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.

[1]  S. Mitchell.  Biological Interactions Of Sulfur Compounds , 1996 .

[2]  B. Touraine,et al.  Demand-Driven Control of Root ATP Sulfurylase Activity and SO42- Uptake in Intact Canola (The Role of Phloem-Translocated Glutathione) , 1996, Plant physiology.

[3]  M. Murillo,et al.  Cloning of a cDNA Encoding ATP Sulfurylase from Arabidopsis thaliana by Functional Expression in Saccharomyces cerevisiae , 1994, Plant physiology.

[4]  H. C. Patel,et al.  ATP sulfurylase from higher plants: kinetic and structural characterization of the chloroplast and cytosol enzymes from spinach leaf. , 1993, Archives of biochemistry and biophysics.

[5]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[6]  T. Leustek,et al.  Sulfate transport and assimilation in plants , 1999, Plant physiology.

[7]  S. Ravanel,et al.  The specific features of methionine biosynthesis and metabolism in plants. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[8]  J. Lunn,et al.  Localization of ATP Sulfurylase and O-Acetylserine(thiol)lyase in Spinach Leaves. , 1990, Plant physiology.

[9]  M. Murillo,et al.  Adenosine-5'-triphosphate-sulfurylase from Arabidopsis thaliana and Escherichia coli are functionally equivalent but structurally and kinetically divergent: nucleotide sequence of two adenosine-5'-triphosphate-sulfurylase cDNAs from Arabidopsis thaliana and analysis of a recombinant enzyme. , 1995, Archives of biochemistry and biophysics.

[10]  A. Grossman,et al.  Sulfur Availability and the SAC1 Gene Control Adenosine Triphosphate Sulfurylase Gene Expression in Chlamydomonas reinhardtii , 1996, Plant physiology.

[11]  R. Schiestl,et al.  Improved method for high efficiency transformation of intact yeast cells. , 1992, Nucleic acids research.

[12]  N. M. Kredich,et al.  The molecular basis for positive regulation of cys promoters in Salmonella typhimurium and Escherichia coli , 1992, Molecular microbiology.

[13]  Thomas L. Madden,et al.  Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. , 1997, Nucleic acids research.

[14]  A. Feinberg,et al.  A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. , 1983, Analytical biochemistry.

[15]  I. H. Segel,et al.  ATP sulfurylase from higher plants : purification and preliminary kinetics studies on the cabbage leaf enzyme. , 1982, Plant physiology.

[16]  M. Van Montagu,et al.  Regulation of sulfur assimilation in higher plants: a sulfate transporter induced in sulfate-starved roots plays a central role in Arabidopsis thaliana. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[17]  L. Willmitzer,et al.  Isolation and characterization of two cDNA clones encoding ATP-sulfurylases from potato by complementation of a yeast mutant. , 1994, The Plant journal : for cell and molecular biology.

[18]  T. Bisseling,et al.  Isolation of total, poly(A) and polysomal RNA from plant tissue. , 1994 .

[19]  M. Hirai,et al.  Role of O-acetyl-l-serine in the coordinated regulation of the expression of a soybean seed storage-protein gene by sulfur and nitrogen nutrition , 1999, Planta.

[20]  F. W. Smith,et al.  Regulation of expression of a cDNA from barley roots encoding a high affinity sulphate transporter. , 1997, The Plant journal : for cell and molecular biology.

[21]  F. Skoog,et al.  A revised medium for rapid growth and bio assays with tobacco tissue cultures , 1962 .

[22]  D. Voytas,et al.  The structure, distribution and evolution of the Ta1 retrotransposable element family of Arabidopsis thaliana. , 1990, Genetics.

[23]  J. Davidian,et al.  Cloning of a cDNA Encoded by a Member of the Arabidopsis thaliana ATP Sulfurylase Multigene Family , 1996, The Journal of Biological Chemistry.

[24]  Takanori Hirano,et al.  Engineered GFP as a vital reporter in plants , 1996, Current Biology.

[25]  C. Glackin,et al.  Organ-specific transcripts of different size and abundance derive from the same pyruvate, orthophosphate dikinase gene in maize. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[26]  I. Small,et al.  The same Arabidopsis gene encodes both cytosolic and mitochondrial alanyl-tRNA synthetases. , 1996, The Plant cell.

[27]  M. Murillo,et al.  Siroheme Biosynthesis in Higher Plants , 1997, The Journal of Biological Chemistry.

[28]  G. Marzluf.  Molecular genetics of sulfur assimilation in filamentous fungi and yeast. , 1997, Annual review of microbiology.

[29]  H. Rennenberg,et al.  Sulfur Nutrition and Sulfur Assimilation in Higher Plants: Molecular, Biochemical and Physiological Aspects , 2000 .

[30]  K. Saito,et al.  Isoform-dependent Differences in Feedback Regulation and Subcellular Localization of Serine Acetyltransferase Involved in Cysteine Biosynthesis from Arabidopsis thaliana * , 1998, The Journal of Biological Chemistry.