- Email: [email protected]
Cysteine biosynthesis in higher plants: a new member of the Arabidopsis thaliana serine acetyltransferase small gene-family obtained by functional complementation of an Escherichia coli cysteine auxotroph1
Biochimica et Biophysica Acta 1350 Ž1997. 123–127
Short sequence-paper
Cysteine biosynthesis in higher plants: a new member of the Arabidopsis thaliana serine acetyltransferase small gene-family obtained by functional complementation of an Escherichia coli cysteine auxotroph 1 Jonathan R. Howarth 2 , Michael A. Roberts 3, John L. Wray
)
Plant Sciences Laboratory, Research DiÕision of EnÕironmental and EÕolutionary Biology, School of Biological and Medical Sciences, Sir Harold Mitchell Building, UniÕersity of St. Andrews, St Andrews, Fife KY16 9TH, UK Received 13 September 1996; revised 5 November 1996; accepted 5 November 1996
Abstract A cDNA clone, Sat-52, encoding a novel isoform of serine acetyltransferase ŽEC 2.3.1.30. was isolated by functional complementation of an Escherichia coli cysE mutant defective in serine acetyltransferase. The 1158 base pair clone contains a full-length open reading frame encoding a deduced protein of 312 amino acids with an M r of 32.77 kDa. Northern analysis revealed a single transcript of ca 1.19 kb that did not increase in abundance under sulfate limitation. Genomic Southern hybridization suggests the presence of a single copy of the Sat-52 gene. Keywords: Serine acetyltransferase; Cysteine biosynthesis; Nucleotide sequence; Sulfate regulation
Serine acetyltransferase Ž SAT. ŽEC 2.3.1.30. catalyses the formation of O-acetyl-L-serine from acetylCoA and L-serine. O-acetyl-L-serine then combines with sulfide, produced by the sulfate assimilation pathway, in a reaction catalyzed by O-acetyl-L-serine Žthiol. lyase ŽOASTL. ŽEC 4.2.99.8. to form the amino acid L-cysteine. Thus cysteine biosynthesis is a )
Corresponding author. Fax: q44 1334 463366. E-mail: [email protected] 1 The nucleotide sequence data reported in this paper have been submitted to the EMBLrGenBankrDDJB Data Libraries under the accession number U30298. 2 Authors’ names are in alphabetical order. The first two named authors made equal but separate contributions to the development and execution of this work. 3 Present address: Cereals Research Department, John Innes Centre, Norwich Research Park, Colney, Norwich, NR4 7UH, UK.
key step in the biological sulfur cycle since it is the point at which inorganic sulfur enters into organic combination. As well as being a precursor for L-cysteine biosythesis, O-acetyl-L-serine has been shown to have a regulatory role in sulfate assimilation w1,2x. Biochemical studies have located SAT w3x and OASTL w4x to the chloroplast, mitochondrion and cytosol of higher plants, suggesting each intracellular compartment has the ability to synthesize L-cysteine. However, why this should be necessary, and the relative extent to which these compartments contribute towards L-cysteine biosynthesis, is unknown. Molecular studies have identified cDNAs encoding putative cytoplasmic forms of SAT from Citrullus Õulgaris Žwatermelon. ŽSat2. w5x and Arabidopsis thaliana ŽSat5 w3x and SAT1 w6x. and a putative chloroplastic form from Arabidopsis thaliana ŽSat-1. w7x. A further Žpartial. cDNA ŽSAT1-6. w8x with
0167-4781r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 7 - 4 7 8 1 Ž 9 6 . 0 0 2 1 3 - 8
124
J.R. Howarth et al.r Biochimica et Biophysica Acta 1350 (1997) 123–127
