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. 1988 Jan 1;106(1):29–37. doi: 10.1083/jcb.106.1.29

Purification and biosynthesis of a derepressible periplasmic arylsulfatase from Chlamydomonas reinhardtii

PMCID: PMC2114941  PMID: 3339089

Abstract

The unicellular green alga Chlamydomonas reinhardtii responds to sulfate deprivation by producing an arylsulfatase (Lien, T., and O. Schreiner. 1975. Biochim. Biophys. Acta. 384:168-179; Schreiner, O., 1975. Biochim. Biophys. Acta. 384:180-193) and by developing the capacity to transport sulfate more rapidly (our unpublished data). The arylsulfatase activity, detectable 3 h after the transfer of the cells to low sulfate medium (less than or equal to 10 microM sulfate), is a periplasmic protein released into the culture medium by cw15, a cell wall-less mutant of C. reinhardtii. We have purified the derepressible arylsulfatase to homogeneity and have raised monospecific antibodies to it. The protein monomer (67.6 kD) associates into a dimer, and the enzyme activity shows an alkaline pH optimum and a Km of 0.3 mM for p- nitrophenylsulfate. Studies focused on arylsulfatase biosynthesis demonstrate that it is glycosylated and synthesized as a higher molecular mass precursor. The mature protein contains complex N-linked oligosaccharides and the primary translation product has an apparent molecular mass approximately 5 kD larger than the deglycosylated monomer. Since translatable RNA encoding the arylsulfatase can only be detected in cells after sulfate starvation, it is likely that accumulation of the enzyme is regulated at the level of transcription, although posttranscriptional processes may also be involved.

