Skip to main content
SpringerLink
Log in

Increased transcript levels of a methionine synthase during adhesion-induced activation of Chlamydomonas reinhardtii gametes

  • Research article
  • Published:
Plant Molecular Biology Aims and scope Submit manuscript

Abstract

Chlamydomonas gametes of opposite mating types interact through flagellar adhesion molecules called agglutinins leading to a signal transduction cascade that induces cell wall loss and activation of mating structures along with other cellular responses that ultimately result in zygote formation. To identify molecules involved in these complex cellular events, we have employed subtractive and differential hybridization with cDNA from mt+ gametes activated for fertilization and non-signaling, vegetative (non-gametic) cells. We identified 55 cDNA clones whose transcripts were regulated in activated gametes. Here we report the molecular cloning and characterization of the complementary DNA (cDNA) for one clone whose transcripts in activated gametes were several-fold higher than in normal gametes. Regulation of the transcript was not related simply to protein synthesis because it was not increased in cells synthesizing new cell wall proteins. The cDNA contained a single open reading frame (ORF) of 815 amino acids encoding a polypeptide of calculated relative mass of 87 kDa. Database search analysis and sequence alignment indicated that the deduced amino acid sequence exhibited 42% identity and 62% similarity to a class of prokaryotic methyl transferases (5-methyltetrahydrofolate-homocysteine methyl transferase; EC 2.1.1.14) known to be involved in the terminal step of de novo biosynthesis of methionine. This enzyme catalyzes transfer of a methyl group from 5-methyltetrahydrofolate to homocysteine resulting in methionine formation. Affinity-purified polyclonal antibodies raised against a bacterially produced GST-fusion protein identified a 85 kDa soluble protein in Chlamydomonas gametes. Southern blot hybridization indicated that the enzyme is encoded by a single-copy gene. The evidence presented in this paper raises the possibility that, in addition to its participation in de novo biosynthesis and regeneration of methionine, Chlamydomonas methionine synthase may play a role in adhesion-induced events during fertilization.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Adair WS, Apt KE: Cell wall regeneration in Chlamydomonas: accumulation of mRNAs encoding cell wall hydroxyproline-rich glycoproteins. Proc Natl Acad Sci USA 87: 7355–7359 (1990).

    Google Scholar 

  2. Banerjee RV, Matthews RG: Cobalamin-dependent methionine synthase. FASEB J 4: 1450–1459 (1990).

    Google Scholar 

  3. Buchanan MJ, Snell WJ: Biochemical studies on lysin, a cell wall degrading enzyme released during fertilization in Chlamydomonas. Exp Cell Res 179: 181–193 (1988).

    Google Scholar 

  4. Clarke S: Protein isoprenylation and methylation at carboxy-terminal cysteine residues. Annu Rev Biochem 61: 355–386 (1992).

    Google Scholar 

  5. Dobberstein B, Blobel G, Chua N: In vitro synthesis and processing of a putative precursor for the small subunit of ribulose-1,5-bisphosphate carboxylase of Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 74: 1082–1085 (1977).

    Google Scholar 

  6. Drennan CL, Huang S, Drummond JT, Matthews RG, Ludwig ML: How a protein binds B12 A 3.0 Å X-ray structure of B12-binding domains of methionine synthase. Science 266: 1669–1674 (1994).

    Google Scholar 

  7. Dworkin MB, Dawid IB: Use of a cloned library for the study of abundant poly(A)+ RNA during Xenopus laevis development. Devel Biol 76: 449–464 (1980).

    Google Scholar 

  8. Emini EA, Hughes JV, Perlow DS, Boger J: Induction of hepatitis A virus-neutralizing antibody by a virus-specific synthetic peptide. J Virol 55: 836–839 (1985).

    Google Scholar 

  9. Feinberg AP, Vogelstein B: A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132: 6–13 (1984).

    Google Scholar 

  10. Ferris PJ, Goodenough UW: Transcription of novel genes including a gene linked to the mating type locus induced by Chlamydomonas fertilization. Mol Cell Biol 7: 2360–2366 (1987).