highest homology to the putative chloroplastic form Sat-1 w7x has also been reported from Arabidopsis thaliana. In this paper we have attempted to isolate novel A. thaliana SAT cDNAs by functional complementation of the Escherichia coli cysE mutant strain JM15, deficient in SAT activity and unable to grow with sulfate as sole sulfur source w9x, to prototrophy. The availability of cDNA sequences for different isoforms of SAT in Arabidopsis thaliana should provide the key to the analysis of the role of O-acetyl-L-serine in sulfur metabolism and to the regulation of L-cysteine biosynthesis. JM15 was lysogenized with lKC, and then transfected with an Arabidopsis thaliana ŽColumbia ecotype. cDNA library in the expression vector lYES w10x. Colonies that grew on minimal medium containing sulfate as sole sulfur source and also 50 m grml ampicillin Žampicillin resistance is conferred by lYES. were selected and replated onto fresh medium. Complemented cells, that retained the ability to grow with sulfate as sole sulfur source and which were also ampicillin-resistant, were obtained at a frequency of 105 from 10 9 cells transfected with 6 = 10 7 lYES bacteriophage. Partial sequencing of the inserts of 10 of the longest complementing clones revealed one cDNA clone, Sat-52, that encoded a novel isoform of SAT. Functionality of the cDNA clone Sat-52 was confirmed by demonstrating that retransformation of mutant JM15 with plasmid pSAT52, isolated and purified from the original complemented cells, conferred prototrophy on the mutant. In contrast mutant JM15 retranformed with the empty vector pYES was unable to grow with sulfate as sole sulfur source. SAT activity, assayed by the method of Kredich and Tomkins w11x, was detected in JM15rpSAT52 cell extracts Ž157 " 19.5 nmol acetyl CoA cleaved miny1 P mg protein w n s 10x. but was absent from extracts of mutant JM15 and JM15rpYES. Activity was dependent on the two substrates, acetyl CoA and Lserine, and SAT activity could be eliminated by boiling the JM15rpSAT52 cell extract. Plasmid pSAT52 contains a cDNA insert, Sat-52, of 1158 nucleotides with an open reading frame of 936 nucleotides, a 56-nucleotide 5X untranslated region and a 147-nucleotide non-coding 3X end, followed by a polyadenylylation stretch of 19 nu-
cleotides ŽFig. 1. . The open reading frame shows codon usage typical of A. thaliana genes w12x. Conceptual translation indicates a polypeptide of 312 amino acids with a M r of 32 770, the size expected of a plant SAT w13x, and a predicted isoelectric point of 7.16. In-frame stop codons at both the N-terminus and C-terminus of the deduced amino acid sequence ŽFig. 1. indicate that the Sat-52 cDNA encodes a full-length A. thaliana SAT protein. The deduced amino acid sequence of the Sat-52 open reading frame was compared with the other published A. thaliana, C. Õulgaris and E. coli sequences using the PILEUP alignment program from the UWGCG software package w14x. The deduced SAT52 amino acid sequence was shown to be 51.8% and 54.9% identical Ž67.5% and 69.8% similar. to those encoded by the cDNA clones Sat5 w3x and Sat-1 w7x that encode
Fig. 1. Nucleotide sequence and deduced amino acid sequence of the serine acetyltransferase clone Sat-52. The longest open reading frame of 312 amino acids from the 1.16 kb Arabidopsis thaliana Sat-52 clone is shown beneath the nucleotide sequence. The in-frame stop codons are indicated by asterisks Ž ) ..
J.R. Howarth et al.r Biochimica et Biophysica Acta 1350 (1997) 123–127
putative cytoplasmic and chloroplastic forms of SAT respectively ŽFig. 2.. The deduced SAT52 amino acid sequence was 50.9% identical Ž 69.4% similar. with the protein encoded by the Escherichia coli cysE gene. The region of greatest identity between the bacterial and plant proteins is found towards the carboxyl-terminus ŽFig. 2. and presumably carries the putative active site of SAT. Dot-plot analysis revealed the presence of internal repeats within the carboxy-terminal region of the deduced proteins encoded by the A. thaliana Sat-52 Žthis paper., Sat-1 w7x and Sat5 w3x, and the C. Õulgaris Sat2 w5x cDNAs, and the E. coli cysE w15x gene. Analysis of these internal repeats revealed an imperfect consensus hexad motif wŽ IrVrL . GXXXXx 5ŽIrVrL. common to a family of bacterial acylracetyltransferases w16x. Sequence analysis of the N-terminus of the deduced SAT proteins has suggested that clones Sat5 w3x and Sat-1 w7x encode isoforms of SAT destined for cytoplasmic and chloroplastic locations respectively.
