1887

Microbiology Society logo

Volume 139, Issue 12

Calcium homeostasis, signalling and protein phosphorylation during calcium-induced conidiation in Free

Abstract

Cytosolic free calcium concentration [Ca] of protoplasts from was measured using the permeant acetoxy ester (quin-2-AM) of the calcium-chelating fluorescent dye quin-2. Low uptake of the ester occurred at pH 5·8–7·0 and its subsequent hydrolysis was low. Uptake was promoted by an external pH of 5·0 and significant hydrolysis to quin-2 achieved by adjustment of the internal pH to 7·2, which was near the optimum of the carboxylic esterases responsible for the hydrolysis. Uptake of Ca was biphasic with the average cell calcium concentration of protoplasts increasing from an initial value of 2 μmol to 50 μmol (kg cell water), during attainment of the steady state after 30 min, at which time [Ca] was unchanged at 20 n but increased to 182 n at 2–6 h exposure to 2·5 m-Ca. Broadly similar changes in [Ca] were found in protoplasts derived from mycelium samples exposed to Ca over the same period of time. The location of Ca was determined in subfractionated organelles and characterized using enzyme markers and electron microscopy. In 32 h mycelium preloaded with Ca for 6 h, Ca was located principally in the mitochondria with lower concentrations associated with the endoplasmic reticulum, Golgi, vacuoles and plasma membrane components. Calcium was not released by inositol 1,4,5-trisphosphate or the calcium ionophore A23187 from any subcellular fractions obtained from mycelium on Percoll gradients, nor from preparations of vacuoles or plasmalemma vesicles, except in the case of mitochondria where rapid release of the ion was achieved by the addition of 2–5 μ-A23187. The anti-calmodulin agent calmidazolium (R24571) greatly reduced sporulation when addition preceded that of Ca. Calcium-induced cultures showed massive novel protein phosphorylation 2 h after addition of the ion which was virtually eliminated by R24571, whilst 1 h and 4–6 h protein phosphorylations, which were also present to some degree in vegetative controls, were substantially reduced. Two-dimensional SDS-PAGE analysis of phosphoproteins confirmed that Ca-induced mycelium had enhanced capacity for calmodulin-mediated phosphorylation relative to corresponding vegetative cells and that complex differential changes in such phosphorylations occurred during Ca-induction of the sporulation process.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
© Society for General Microbiology, 1993
Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-139-12-3053
1993-12-01
2024-04-28
Loading full text...

Full text loading...

/deliver/fulltext/micro/139/12/mic-139-12-3053.html?itemId=/content/journal/micro/10.1099/00221287-139-12-3053&mimeType=html&fmt=ahah