    Google Scholar 

  11. Frangioni JV, Neel BG: Solubilization and purification of enzymatically active glutathione S-transferase (pGEX) fusion proteins. Anal Biochem 210: 179–187 (1993).

    Google Scholar 

  12. Frohman MA, Dush MK, Martin GR: Rapid production of full-length cDNAs from rare transcripts amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci USA 85: 8998–9002 (1988).

    Google Scholar 

  13. Goodenough UW: Mating interactions in Chlamydomonas. In: Reissig JL (ed) Microbial Interactions, pp. 323–350. Chapman and Hall, London (1977).

    Google Scholar 

  14. Goodenough UW, Adair WS, Collin-Osdoby P, Heuser JE: Chlamydomonas cells in contact. In: Edelman GM, Thiery JP (eds) The Cell in Contact: Adhesions and Junctions as Morphogenetic Determinants, pp. 111–135. John Wiley, New York, (1985).

    Google Scholar 

  15. Goodenough UW: Green yeast. Cell 70: 533–538 (1992).

    Google Scholar 

  16. Gonzalez JC, Banerjee RV, Huang S, Sumner JS, Matthews RG: Comparison of cobalamin-independent and cobalamin-dependent methionine synthases from Escherichia coli: two solutions to the same chemical problem. Biochemistry 31: 6045–6056 (1992).

    Google Scholar 

  17. Harris EH: The Chlamydomonas sourcebook. A comprehensive guide to biology and laboratory use. Academic Press, San Diego, CA (1989).

    Google Scholar 

  18. Horwich AL, Low KB, Fenton WA, Hirshfield IN, Furtak K: Folding in vivo of bacterial cytoplasmic proteins: role for GroEL. Cell 74: 909–917 (1993).

    Google Scholar 

  19. Klar A, Baldassare M, Jessell TM: F-spondin: A gene expressed at high levels in the floor plate encodes a secreted protein that promotes neural cell adhesion and neurite extension. Cell 69: 95–110 (1992).

    Google Scholar 

  20. Kurvari V, Zhang Y, Luo Y, Snell W: A novel protein kinase in Chlamydomonas: An element of a new signaling pathway in fertilization Proc Natl Acad Sci USA, in press.

  21. Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685 (1974).

    Google Scholar 

  22. Lefebvre PA, Silflow CD, Wieben ED, Rosenbaum JL: Increased levels of mRNAs for tubulin and other flagellar proteins after amputation or shortening of Chlamydomonas flagella. Cell 20: 469–477 (1980).

    Google Scholar 

  23. Lelias J, Adra CN, Wulf GM, Gruillemot J, Khagad M, Caput D, Lim B: cDNA cloning of a human mRNA preferentially expressed in hematopoietic cells and with homology to a GDP-dissociation inhibitor for the rho GTP-binding proteins. Proc Natl Acad Sci USA 90: 1479–1483 (1993).

    Google Scholar 

  24. Mathur M, Saluja D, Sachar RC: Post-trascriptional regulation of S-adenosylmethionine synthetase from its stored mRNA in germinated wheat embryos. Biochim Biophys Acta 1078: 161–170 (1991).

    Google Scholar 

  25. Matthews RG: Methionine biosynthesis. In: Blakley RL, Benkovic SJ (eds) Folates and Pterins, pp. 497–554, John Wiley, New York (1984).

    Google Scholar 

  26. Pasquale SM, Goodenough UW: Cyclic AMP functions as a primary sexual signal in gametes of Chlamydomonas reinhardtii. J Cell Biol 105: 2279–2292 (1987).

    Google Scholar 

  27. Peleman J, Boerjan W, Engler G, Seurinck J, Botterman J, Alliotte T, Van Montagu M, Inze D: Strong cellular preference in the expression of a housekeeping gene of Arabidopsis thaliana encoding S-adenosylmethionine synthetase. Plant Cell 1: 81–93 (1989).