125
It was therefore of great interest to attempt to identify a putative cellular location for the SAT52 protein. Assessment of the first ca. 40 amino acids of SAT52, chosen for analysis since homology with the other SAT protein sequences begins after this point and also this region represents an N-terminal extension when compared to the E. coli CYSE protein ŽFig. 2., revealed the absence of all the structural characteristics expected of both chloroplast w17,18x and peroxisomalrglyoxisomal transit peptides w19x. However, secondary structure predictions using Predict Protein software w20x suggested a mitochondrial location for the SAT52 isoform. Although no cleavage-site could be identified, a characteristic a-helical structure is formed between amino acids 22 to 57 of the deduced SAT52 protein, and the amino acid residues alanine, leucine, arginine and serine, also characteristic of mitochondrial transit peptides, represent 21 of the first 40 amino acids. In contrast they comprise only seven of the first 40 amino acids of the putatively-cytoplasmic SAT5 protein w3x. We note, however, that
Fig. 2. Comparison of all available plant serine acetyltransferase deduced amino acid sequences with each other and with the deduced amino acid sequence of the E. coli CYSE protein. Sequences were aligned using the PILEUP program w14x. Breaks in the alignment are represented by a dot. Amino acid residues identical to SAT52 are represented by a dash. SAT sequences compared are: A. thaliana Sat-52 Žthis paper, GenBank accession no. U30298.; A. thaliana Sat-1 ŽRef. w7x, GenBank accession no. U22964.; A. thaliana SAT1-6 ŽRef. w8x, GenBank accession no. X82888.; A. thaliana SAT1 ŽRef. w6x, GenBank accession no. L42212.; A. thaliana Sat5 ŽRef. w3x, GenBank accession Z34888.; Citrullus Õulgaris Žwatermelon. Sat2 ŽRef. w5x, GenBank accession no. D49535; E. coli cysE ŽRef. w15x, GenBank accession no. M15745..
126
J.R. Howarth et al.r Biochimica et Biophysica Acta 1350 (1997) 123–127
the SAT52 protein is not longer than the SAT5 protein and thus it may represent a second cytosolic species of SAT. Further studies are clearly needed to establish unequivocally the subcellular location of the three SAT species so far identified. Southern hybridization, using radiolabelled fulllength Sat-52 as a probe, was performed on Arabidopsis thaliana genomic DNA digested with restriction enzymes BamHI, BanII and BstEI which do not cut within the Sat-52 cDNA sequence and HindIII and Pst I which cut twice and once respectively ŽFig. 3A. . BamHI, BanII and Bst EI-digested DNA showed single strongly-hybridizing bands. HindIII and Pst I-digested DNA showed three and two strongly-hybridizing bands respectively. The data suggest the presence of a single copy of the Sat-52 gene. Earlier observations have shown increases in the capacity of the sulfate transporter w21x and in the activity of ATP sulfurylase w22x in response to sulfate limitation. More recent data demonstrate an increase in the abundance of transcripts for the sulfate transporter w23x, for O-acetylserine Žthiol.lyase w24x, for ‘APS reductase’ w25x and also the putatively chloroplastic Sat-1-type transcript SAT1-6 w8x when sulfate limitation is imposed. Thus it appears that under S limitation the capacity of the sulfate assimilation pathway may increase in an attempt both to scavenge for S and to maintain S flux through the pathway. We therefore looked to see whether this also held true for Sat-52 transcripts. When a gene-specific probe, derived from the 5X end of Sat-52 and unable to crosshybridize with either Sat-1 or Sat-5 transcripts, was used in Northern analysis against total RNA extracted from Arabidopsis thaliana plants w26x grown in liquid shake-culture, a single transcript of approx. 1.19 kb was detected. However, its abundance did not increase on imposition of sulfate limitation Ž Fig. 3B. under conditions where transcript abundance of ‘APS reductase’ did increase w25x. We conclude that SAT transcript levels are differentially regulated by sulfate limitation and that this differential regulation is related in some way to the roles the different SAT isoforms play in intracellular cysteine biosynthesis. The A. thaliana ŽColumbia ecotype. cDNA library constructed in the lYES yeast-Escherichia coli expression vector and E. coli strain BNN132 contain-
Fig. 3. A Southern blot of Arabidopsis thaliana genomic DNA Ž10 m g. digested with BamHI Žlane 1., BanII Žlane 2., BstEI Žlane 3., HindIII Žlane 4. and Pst I Žlane 5.. Digests were fractionated on a 1% agarose gel, blotted onto nylon membrane and probed with the radiolabelled full-length Sat-52 cDNA insert. The membrane was washed to 0.2=SSPE, 0.1% SDS at 428C. B. Northern analysis of Arabidopsis thaliana RNA extracted from Ža. sulfur-fed and Žb. 48 h sulfur-starved plants grown in vitro. Plants were grown in complete Murashige-Skoog w27x medium and then either transferred to the same medium or to medium in which sulfate salts had been replaced by the equivalent chloride salt. Total RNA Ž30 m g. was fractionated on a formaldehyderagarose gel, blotted onto nylon membrane and probed with the radiolabelled gene-specific Sat-52 cDNA probe. The membrane was washed to 0.2=SSPE, 0.1% SDS at 428C. Band size in kb is shown. Constitutive actin cDNA was used to probe the same filter.
