Planta

, Volume 197, Issue 2, pp 343–351 | Cite as

Calcium modulates rapid protein phosphorylation/dephosphorylation in isolated eyespot apparatuses of the green alga Spermatozopsis similis

  • Lars Linden
  • Georg Kreimer
Article

Abstract

We present an initial characterization of protein kinase and phosphatase activities associated with isolated eyespot apparatuses, the organelle involved in blue/ green-light-mediated behavioural responses of flagellate green algae. In the presence of the phosphatase inhibitors okadaic acid and vanadate, rapid overall protein phosphorylation (t0.5 ≈ 10 s) was observed. The majority of protein kinase activities and their substrates were identified as integral or tightly-bound peripheral membrane proteins. While vanadate generally increased the phosphate incorporation into all phosphoproteins, okadaic acid specifically enhanced phosphorylation of proteins in the range of 39–43 kDa. In contrast to all other phosphoproteins in this subcellular fraction, two proteins with apparent molecular masses of 83 and 16 kDa shared remarkable similarities: (i) They exhibited a fast turnover of the 32P-label, even in the presence of phosphatase inhibitors, and (ii) their dephosphorylation was delayed at 10−8 M free Ca2+. In addition, the 16-kDa protein underwent thiophosphorylation. The general in-vitro phosphorylation pattern was strongly influenced by alterations of free Ca2+ in a concentration range known to affect responses related to phototactic and photophobic behaviour of this alga (10−8 M to 10−7 M). However, characteristics of Ca2+-calmodulin-dependent protein kinases were not observed, i.e. exogenous calmodulin and trifluoperazine had no significant effect on protein phosphorylation. Also exogenous lipids (phosphatidylserine, diacylglycerol), inhibitors of cGMP and cAMPdependent protein kinases and protein kinase C (H-7 and HA1004) as well as exogenously added cGMP and cAMP did not potentiate or inhibit protein phosphorylation. These characteristics of the kinase activity in our fraction most closely resemble those of the plant- and protist-specific group of Ca2+-dependent, calmodulin-independent protein kinases. In-situ phosphorylation experiments following electrophoretic separation revealed the presence of three putative Ca2+-dependent kinases or their catalytic subunits (77,48 and 47 kDa) in the eyespot preparation. In addition, a Ca2+-independent activity at 28 kDa was detected. Possible roles of reversible protein phosphorylation in eyespot apparatuses are discussed.

Key words

Calcium dependent protein phosphorylation Eyespot apparatus Phototaxis Protein kinase Protein phosphatase Spermatozopsis 