References

  1. Anraku Y., Ohya Y., Iida H. 1991; Cell cycle control by calcium and calmodulin in Saccharomyces cerevisiae. Biochimica et Biophysica Acta 1093:169–177
     [Google Scholar]
  2. Berridge M.J. 1987; Inositol trisphosphate and diacylglycerol: two interacting second messengers. Annual Review of Biochemistry 56:159–193
     [Google Scholar]
  3. Berridge M.J., Irvine R.F. 1984; Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature; London: 312315–321
     [Google Scholar]
  4. Borle A.B., Struder B. 1978; Effects of calcium ionophores on the transport and distribution of calcium in isolated cells and in liver and kidney slices. Journal of Membrane Biology 38:51–58
     [Google Scholar]
  5. Bowman E.J. 1983; Comparison of the vacuolar membrane ATPase of Neurospora crassa with the mitochondrial and plasma membrane ATPases. Journal of Biological Chemistry 258:15238–15244
     [Google Scholar]
  6. Bowman E.J., Bowman B.J., Slayman C.W. 1981; Isolation and characterization of plasma membranes from wild type Neuro-spora crassa. Journal of Biological Chemistry 256:12336–12342
     [Google Scholar]
  7. Bowman B.J., Borgeson C.E., Bowman E.J. 1987; Composition of Neurospora crassa vacuolar membranes and comparison to endoplasmic reticulum, plasma membranes, and mitochondrial membranes. Experimental Mycology 11:197–205
     [Google Scholar]
  8. Brunton A.H., Gadd G.M. 1991; Evidence for an inositol lipid signal pathway in the yeast-mycelium transition of Ophiostoma ulmi, the Dutch elm disease fungus. Mycological Research 95:484–491
     [Google Scholar]
  9. Clapper D.L., Lee H.C. 1985; Inositol trisphosphate induces calcium release from non-mitochondrial stores in sea urchin egg homogenates. Journal of Biological Chemistry 260:13947–13954
     [Google Scholar]
  10. Cooperstein S.J., Lazarow A. 1951; A microspectrophotometric method for the determination of cytochrome oxidase. Journal of Biological Chemistry 189:665–670
     [Google Scholar]
  11. Cornelius G., Nakashima H. 1987; Vacuoles play a decisive role in calcium homeostasis in Neurospora crassa. Journal of General Microbiology 133:2341–2347
     [Google Scholar]
  12. Cornelius G., Gebauer G., Techel D. 1989; Inositol trisphosphate induces calcium release from Neurospora crassa vacuoles. Biochemical and Biophysical Research Communications 162:852–856
     [Google Scholar]
  13. Cramer C.L., Ristow J.L., Paulus T.J., Davis R.H. 1983; Methods for mycelial breakage and isolation of mitochondria and vacuoles of Neurospora. Analytical Biochemistry 128:384–392
     [Google Scholar]
  14. Dawson A.P., Comerford J.G., Fulton D.V. 1986; The effect of GTP on inositol 1,4,5-trisphosphate-stimulated Ca2+ efflux from a rat liver microsomal fraction. Biochemical Journal 234:311–315
     [Google Scholar]
  15. Elliott C.G. 1986; Inhibition of reproduction by Phytophthora by calmodulin-interacting compounds trifluoperazine and ophiobolin A. Journal of General Microbiology 132:1781–1785
     [Google Scholar]
  16. Favre B., Turian G. 1987; Identification of a calcium- and phospholipid-dependent protein kinase (protein kinase C) in Neurospora crassa. Plant Science 49:15–21
     [Google Scholar]
  17. Fiske C.H., Subbarow Y. 1925; The colorimetric determination of phosphorus. Journal of Biological Chemistry 66:375–400
     [Google Scholar]
  18. Freedman M.H., Raff M.C. 1975; Induction of increased calcium uptake in mouse T lymphocytes by concanavalin A and its modulation by cyclic nucleotides. Nature; London: 225378–382
     [Google Scholar]
  19. Gadd G.M., Brunton A.H. 1992; Calcium involvement in dimorphism of Ophiostoma ulmi, the Dutch elm disease fungus, and characterization of calcium uptake by yeast cells and germ tubes. Journal of General Microbiology 138:1561–1571
     [Google Scholar]
  20. Gietzen K., Wüthrich A., Bader H. 1981; R24571: a new powerful inhibitor of red blood cell Ca2+-transport ATPase and of calmodulin-regulated functions. Biochemical and Biophysical Research Communications 101:418–425
     [Google Scholar]
  21. Gilroy S., Hughes W.A., Trewavas A.J. 1986; The measurement of intracellular calcium levels in protoplasts from higher plant cells. FEBS Letters 199:217–222
     [Google Scholar]