ing lKC were gifts from Dr J.T. Mulligan, Stanford University School of Medicine, USA. J.R.H. was supported by a BBSRC Research Studentship. M.A.R. was supported by a University Research Studentship and by an Overseas Research Student Award. This work was supported by BBSRC Research Grant GRrJ65396 to J.L.W.
J.R. Howarth et al.r Biochimica et Biophysica Acta 1350 (1997) 123–127
References w1x Neuenschwander, U., Suter, M. and Brunold, C. Ž1991. Plant Physiol. 97, 253–258. w2x Saito, K., Kurosawa, M., Tatsuguchi, K., Takagi, Y. and Murakoshi, I. Ž1994. Plant Physiol. 106, 887–895. w3x Ruffet, M.-L., Lebrun, M., Droux, M. and Douce, R. Ž1995. Eur. J. Biochem. 227, 500–509. w4x Lunn, J.E., Droux, M., Martin, J. and Douce, R. Ž1990. Plant Physiol. 94, 1345–1352. w5x Saito, K., Yokoyama, H., Noji, M. and Murakoshi, I. Ž1995. J. Biol. Chem. 270, 16321–16326. w6x Murillo, M., Foglia, R., Diller, A., Lee, S. and Leustek, T. Ž1995. Cell. Mol. Biol. Res. 41, 425–433. w7x Roberts, M.A. and Wray, J.L. Ž1996. Plant Mol. Biol. 30, 1041–1049. w8x Bogdanova, N., Bork, and Hell, R. Ž1995. FEBS Lett. 358, 43–47. w9x Jones-Mortimer, M.C. Ž1968. Biochem. J. 110, 589–595. w10x Elledge, S.J., Mulligan, J.T., Ramer, S.W., Spottswood, M. and Davis, R.W. Ž1991. Proc. Natl. Acad. Sci. USA 88, 1731–1735. w11x Kredich, N.M. and Tomkins, G.M. Ž1966. J. Biol. Chem. 241, 4955–4965. w12x Wada, K.-N., Wada, Y., Ishibashi, F., Gojobori, T. and Ikemura, T. Ž1992. Nucleic Acids Res. 20, 2111–2118. w13x Ruffet, M.-L., Droux, M. and Douce, R. Ž1994. Plant Physiol. 104, 597–604.
127
w14x Devereux, J., Haeberli, P. and Smithies, O. Ž1984. Nucleic Acids Res. 12, 387–395. w15x Denk, D. and Bock, A. Ž1987. J. Gen. Microbiol. 133, ¨ 515–525. w16x Vuorio, R. Harkonen, T., Tolvanen, M. and Vaara, M. ¨ ¨ Ž1994. FEBS Lett. 337, 289–292. w17x Von Heijne, G., Steppuhn, J. and Herrmann, R.G. Ž1989. Eur. J. Biochem. 180, 535–545. w18x Von Heijne, G. Ž1992. in Genetic Engineering ŽSetlow, J.K., Ed.., Vol. 14, Plenum Press, New York, 1992, pp. 1–11. w19x De Hoop, M.J. and Ab, G. Ž1992. Biochem. J. 286, 657–669. w20x Rost, B. and Sander, C. Ž1993. J. Mol. Biol. 232, 584–599. w21x Hawkesford, M.J. and Belcher, A.R. Ž1991. Planta 185, 323–329. w22x Reuveny, Z. and Filner, P. Ž1977. J. Biol. Chem. 252, 1858–1864. w23x Smith, F.W., Ealing, P.M., Hawkesford, M.J.and Clarkson, D.T. Ž1995. Proc. Natl. Acad. Sci. USA 92, 9373–9377. w24x Barroso, C., Vega, J.M. and Gotor, C.Ž1995. FEBS Lett. 363, 1–5. w25x Gutierrez-Marcos, J.F., Roberts, M.A., Campbell, E.I. and Wray, J.L. Ž1996. Proc. Natl. Acad. Sci. USA, in press w26x Verwoerd, T.C., Dekker, B.M.M. and Hoekma, A. Ž1989. Nucleic Acids Res. 117, 2362. w27x Murashige, T. and Skoog, F. Ž1962. Physiol. Plant. 15, 473–497.