    Google Scholar 

  28. Peleman J, Saito K, Cottyn B, Engler G, Seurinck J, Van Montagu M, Inze D: Structure and expression of the S-adenosylmethionine synthetase gene family in Arabidopsis thaliana. Gene 84: 359–369 (1989).

    Google Scholar 

  29. Pijst HLA, van Driel R, Janssens PMW, Musgrave A, van den Ende H: Cyclic AMP is involved in sexual reproduction of Chlamydomonas eugametos. FEBS Lett 174: 132–136 (1984).

    Google Scholar 

  30. Rognes SE, Lea PJ, Miflin BJ: S-adenosylmethionine, a novel regulator of aspartate kinase. Nature 287: 257–359 (1980).

    Google Scholar 

  31. Rutherford SL, Zuker CS: Protein folding and the regulation of signaling pathways. Cell 79: 1129–1132 (1994).

    Google Scholar 

  32. Sager R, Granick S: Nutritional control of sexuality in Chlamydomonas reinhardi. J Gen Physiol 37: 729–742 (1954).

    Google Scholar 

  33. Saito T, Small L, Goodenough UW: Activation of adenylyl cyclase in Chlamydomonas reinhardtii by adhesion and by heat. J Cell Biol 122: 137–147 (1993).

    Google Scholar 

  34. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, NY (1989).

    Google Scholar 

  35. Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain-terminating inhibitors Proc Natl Acad Sci USA 74: 5463–5467 (1977).

    Google Scholar 

  36. Sapperstein S, Berkower C, Michaelis S: Nucleotide sequence of the yeast STE14 gene which encodes farnesyl-cysteine carboxyl methyltransferase and demonstration of its essential role in a-Factor export. Mol Cell Biol 14: 1438–1449 (1994).

    Google Scholar 

  37. Schloss JA, Silflow CD, Rosenbaum JL: mRNA abundance changes during flagellar regeneration in Chlamydomonas reinhardtii. Mol Cell Biol 4: 424–434 (1984).

    Google Scholar 

  38. Schmidt A, Rennenberg H, Wilson LG, Filner P: Formation of methanethiol from methionine by leaf tissue. Phytochemistry 24: 1181–1185 (1985).

    Google Scholar 

  39. Silflow CD, Chisholm RL, Conner TW, Ranum LPW: The two alpha-tubulin genes of Chlamydomonas reinhardtii code for slightly different proteins. Mol Cell Biol 5: 2389–2398 (1985).

    Google Scholar 

  40. Sive HL, John TS: A simple subtractive hybridization technique employing photoactivatable biotin and phenol extraction. Nucl Acids Res 16: 10937 (1988).

    Google Scholar 

  41. Snell WJ: Gamete induction and flagellar adhesion in Chlamydomonas reinhardi In: Gantt E (ed) Developmental and Cytological Methods, pp. 37–45. Cambridge University Press, Cambridge, UK (1976).

    Google Scholar 

  42. Snell WJ: Study of the release of cell wall degrading enzymes during adhesion of Chlamydomonas gametes. Exp Cell Res 138: 109–119 (1982).

    Google Scholar 

  43. Snell WJ: Adhesion and signaling in multicellular and unicellular organisms. Curr Opin Cell Biol 2: 821–832 (1990).

    Google Scholar 

  44. Snell WJ: Signal transduction during fertilization in Chlamydomonas. In: Kurjan J, Taylor BL (eds) Signal Transduction: Procaryotic and Simple Eucaryotic Systems, pp. 255–277, Academic Press, New York (1993).

    Google Scholar 

  45. Snell WJ, Moore WS: Aggregation-dependent turnover of flagellar adhesion molecules in Chlamydomonas gametes. J Cell Biol 84: 203–210 (1980).

