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.
Similar content being viewed by others
References
Beckmann M, Hegemann P (1991) In vitro identification of rhodopsin in the green alga Chlamydomonas. Biochemistry 30: 3692–3697
Cohen P (1989) The structure and regulation of protein phosphatases. Annu Rev Biochem 58: 453–508
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: 1017–1021
Dolphin AC (1991) Regulation of calcium channel activity by GTP binding proteins and second messengers. Biochim Biophys Acta 1091: 68–80
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: 387–389
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 53–64
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 179–217
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: 83–88
Foster KW, Smyth RD (1980) Light antennas in phototactic algae. Microbiol Rev 44: 572–630
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: 756–759
Fowles C, Akhtar M (1989) Interplay of phosphorylation and de-phosphorylation in vision: protein phosphatases of bovine rod outer segments. Biochemistry 28: 9385–9391
Goodno CC (1982) Myosin active-site trapping with vanadate ion. Methods Enzymol 85: 116–123
Gordon SE, Brautigan DL, Zimmerman AL (1992) Protein phosphatases modulate apparent agonist affinity of the light-regulated ion channel in retinal rods. Neuron 9: 739–748
Guo YL, Roux SJ (1990) Partial purification and characterization of a Ca2+-dependent protein kinase from the green alga Dunaliella salina. Plant Physiol 94: 143–150
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: 120–126
Hall A (1993) Ras-related proteins. Curr Opin Cell Biol 5: 265–268
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: 951–954
Harz H, Hegemann P (1991) Rhodopsin-regulated calcium currents in Chlamydomonas. Nature 351: 489–491
Harz H, Nonnengässer C, Hegemann P (1992) The photoreceptor current of the green alga Chlamydomonas. Philos Trans R Soc Lond B 338: 39–52
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 279–307
Hidaka H, Kobayashi R (1992) Pharmacology of protein kinase inhibitors. Annu Rev Pharmacol Toxicol 32: 377–397
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: 5036–5041
Huber S, Huber JL, McMichael Jr RW (1994) Control of plant enzyme activity by reversible protein phosphorylation. Int Rev Cytol 149: 47–98
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: 97–107
Kaupp UB, Koch KW (1992) Role of cGMP and Ca2+ in vertebrate photoreceptor excitation and adaptation. Annu Rev Physiol 54: 153–175
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. 339–342
Klimczak LJ, Hind G (1990) Biochemical similarities between soluble and membrane-bound calcium-dependent protein kinases of barley. Plant Physiol 92: 919–923
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: 132–134
Kreimer G (1994) Cell biology of phototaxis in flagellate algae. Int Rev Cytol 148: 229–310
Kreimer G, Witman GB (1994) Novel touch-induced, Ca2+-dependent phobic response in a flagellate green alga. Cell Motil Cytoskeleton 29: 97–109
Kreimer G, Melkonian M, Holtum JAM, Latzko E (1985) Characterization of calcium fluxes across the envelope of intact spinach chloroplasts. Planta 166: 515–523
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: 318–327
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: 49–52
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: 513–521
Litvin FF, Sineshchekov OA, Sineshchekov VA (1978) Photoreceptor electric potential in the phototaxis of the alga Haematococcus pluvialis. Nature 271: 476–478
MacKintosh C, Cohen P (1989) Identification of high levels of type 1 and type 2A protein phosphatases in higher plants. Biochem J 262: 335–339
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. 197–212
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: 903–912
Melkonian M, Robenek H (1984) The eyespot apparatus of flagellated green algae: a critical review. Prog Phycol Res 3: 193–268
Miller AJ, Sanders D (1987) Depletion of cytosolic free calcium induced by photosynthesis. Nature 326: 397–400
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: 1401–1409
Morel-Laurens N (1987) Calcium control of phototactic orientation in Chlamydomonas reinhardtii: sign and strength of response. Photochem Photobiol 45: 119–128
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: 459–467
Muto S (1992) Intracellular Ca2+ messenger system in plants. Int Rev Cytol 142: 305–345
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: 1657–1670
Nomura T, Fukui T, Ichikawa A (1991) Purification and characterization of nucleotide diphosphate kinase from spinach. Biochim Biophys Acta 1077: 47–55
Nultsch W (1979) Effect of external factors on phototaxis of Chlamydomonas reinhardtii. III. Cations Arch Microbiol 123: 93–99
Nultsch W (1983) The photocontrol of movement of Chlamydomonas Symp. Soc Exp Biology 36: 521–539
Pennington SR (1994) GTP-binding proteins: heterotrimeric G proteins. Protein Profile 1: 169–342
Polya GM, Miccuci V (1985) Interaction of wheat germ Ca2+-dependent protein kinases with calmodulin antagonists and polyamides. Plant Physiol 79: 968–972
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: 18182–18189
Rayer B, Naynert M, Stieve H (1990) Phototransduction: Different mechanisms in vertebrates and invertebrates. J Photochem Photobiol B: Biol 7: 107–148
Roberts DM (1993) Protein kinases with calmodulin-like domains: novel targets of calcium signals in plants. Curr Opin Cell Biol 5: 242–246
Roberts DM, Harmon AC (1992) Calcium-modulated proteins: targets of intracellular calcium signals in higher plants: Annu Rev Plant Physiol Plant Mol Biol 43: 375–414
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: 1721–1727
Schlösser UG (1986) Sammlung von Algenkulturen Göttingen: additions to the collection since 1984. Ber Dtsch Bot Ges 99: 161–168
Schmidt JA, Eckert R (1976) Calcium couples flagellar reversal to photostimulation in Chlamydomonas reinhardtii. Nature 262: 713–715
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: 1416–1421
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. 523–532
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: 119–134
Smith JA, Francis SH, Corbin JD (1993) Autophosphorylation: a salient feature of protein kinases. Mol Cell Biochem 127/128: 51–70
Stavis RL, Hirschberg R (1973) Phototaxis in Chlamydomonas. J Cell Biol 59: 367–377
Terryn N, Van Montagu M, Inzé D (1993) GTP-binding proteins in plants. Plant Mol Biol 22: 143–152
Trewavas AJ, Blowers DP (1990) Protein kinases in the plant plasma membrane. Curr Top Plant Biochem Physiol 9: 153–163
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: 8925–8929
Author information
Authors and Affiliations
Additional information
This study was supported by the Deutsche Forschungsgemeinschaft. We wish to thank Prof. Dr. M. Melkonian (Botanisches Institut, Universität zu Köln) for critical reading of the manuscript.
Rights and permissions
About this article
Cite this article
Linden, L., Kreimer, G. Calcium modulates rapid protein phosphorylation/dephosphorylation in isolated eyespot apparatuses of the green alga Spermatozopsis similis . Planta 197, 343–351 (1995). https://doi.org/10.1007/BF00202656
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00202656