Short sequence-paper
Cysteine biosynthesis in higher plants: a new member of the Arabidopsis thaliana serine acetyltransferase small gene-family obtained by functional complementation of an Escherichia coli cysteine auxotroph 1 Jonathan R. Howarth 2 , Michael A. Roberts 3, John L. Wray
)
Plant Sciences Laboratory, Research DiÕision of EnÕironmental and EÕolutionary Biology, School of Biological and Medical Sciences, Sir Harold Mitchell Building, UniÕersity of St. Andrews, St Andrews, Fife KY16 9TH, UK Received 13 September 1996; revised 5 November 1996; accepted 5 November 1996
Abstract A cDNA clone, Sat-52, encoding a novel isoform of serine acetyltransferase ŽEC 2.3.1.30. was isolated by functional complementation of an Escherichia coli cysE mutant defective in serine acetyltransferase. The 1158 base pair clone contains a full-length open reading frame encoding a deduced protein of 312 amino acids with an M r of 32.77 kDa. Northern analysis revealed a single transcript of ca 1.19 kb that did not increase in abundance under sulfate limitation. Genomic Southern hybridization suggests the presence of a single copy of the Sat-52 gene. Keywords: Serine acetyltransferase; Cysteine biosynthesis; Nucleotide sequence; Sulfate regulation
Serine acetyltransferase Ž SAT. ŽEC 2.3.1.30. catalyses the formation of O-acetyl-L-serine from acetylCoA and L-serine. O-acetyl-L-serine then combines with sulfide, produced by the sulfate assimilation pathway, in a reaction catalyzed by O-acetyl-L-serine Žthiol. lyase ŽOASTL. ŽEC 4.2.99.8. to form the amino acid L-cysteine. Thus cysteine biosynthesis is a )
Corresponding author. Fax: q44 1334 463366. E-mail: [email protected] 1 The nucleotide sequence data reported in this paper have been submitted to the EMBLrGenBankrDDJB Data Libraries under the accession number U30298. 2 Authors’ names are in alphabetical order. The first two named authors made equal but separate contributions to the development and execution of this work. 3 Present address: Cereals Research Department, John Innes Centre, Norwich Research Park, Colney, Norwich, NR4 7UH, UK.
key step in the biological sulfur cycle since it is the point at which inorganic sulfur enters into organic combination. As well as being a precursor for L-cysteine biosythesis, O-acetyl-L-serine has been shown to have a regulatory role in sulfate assimilation w1,2x. Biochemical studies have located SAT w3x and OASTL w4x to the chloroplast, mitochondrion and cytosol of higher plants, suggesting each intracellular compartment has the ability to synthesize L-cysteine. However, why this should be necessary, and the relative extent to which these compartments contribute towards L-cysteine biosynthesis, is unknown. Molecular studies have identified cDNAs encoding putative cytoplasmic forms of SAT from Citrullus Õulgaris Žwatermelon. ŽSat2. w5x and Arabidopsis thaliana ŽSat5 w3x and SAT1 w6x. and a putative chloroplastic form from Arabidopsis thaliana ŽSat-1. w7x. A further Žpartial. cDNA ŽSAT1-6. w8x with
0167-4781r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 7 - 4 7 8 1 Ž 9 6 . 0 0 2 1 3 - 8
124
J.R. Howarth et al.r Biochimica et Biophysica Acta 1350 (1997) 123–127
highest homology to the putative chloroplastic form Sat-1 w7x has also been reported from Arabidopsis thaliana. In this paper we have attempted to isolate novel A. thaliana SAT cDNAs by functional complementation of the Escherichia coli cysE mutant strain JM15, deficient in SAT activity and unable to grow with sulfate as sole sulfur source w9x, to prototrophy. The availability of cDNA sequences for different isoforms of SAT in Arabidopsis thaliana should provide the key to the analysis of the role of O-acetyl-L-serine in sulfur metabolism and to the regulation of L-cysteine biosynthesis. JM15 was lysogenized with lKC, and then transfected with an Arabidopsis