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References

  1. Beckmann M, Hegemann P (1991) In vitro identification of rhodopsin in the green alga Chlamydomonas. Biochemistry 30: 3692–3697Google Scholar
  2. Cohen P (1989) The structure and regulation of protein phosphatases. Annu Rev Biochem 58: 453–508Google Scholar
  3. Derguini F, Mazur P, Nakanishi K, Starace DM, Saranak J, Foster KW (1991) All-trans-retinal is the chromophore bound to the photoreceptor of the alga Chlamydomonas reinhardtii. Photochem Photobiol 54: 1017–1021Google Scholar
  4. Dolphin AC (1991) Regulation of calcium channel activity by GTP binding proteins and second messengers. Biochim Biophys Acta 1091: 68–80Google Scholar
  5. Dumler IL, Korolkov SN, Garnovskaya MN, Parfenova DV, Etinghof RN (1989) The systems of photo- and pheromone signals transduction in unicellular eukaryotes. J Protein Chem 8: 387–389Google Scholar
  6. Fallon KM, Trewavas AJ (1993) The significance of post-translational modification of proteins by phosphorylation in the regulation of plant development and metabolism. In: Battey NH, Dickinson HG, Hetherington AM (eds) Post-translational modifications in plants. (Soc Expt Biol Seminar Ser, vol 53). University Press, Cambridge, pp 53–64Google Scholar
  7. Findlay JBC (1987) The isolation and labelling of membrane proteins and peptides. In: Findlay JB, Evans WH (eds) Biological membranes—a practical approach. IRL Press, Oxford, pp 179–217Google Scholar
  8. Fling SP, Gregerson DS (1986) Peptide and protein molecular weight determination by electrophoresis using a high-molarity Tris buffer system without urea. Anal Biochem 155: 83–88Google Scholar
  9. Foster KW, Smyth RD (1980) Light antennas in phototactic algae. Microbiol Rev 44: 572–630Google Scholar
  10. Foster KW, Saranak J, Patel N, Zarilli G, Okabe M, Kline T, Nakanishi K (1984) A rhodopsin is the functional photoreceptor for phototaxis in the unicellular eukaryote Chlamydomonas. Nature 311: 756–759Google Scholar
  11. Fowles C, Akhtar M (1989) Interplay of phosphorylation and de-phosphorylation in vision: protein phosphatases of bovine rod outer segments. Biochemistry 28: 9385–9391Google Scholar
  12. Goodno CC (1982) Myosin active-site trapping with vanadate ion. Methods Enzymol 85: 116–123Google Scholar
  13. Gordon SE, Brautigan DL, Zimmerman AL (1992) Protein phosphatases modulate apparent agonist affinity of the light-regulated ion channel in retinal rods. Neuron 9: 739–748Google Scholar
  14. Guo YL, Roux SJ (1990) Partial purification and characterization of a Ca2+-dependent protein kinase from the green alga Dunaliella salina. Plant Physiol 94: 143–150Google Scholar
  15. Hager A, Brich M, Bazlen I (1993) Redox dependence of the blue-light-induced phosphorylation of a 100-kDa protein of isolated plasma membranes from tips of coleoptiles. Planta 190: 120–126Google Scholar
  16. Hall A (1993) Ras-related proteins. Curr Opin Cell Biol 5: 265–268Google Scholar
  17. Harper JF, Sussman MR, Schaller EG, Putnam-Evans C, Charbonneau H, Harmon AC (1991) A calcium-dependent protein kinase with a regulatory domain similar to calmodulin. Science 252: 951–954Google Scholar
  18. Harz H, Hegemann P (1991) Rhodopsin-regulated calcium currents in Chlamydomonas. Nature 351: 489–491Google Scholar
  19. Harz H, Nonnengässer C, Hegemann P (1992) The photoreceptor current of the green alga Chlamydomonas. Philos Trans R Soc Lond B 338: 39–52Google Scholar
  20. Hegemann P, Harz H (1993) Photoreception in Chlamydomonas. In: Kurjan J, Taylor BL (eds) Signal transduction: Prokaryotic and simple eukaryotic systems. Academic Press, San Diego, pp 279–307Google Scholar
  21. Hidaka H, Kobayashi R (1992) Pharmacology of protein kinase inhibitors. Annu Rev Pharmacol Toxicol 32: 377–397CrossRefPubMedGoogle Scholar
  22. Hidaka H, Inagaki M, Kawamoto S, Sasaki Y (1984) Isoquinolinesulfonamides, novel and potent inhibitors of cyclic nucleotide-dependent protein kinase and protein kinase C. Biochemistry 23: 5036–5041Google Scholar
