
Leustek
T
T
Saito
K
K
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D002874:Chromosome Mapping; D003600:Cytosol / Q000201:enzymology; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000737:chemistry / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant / Q000235:genetics*; D005816:Genetic Complementation Test; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013430:Sulfate Adenylyltransferase / Q000235:genetics*
D018076:DNA, Complementary; D018744:DNA, Plant; D008164:Luminescent Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D013430:Sulfate Adenylyltransferase