thaliana ŽColumbia ecotype. cDNA library in the expression vector lYES w10x. Colonies that grew on minimal medium containing sulfate as sole sulfur source and also 50 m grml ampicillin Žampicillin resistance is conferred by lYES. were selected and replated onto fresh medium. Complemented cells, that retained the ability to grow with sulfate as sole sulfur source and which were also ampicillin-resistant, were obtained at a frequency of 105 from 10 9 cells transfected with 6 = 10 7 lYES bacteriophage. Partial sequencing of the inserts of 10 of the longest complementing clones revealed one cDNA clone, Sat-52, that encoded a novel isoform of SAT. Functionality of the cDNA clone Sat-52 was confirmed by demonstrating that retransformation of mutant JM15 with plasmid pSAT52, isolated and purified from the original complemented cells, conferred prototrophy on the mutant. In contrast mutant JM15 retranformed with the empty vector pYES was unable to grow with sulfate as sole sulfur source. SAT activity, assayed by the method of Kredich and Tomkins w11x, was detected in JM15rpSAT52 cell extracts Ž157 " 19.5 nmol acetyl CoA cleaved miny1 P mg protein w n s 10x. but was absent from extracts of mutant JM15 and JM15rpYES. Activity was dependent on the two substrates, acetyl CoA and Lserine, and SAT activity could be eliminated by boiling the JM15rpSAT52 cell extract. Plasmid pSAT52 contains a cDNA insert, Sat-52, of 1158 nucleotides with an open reading frame of 936 nucleotides, a 56-nucleotide 5X untranslated region and a 147-nucleotide non-coding 3X end, followed by a polyadenylylation stretch of 19 nu-
cleotides ŽFig. 1. . The open reading frame shows codon usage typical of A. thaliana genes w12x. Conceptual translation indicates a polypeptide of 312 amino acids with a M r of 32 770, the size expected of a plant SAT w13x, and a predicted isoelectric point of 7.16. In-frame stop codons at both the N-terminus and C-terminus of the deduced amino acid sequence ŽFig. 1. indicate that the Sat-52 cDNA encodes a full-length A. thaliana SAT protein. The deduced amino acid sequence of the Sat-52 open reading frame was compared with the other published A. thaliana, C. Õulgaris and E. coli sequences using the PILEUP alignment program from the UWGCG software package w14x. The deduced SAT52 amino acid sequence was shown to be 51.8% and 54.9% identical Ž67.5% and 69.8% similar. to those encoded by the cDNA clones Sat5 w3x and Sat-1 w7x that encode
Fig. 1. Nucleotide sequence and deduced amino acid sequence of the serine acetyltransferase clone Sat-52. The longest open reading frame of 312 amino acids from the 1.16 kb Arabidopsis thaliana Sat-52 clone is shown beneath the nucleotide sequence. The in-frame stop codons are indicated by asterisks Ž ) ..
J.R. Howarth et al.r Biochimica et Biophysica Acta 1350 (1997) 123–127
putative cytoplasmic and chloroplastic forms of SAT respectively ŽFig. 2.. The deduced SAT52 amino acid sequence was 50.9% identical Ž 69.4% similar. with the protein encoded by the Escherichia coli cysE gene. The region of greatest identity between the bacterial and plant proteins is found towards the carboxyl-terminus ŽFig. 2. and presumably carries the putative active site of SAT. Dot-plot analysis revealed the presence of internal repeats within the carboxy-terminal region of the deduced proteins encoded by the A. thaliana Sat-52 Žthis paper., Sat-1 w7x and Sat5 w3x, and the C. Õulgaris Sat2 w5x cDNAs, and the E. coli cysE w15x gene. Analysis of these internal repeats revealed an imperfect consensus hexad motif wŽ IrVrL . GXXXXx 5ŽIrVrL. common to a family of bacterial acylracetyltransferases w16x. Sequence analysis of the N-terminus of the deduced SAT proteins has suggested that clones Sat5 w3x and Sat-1 w7x encode isoforms of SAT destined for cytoplasmic and chloroplastic locations respectively.