    Google Scholar 

  46. Stock J, Surette MG, McCleary WR, Stock AM: Signal transduction in bacteria. J Biol Chem 267: 19753–19756 (1992).

    Google Scholar 

  47. Tabor CW, Tabor H: Polyamines. Annu Rev Biochem 53: 749–790 (1984).

    Google Scholar 

  48. Thomas D, Rothstein R, Rosenberg N, Surdin-Kerjan Y: SAM2 encodes the second methionine S-adenosyltransferase in Saccharomyces cerevisiae: physiology and regulation of both enzymes. Mol Cell Biol 8: 5132–5139 (1988).

    Google Scholar 

  49. Travis GH, Milner RJ, Sutcliffe JG: Preparation and use of subtractive cDNA hybridization probes for cDNA cloning. In: Boulton AA, Baker GB, Campagnoni AT (eds) Neuromethods, pp. 49–78. Humana Press, Clifton, NJ (1989).

    Google Scholar 

  50. Urbanowski ML, Stauffer LT, Plamann LS, Stauffer GV: A new methionine locus metR that encodes a trans-acting protein required for activation of metE and metH in Escherichia coli and Salmonella typhimurium. J Bact 169: 1391–1397 (1987).

    Google Scholar 

  51. Urbanowski ML, Stauffer GV: The metH gene from Salmonella typhimurium LT2: cloning and initial characterization. Gene 44: 211–217 (1986).

    Google Scholar 

  52. van den Ende H: Sexual signaling in Chlamydomonas. In: Callow JA, Green JR (eds) Perspectives in Plant Cell Recognition, pp. 1–19, Cambridge University Press, London, (1992).

    Google Scholar 

  53. Weeks DP: Chlamydomonas: an increasingly powerful model plant cell system. Plant Cell 3: 451–456 (1992).

    Google Scholar 

  54. Wegener D, Beck CF: Identification of novel genes specifically expressed in Chlamydomonas reinhardtii zygotes. Plant Mol Biol 16: 937–946 (1991).

    Google Scholar 

  55. Yang SF, Hoffman NE: Ethylene biosynthesis and its regulation in higher plants. Annu Rev Plant Physiol 35: 155–189 (1984).

    Google Scholar 

  56. Youngblom J, Schloss JA, Silflow CD: The two β-tubulin genes of Chlamydomonas reinhardtii code for identical proteins. Mol Cell Biol 4: 2686–2696 (1984).

    Google Scholar 

  57. Yu LM, Selman BR: cDNA sequence and predicted primary structure of the γ subunit from the ATP synthase from Chlamydomonas reinhardtii. J Biol Chem 263: 19342–19345 (1988).

    Google Scholar 

  58. Zhang Y, Ross EM, Snell WJ: ATP-dependent regulation of flagellar adenylylcyclase in gametes of Chlamydomonas reinhardtii. J Biol Chem 266: 22954–22959 (1991).

    Google Scholar 

  59. Zhang Y, Snell WJ: Differential regulation of adenylylcyclases in vegetative and gametic flagella of Chlamydomonas. J Biol Chem 268: 1786–1791 (1993).

    Google Scholar 

  60. Zhang Y, Snell WJ: Flagellar adhesion-dependent regulation of Chlamydomonas adenylyl cyclase in vitro: a possible role for protein kinases in sexual signaling. J Cell Biol 125: 617–624 (1994).

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Cell Biology and Neuroscience, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, 75235-9039, Dallas, TX, USA

    Venkatesh Kurvari, Fang Qian & William J. Snell

Authors
  1. Venkatesh Kurvari
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Fang Qian
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. William J. Snell
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kurvari, V., Qian, F. & Snell, W.J. Increased transcript levels of a methionine synthase during adhesion-induced activation of Chlamydomonas reinhardtii gametes. Plant Mol Biol 29, 1235–1252 (1995). https://doi.org/10.1007/BF00020465

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00020465

Key words

Search

Navigation