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Selected References

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  1. Ahmad A., Surolia A., Bachhawat B. K. Affinity chromatographic separation of arylsulfatase A and B using Cibacron Blue-Sepharose. Biochim Biophys Acta. 1977 Apr 12;481(2):542–548. doi: 10.1016/0005-2744(77)90286-8. [DOI] [PubMed] [Google Scholar]
  2. Apte B. N., Bhavsar P. N., Siddiqi O. The regulation of aryl sulphatase in Aspergillus nidulans. J Mol Biol. 1974 Jul 5;86(3):637–648. doi: 10.1016/0022-2836(74)90186-7. [DOI] [PubMed] [Google Scholar]
  3. Bostian K. A., Lemire J. M., Halvorson H. O. Physiological control of repressible acid phosphatase gene transcripts in Saccharomyces cerevisiae. Mol Cell Biol. 1983 May;3(5):839–853. doi: 10.1128/mcb.3.5.839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  5. Cashmore A. R., Broadhurst M. K., Gray R. E. Cell-free synthesis of leaf protein: Identification of an apparent precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase. Proc Natl Acad Sci U S A. 1978 Feb;75(2):655–659. doi: 10.1073/pnas.75.2.655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chua N. H., Blomberg F. Immunochemical studies of thylakoid membrane polypeptides from spinach and Chlamydomonas reinhardtii. A modified procedure for crossed immunoelectrophoresis of dodecyl sulfate.protein complexes. J Biol Chem. 1979 Jan 10;254(1):215–223. [PubMed] [Google Scholar]
  7. Coleman J. R., Berry J. A., Togasaki R. K., Grossman A. R. Identification of Extracellular Carbonic Anhydrase of Chlamydomonas reinhardtii. Plant Physiol. 1984 Oct;76(2):472–477. doi: 10.1104/pp.76.2.472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Coleman J. R., Grossman A. R. Biosynthesis of carbonic anhydrase in Chlamydomonas reinhardtii during adaptation to low CO(2). Proc Natl Acad Sci U S A. 1984 Oct;81(19):6049–6053. doi: 10.1073/pnas.81.19.6049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Goodenough U. W., Heuser J. E. The Chlamydomonas cell wall and its constituent glycoproteins analyzed by the quick-freeze, deep-etch technique. J Cell Biol. 1985 Oct;101(4):1550–1568. doi: 10.1083/jcb.101.4.1550. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gorman D. S., Levine R. P. Cytochrome f and plastocyanin: their sequence in the photosynthetic electron transport chain of Chlamydomonas reinhardi. Proc Natl Acad Sci U S A. 1965 Dec;54(6):1665–1669. doi: 10.1073/pnas.54.6.1665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Grant W. D. Cell wall teichoic acid as a reserve phosphate source in Bacillus subtilis. J Bacteriol. 1979 Jan;137(1):35–43. doi: 10.1128/jb.137.1.35-43.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Imam S. H., Buchanan M. J., Shin H. C., Snell W. J. The Chlamydomonas cell wall: characterization of the wall framework. J Cell Biol. 1985 Oct;101(4):1599–1607. doi: 10.1083/jcb.101.4.1599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kobata A. Use of endo- and exoglycosidases for structural studies of glycoconjugates. Anal Biochem. 1979 Nov 15;100(1):1–14. doi: 10.1016/0003-2697(79)90102-7. [DOI] [PubMed] [Google Scholar]
  14. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  15. Lemire J. M., Willcocks T., Halvorson H. O., Bostian K. A. Regulation of repressible acid phosphatase gene transcription in Saccharomyces cerevisiae. Mol Cell Biol. 1985 Aug;5(8):2131–2141. doi: 10.1128/mcb.5.8.2131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lien T., Schreiner O., Steine M. Purification of a derepressible arylsulfatase from Chlamydomonas reinhardti. Properties of the enzyme in intact cells and in purified state. Biochim Biophys Acta. 1975 Mar 28;384(1):168–179. doi: 10.1016/0005-2744(75)90106-0. [DOI] [PubMed] [Google Scholar]
  17. Loppes R., Matagne R. F. Acid phosphatase mutants in Chlamydomonas: isolation and characterization by biochemical, electrophoretic and genetic analysis. Genetics. 1973 Dec;75(4):593–604. doi: 10.1093/genetics/75.4.593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Moroney J. V., Husic H. D., Tolbert N. E. Effect of Carbonic Anhydrase Inhibitors on Inorganic Carbon Accumulation by Chlamydomonas reinhardtii. Plant Physiol. 1985 Sep;79(1):177–183. doi: 10.1104/pp.79.1.177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Moroney J. V., Kitayama M., Togasaki R. K., Tolbert N. E. Evidence for Inorganic Carbon Transport by Intact Chloroplasts of Chlamydomonas reinhardtii. Plant Physiol. 1987 Mar;83(3):460–463. doi: 10.1104/pp.83.3.460. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. REISFELD R. A., LEWIS U. J., WILLIAMS D. E. Disk electrophoresis of basic proteins and peptides on polyacrylamide gels. Nature. 1962 Jul 21;195:281–283. doi: 10.1038/195281a0. [DOI] [PubMed] [Google Scholar]
  21. Schmidt G. W., Devillers-Thiery A., Desruisseaux H., Blobel G., Chua N. H. NH2-terminal amino acid sequences of precursor and mature forms of the ribulose-1,5-bisphosphate carboxylase small subunit from Chlamydomonas reinhardtii. J Cell Biol. 1979 Dec;83(3):615–622. doi: 10.1083/jcb.83.3.615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Schreiner O., Lien T., Knutsen G. The capacity for arylsulfatase synthesis in synchronous and synchronized cultures of Chlamydomonas reinhardti. Biochim Biophys Acta. 1975 Mar 28;384(1):180–193. doi: 10.1016/0005-2744(75)90107-2. [DOI] [PubMed] [Google Scholar]
  23. Scott W. A., Metzenberg R. L. Location of Aryl Sulfatase in Conidia and Young Mycelia of Neurospora crassa. J Bacteriol. 1970 Dec;104(3):1254–1265. doi: 10.1128/jb.104.3.1254-1265.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Spalding M. H., Spreitzer R. J., Ogren W. L. Reduced Inorganic Carbon Transport in a CO(2)-Requiring Mutant of Chlamydomonas reinhardii. Plant Physiol. 1983 Oct;73(2):273–276. doi: 10.1104/pp.73.2.273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]

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