125
It was therefore of great interest to attempt to identify a putative cellular location for the SAT52 protein. Assessment of the first ca. 40 amino acids of SAT52, chosen for analysis since homology with the other SAT protein sequences begins after this point and also this region represents an N-terminal extension when compared to the E. coli CYSE protein ŽFig. 2., revealed the absence of all the structural characteristics expected of both chloroplast w17,18x and peroxisomalrglyoxisomal transit peptides w19x. However, secondary structure predictions using Predict Protein software w20x suggested a mitochondrial location for the SAT52 isoform. Although no cleavage-site could be identified, a characteristic a-helical structure is formed between amino acids 22 to 57 of the deduced SAT52 protein, and the amino acid residues alanine, leucine, arginine and serine, also characteristic of mitochondrial transit peptides, represent 21 of the first 40 amino acids. In contrast they comprise only seven of the first 40 amino acids of the putatively-cytoplasmic SAT5 protein w3x. We note, however, that
Fig. 2. Comparison of all available plant serine acetyltransferase deduced amino acid sequences with each other and with the deduced amino acid sequence of the E. coli CYSE protein. Sequences were aligned using the PILEUP program w14x. Breaks in the alignment are represented by a dot. Amino acid residues identical to SAT52 are represented by a dash. SAT sequences compared are: A. thaliana Sat-52 Žthis paper, GenBank accession no. U30298.; A. thaliana Sat-1 ŽRef. w7x, GenBank accession no. U22964.; A. thaliana SAT1-6 ŽRef. w8x, GenBank accession no. X82888.; A. thaliana SAT1 ŽRef. w6x, GenBank accession no. L42212.; A. thaliana Sat5 ŽRef. w3x, GenBank accession Z34888.; Citrullus Õulgaris Žwatermelon. Sat2 ŽRef. w5x, GenBank accession no. D49535; E. coli cysE ŽRef. w15x, GenBank accession no. M15745..
126
J.R. Howarth et al.r Biochimica et Biophysica Acta 1350 (1997) 123–127
the SAT52 protein is not longer than the SAT5 protein and thus it may represent a second cytosolic species of SAT. Further studies are clearly needed to establish unequivocally the subcellular location of the three SAT species so far identified. Southern hybridization, using radiolabelled fulllength Sat-52 as a probe, was performed on Arabidopsis thaliana genomic DNA digested with restriction enzymes BamHI, BanII and BstEI which do not cut within the Sat-52 cDNA sequence and HindIII and Pst I which cut twice and once respectively ŽFig. 3A. . BamHI, BanII and Bst EI-digested DNA showed single strongly-hybridizing bands. HindIII and Pst I-digested DNA showed three and two strongly-hybridizing bands respectively. The data suggest the presence of a single copy of the Sat-52 gene. Earlier observations have shown increases in the capacity of the sulfate transporter w21x and in the activity of ATP sulfurylase w22x in response to sulfate limitation. More recent data demonstrate an increase in the abundance of transcripts for the sulfate transporter w23x, for O-acetylserine Žthiol.lyase w24x, for ‘APS reductase’ w25x and also the putatively chloroplastic Sat-1-type transcript SAT1-6 w8x when sulfate limitation is imposed. Thus it appears that under S limitation the capacity of the sulfate assimilation pathway may increase in an attempt both to scavenge for S and to maintain S flux through the pathway. We therefore looked to see whether this also held true for Sat-52 transcripts. When a gene-specific probe, derived from the 5X end of Sat-52 and unable to crosshybridize with either Sat-1 or Sat-5 transcripts, was used in Northern analysis against total RNA extracted from Arabidopsis thaliana plants w26x grown in liquid shake-culture, a single transcript of approx. 1.19 kb was detected. However, its abundance did not increase on imposition of sulfate limitation Ž Fig. 3B. under conditions where transcript abundance of ‘APS reductase’ did increase w25x. We conclude that SAT transcript levels are differentially regulated by sulfate limitation and that this differential regulation is related in some way to the roles the different SAT isoforms play in intracellular cysteine biosynthesis. The A. thaliana ŽColumbia ecotype. cDNA library constructed in the lYES yeast-Escherichia coli expression vector and E. coli strain BNN132 contain-
Fig. 3. A Southern blot of Arabidopsis thaliana genomic DNA Ž10 m g. digested with BamHI Žlane 1., BanII Žlane 2., BstEI Žlane 3., HindIII Žlane 4. and Pst I Žlane 5.. Digests were fractionated on a 1% agarose gel, blotted onto nylon membrane and probed with the radiolabelled full-length Sat-52 cDNA insert. The membrane was washed to 0.2=SSPE, 0.1% SDS at 428C. B. Northern analysis of Arabidopsis thaliana RNA extracted from Ža. sulfur-fed and Žb. 48 h sulfur-starved plants grown in vitro. Plants were grown in complete Murashige-Skoog w27x medium and then either transferred to the same medium or to medium in which sulfate salts had been replaced by the equivalent chloride salt. Total RNA Ž30 m g. was fractionated on a formaldehyderagarose gel, blotted onto nylon membrane and probed with the radiolabelled gene-specific Sat-52 cDNA probe. The membrane was washed to 0.2=SSPE, 0.1% SDS at 428C. Band size in kb is shown. Constitutive actin cDNA was used to probe the same filter.
ing lKC were gifts from Dr J.T. Mulligan, Stanford University School of Medicine, USA. J.R.H. was supported by a BBSRC Research Studentship. M.A.R. was supported by a University Research Studentship and by an Overseas Research Student Award. This work was supported by BBSRC Research Grant GRrJ65396 to J.L.W.