  23. Huber S, Huber JL, McMichael Jr RW (1994) Control of plant enzyme activity by reversible protein phosphorylation. Int Rev Cytol 149: 47–98Google Scholar
  24. Kamiya R, Witman GB (1984) Submicromolar levels of calcium control and balance of beating between the two flagella in demembranated models of Chlamydomonas. J Cell Biol 98: 97–107Google Scholar
  25. Kaupp UB, Koch KW (1992) Role of cGMP and Ca2+ in vertebrate photoreceptor excitation and adaptation. Annu Rev Physiol 54: 153–175Google Scholar
  26. Keszthelyi L (1992) Light excited electric signals from Chlamydomonas. In: Rigaud JL (ed), Structure and functions of retinal proteins (Colloque INSERM, vol 221). John Libbey Eurotext Ltd., Montrouge, pp. 339–342Google Scholar
  27. Klimczak LJ, Hind G (1990) Biochemical similarities between soluble and membrane-bound calcium-dependent protein kinases of barley. Plant Physiol 92: 919–923Google Scholar
  28. Korolkov SN, Garnovskaya MN, Basov AS, Chuanev AS, Dumler IL (1990) The detection and characterization of G-proteins in the eyespot of Chlamydomonas reinhardtii. FEBS Lett 270: 132–134Google Scholar
  29. Kreimer G (1994) Cell biology of phototaxis in flagellate algae. Int Rev Cytol 148: 229–310Google Scholar
  30. Kreimer G, Witman GB (1994) Novel touch-induced, Ca2+-dependent phobic response in a flagellate green alga. Cell Motil Cytoskeleton 29: 97–109Google Scholar
  31. Kreimer G, Melkonian M, Holtum JAM, Latzko E (1985) Characterization of calcium fluxes across the envelope of intact spinach chloroplasts. Planta 166: 515–523Google Scholar
  32. Kreimer G, Brohsonn U, Melkonian M (1991a) Isolation and partial characterization of the photoreceptive organelle for phototaxis of a flagellate green alga. Eur J Cell Biol 55: 318–327Google Scholar
  33. Kreimer G, Marner FJ, Brohsonn U, Melkonian M (1991b) Identification of 11-cis and all-trans-retinal in the photoreceptive organelle of a flagellate green alga. FEBS Lett 293: 49–52Google Scholar
  34. Kreimer G, Overländer C, Sineshchekov OA, Stolzis H, Nultsch W, Melkonian M (1992) Functional analysis of the eyespot in Chlamydomonas reinhardtii mutant ey 627, mt. Planta 188: 513–521Google Scholar
  35. Litvin FF, Sineshchekov OA, Sineshchekov VA (1978) Photoreceptor electric potential in the phototaxis of the alga Haematococcus pluvialis. Nature 271: 476–478Google Scholar
  36. MacKintosh C, Cohen P (1989) Identification of high levels of type 1 and type 2A protein phosphatases in higher plants. Biochem J 262: 335–339Google Scholar
  37. MacKintosh RW, MacKintosh C (1993) Regulation of plant metabolism by reversible protein (serine/threonine) phosphorylation. In: Battey NH, Dickinson HG, Hetherington AM (eds) Posttranslational modifications in plants (Soc Expt Biol Seminar Ser, vol 53). University Press, Cambridge, pp. 197–212Google Scholar
  38. McFadden GI, Schulze D, Surek B, Salisbury JL, Melkonian M (1987) Basal body reorientation mediated by a Ca2+- modulated contractile protein. J Cell Biol 105: 903–912Google Scholar
  39. Melkonian M, Robenek H (1984) The eyespot apparatus of flagellated green algae: a critical review. Prog Phycol Res 3: 193–268Google Scholar
  40. Miller AJ, Sanders D (1987) Depletion of cytosolic free calcium induced by photosynthesis. Nature 326: 397–400Google Scholar
  41. Moisyadi S, Dharmasiri S, Harrington HM, Lukas TJ (1994) Characterization of a low molecular mass autophosphorylating protein in cultured sugarcane cells and its identification as a nucleoside diphosphate kinase. Plant Physiol 104: 1401–1409Google Scholar
  42. Morel-Laurens N (1987) Calcium control of phototactic orientation in Chlamydomonas reinhardtii: sign and strength of response. Photochem Photobiol 45: 119–128Google Scholar
  43. Moréra S, Lascu I, Dumas C, LeBras G, Briozzo P, Véron M, Janin J (1994) Adenosine 5′-diphosphate binding and the active site of nucleoside diphosphate kinase. Biochemistry 33: 459–467Google Scholar
  44. Muto S (1992) Intracellular Ca2+ messenger system in plants. Int Rev Cytol 142: 305–345Google Scholar
  45. Neuhoff V, Philipp K, Zimmer HG, Mesecke S (1979) A simple, versatile, sensitive and volume-independent method for quantitative protein determination which is independent of other external influences. Hoppe-Seylers Z Physiol Chem 360: 1657–1670Google Scholar
  46. Nomura T, Fukui T, Ichikawa A (1991) Purification and characterization of nucleotide diphosphate kinase from spinach. Biochim Biophys Acta 1077: 47–55Google Scholar
  47. Nultsch W (1979) Effect of external factors on phototaxis of Chlamydomonas reinhardtii. III. Cations Arch Microbiol 123: 93–99Google Scholar
  48. Nultsch W (1983) The photocontrol of movement of Chlamydomonas Symp. Soc Exp Biology 36: 521–539Google Scholar
  49. Pennington SR (1994) GTP-binding proteins: heterotrimeric G proteins. Protein Profile 1: 169–342Google Scholar
  50. Polya GM, Miccuci V (1985) Interaction of wheat germ Ca2+-dependent protein kinases with calmodulin antagonists and polyamides. Plant Physiol 79: 968–972Google Scholar
  51. Randazzo PA, Northup JK, Kahn RA (1992) Regulatory GTP- binding proteins (ADP-ribosylation factor, Gt and ras) are not activated directly by nucleoside diphosphate kinase. J Biol Chem 267: 18182–18189Google Scholar
  52. Rayer B, Naynert M, Stieve H (1990) Phototransduction: Different mechanisms in vertebrates and invertebrates. J Photochem Photobiol B: Biol 7: 107–148Google Scholar
  53. Roberts DM (1993) Protein kinases with calmodulin-like domains: novel targets of calcium signals in plants. Curr Opin Cell Biol 5: 242–246Google Scholar
  54. Roberts DM, Harmon AC (1992) Calcium-modulated proteins: targets of intracellular calcium signals in higher plants: Annu Rev Plant Physiol Plant Mol Biol 43: 375–414Google Scholar
  55. Schaller GE, Harmon AC, Sussman MR (1992) Characterization of a calcium- and lipid-dependent protein kinase associated with the plasma membrane of oat. Biochemistry 31: 1721–1727Google Scholar
  56. Schlösser UG (1986) Sammlung von Algenkulturen Göttingen: additions to the collection since 1984. Ber Dtsch Bot Ges 99: 161–168Google Scholar
  57. Schmidt JA, Eckert R (1976) Calcium couples flagellar reversal to photostimulation in Chlamydomonas reinhardtii. Nature 262: 713–715Google Scholar
  58. Shimazaki KI, Kinoshita T, Nishimura M (1992) Involvement of calmodulin and calmodulin-dependent myosin light chain kinase in blue light-dependent H+ pumping by guard cell protoplasts from Vicia faba L. Plant Physiol 99: 1416–1421Google Scholar
  59. Sineshchekov OA (1991) Photoreception in unicellular flagellates: bioelectric phenomena in phototaxis. In: Douglas RH, Moan J, Ronto G (eds) Light in biology and medicine (Proc III Conf Eur Soc Photobiol Budapest 1989, vol 2). Plenum Press, New York, pp. 523–532Google Scholar
  60. Sineshchekov OA, Govorunova EG, Der A, Keszthelyi L, Nultsch W (1992) Photoelectric responses in phototactic flagellated algae measured in cell suspensions. J Photochem Photobiol 13: 119–134Google Scholar
  61. Smith JA, Francis SH, Corbin JD (1993) Autophosphorylation: a salient feature of protein kinases. Mol Cell Biochem 127/128: 51–70Google Scholar
  62. Stavis RL, Hirschberg R (1973) Phototaxis in Chlamydomonas. J Cell Biol 59: 367–377Google Scholar
  63. Terryn N, Van Montagu M, Inzé D (1993) GTP-binding proteins in plants. Plant Mol Biol 22: 143–152Google Scholar
  64. Trewavas AJ, Blowers DP (1990) Protein kinases in the plant plasma membrane. Curr Top Plant Biochem Physiol 9: 153–163Google Scholar
  65. Warpeha KMF, Hamm HE, Rasenick MM, Kaufman LS (1991) A blue-light-activated GTP-binding protein in the plasma membranes of etiolated peas. Proc Natl Acad Sci USA 88: 8925–8929Google Scholar

Copyright information

© Springer-Verlag 1995

Authors and Affiliations

  • Lars Linden
    • 1
  • Georg Kreimer
    • 1
  1. 1.Universität zu Köln, Botanisches InstitutKölnGermany

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