J.R. Howarth et al.r Biochimica et Biophysica Acta 1350 (1997) 123–127
References w1x Neuenschwander, U., Suter, M. and Brunold, C. Ž1991. Plant Physiol. 97, 253–258. w2x Saito, K., Kurosawa, M., Tatsuguchi, K., Takagi, Y. and Murakoshi, I. Ž1994. Plant Physiol. 106, 887–895. w3x Ruffet, M.-L., Lebrun, M., Droux, M. and Douce, R. Ž1995. Eur. J. Biochem. 227, 500–509. w4x Lunn, J.E., Droux, M., Martin, J. and Douce, R. Ž1990. Plant Physiol. 94, 1345–1352. w5x Saito, K., Yokoyama, H., Noji, M. and Murakoshi, I. Ž1995. J. Biol. Chem. 270, 16321–16326. w6x Murillo, M., Foglia, R., Diller, A., Lee, S. and Leustek, T. Ž1995. Cell. Mol. Biol. Res. 41, 425–433. w7x Roberts, M.A. and Wray, J.L. Ž1996. Plant Mol. Biol. 30, 1041–1049. w8x Bogdanova, N., Bork, and Hell, R. Ž1995. FEBS Lett. 358, 43–47. w9x Jones-Mortimer, M.C. Ž1968. Biochem. J. 110, 589–595. w10x Elledge, S.J., Mulligan, J.T., Ramer, S.W., Spottswood, M. and Davis, R.W. Ž1991. Proc. Natl. Acad. Sci. USA 88, 1731–1735. w11x Kredich, N.M. and Tomkins, G.M. Ž1966. J. Biol. Chem. 241, 4955–4965. w12x Wada, K.-N., Wada, Y., Ishibashi, F., Gojobori, T. and Ikemura, T. Ž1992. Nucleic Acids Res. 20, 2111–2118. w13x Ruffet, M.-L., Droux, M. and Douce, R. Ž1994. Plant Physiol. 104, 597–604.
127
w14x Devereux, J., Haeberli, P. and Smithies, O. Ž1984. Nucleic Acids Res. 12, 387–395. w15x Denk, D. and Bock, A. Ž1987. J. Gen. Microbiol. 133, ¨ 515–525. w16x Vuorio, R. Harkonen, T., Tolvanen, M. and Vaara, M. ¨ ¨ Ž1994. FEBS Lett. 337, 289–292. w17x Von Heijne, G., Steppuhn, J. and Herrmann, R.G. Ž1989. Eur. J. Biochem. 180, 535–545. w18x Von Heijne, G. Ž1992. in Genetic Engineering ŽSetlow, J.K., Ed.., Vol. 14, Plenum Press, New York, 1992, pp. 1–11. w19x De Hoop, M.J. and Ab, G. Ž1992. Biochem. J. 286, 657–669. w20x Rost, B. and Sander, C. Ž1993. J. Mol. Biol. 232, 584–599. w21x Hawkesford, M.J. and Belcher, A.R. Ž1991. Planta 185, 323–329. w22x Reuveny, Z. and Filner, P. Ž1977. J. Biol. Chem. 252, 1858–1864. w23x Smith, F.W., Ealing, P.M., Hawkesford, M.J.and Clarkson, D.T. Ž1995. Proc. Natl. Acad. Sci. USA 92, 9373–9377. w24x Barroso, C., Vega, J.M. and Gotor, C.Ž1995. FEBS Lett. 363, 1–5. w25x Gutierrez-Marcos, J.F., Roberts, M.A., Campbell, E.I. and Wray, J.L. Ž1996. Proc. Natl. Acad. Sci. USA, in press w26x Verwoerd, T.C., Dekker, B.M.M. and Hoekma, A. Ž1989. Nucleic Acids Res. 117, 2362. w27x Murashige, T. and Skoog, F. Ž1962. Physiol. Plant. 15, 473–497.