  22. Gomes S.L., Mennucci L., Maia J.C.C. 1979; A calcium dependent protein activator of mammalian cyclic nucleotide phosphodiesterase from Blastocladiella emersonii. FEBS Letters 99:39–42
     [Google Scholar]
  23. Hadley G., Harrold C. E. 1958a; The sporulation of Penicillium notatum Westling in submerged liquid culture. I. The effects of calcium and nutrients on sporulation. Journal of Experimental Botany 9:408–417
     [Google Scholar]
  24. Hadley G., Harrold C. E. 1958b; The sporulation of Penicillium notatum Westling in submerged liquid culture. II. The initial sporulation phase. Journal of Experimental Botany 9:418–425
     [Google Scholar]
  25. Hodges T.K. 1974; Purification of a plasma-membrane-bound adenosine triphosphatase from plant roots. Methods in Enzymology 32:392–407
     [Google Scholar]
  26. Hoshino T., Mizutani A., Hidaka H., Yamane T. 1992; Identification of Ca2+/calmodulin-dependent protein-kinase and endogenous substrate of Fusarium oxysporum. FEMS Microbiology Letters 94:27–30
     [Google Scholar]
  27. Iida H., Yagawa Y., Anraku Y. 1990; Essential röle for induced Ca2+ influx followed by [Ca2+]j rise in maintaining viability of yeast cells late in the mating pheromone response pathway. Journal of Biological Chemistry 265:13391–13399
     [Google Scholar]
  28. Kaile A., Pitt D., Kuhn P.J. 1992; Calcium cytotoxicity, protoplast viability and the role of calcium in soft-rot of Brassica napus due to Botrytis cinerea Pers. Physiological and Molecular Plant Pathology 40:49–62
     [Google Scholar]
  29. Knight M.R., Campbell A.K., Smith S.M., Trewavas A.J. 1991; Transgenic plant aequorin reports the effects of touch and cold-shock and elicitors on cytoplasmic calcium. Nature; London: 352524–526
     [Google Scholar]
  30. Laemmli U.K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature; London: 277680–685
     [Google Scholar]
  31. Mannai M., Cozzone A.J. 1982; Endogenous protein phosphorylation in Escherichia coli extracts. Biochemical and Biophysical Research Communications 107:981–988
     [Google Scholar]
  32. Miller A.J., Sanders D. 1989; The energetics of cytosolic calcium homeostasis in fungal cells. Plant Physiology and Bio-chemistry 27:551–556
     [Google Scholar]
  33. Miller A.J., Vogg G., Sanders D. 1990; Cytosolic calcium homeostasis in fungi: rôles of plasma membrane transport and intracellular sequestration of calcium. Proceedings of the National Academy of Sciences of the United States of America 879348–9352
     [Google Scholar]
  34. Miyakawa T., Youichi O., Tsuchiya E., Fukui S. 1989; Saccharomvces cerevisiae protein kinase dependent on Ca2+ and calmodulin. Journal of Bacteriology 171:1417–1422
     [Google Scholar]
  35. Mosley M.J., Pitt D., Barnes J.C. 1989; Adenine and pyridine nucleotide levels during calcium-induced conidiation in Penicillium notatum. Antonie van Leeuwenhoek 56:191–199
     [Google Scholar]
  36. Muthukumar G., Nickerson A.W., Nickerson K.W. 1987; Calmodulin levels in yeasts and filamentous fungi. FEMS Microbiology Letters 41:253–255
     [Google Scholar]
  37. O’farrell P.Z., Goodman H.M., O’farrell P.H. 1977; High resolution two dimensional electrophoresis of basic as well as acidic proteins. Cell 12:1133–1142
     [Google Scholar]
  38. Pitt D. 1970; Changes in hydrolase activity of Solanum tuber tissues during infection by Phytophthora erythroseptica. Transactions of the British Mycological Society 55:257–266
     [Google Scholar]
  39. Pitt D., Kaile A. 1990; Transduction of the calcium signal with special reference to Ca2+-induced conidiation in Penicillium notatum. In Biochemistry of Cell Walls and Membranes in Fungi pp. 283–298 Edited by Kuhn P. J., Trinci A. P. J., Jung M. J., Goosey M. W., Copping L. G. Berlin: Springer-Verlag;
     [Google Scholar]
  40. Pitt D., Mosley M. J. 1985a; Enzymes of gluconate metabolism and glycolysis in Penicillium notatum. Antonie van Leeuwenhoek 51:353–364
     [Google Scholar]
  41. Pitt D., Mosley M. J. 1985b; Pathways of glucose catabolism and the origin and metabolism of pyruvate during calcium-induced conidiation of Penicillium notatum. Antonie van Leeuwenhoek 51:365–385
     [Google Scholar]
  42. Pitt D., Mosley M.J. 1986; Oxidation of carbon sources via the tricarboxylic acid cycle during calcium-induced conidiation of Penicillium notatum. Antonie van Leeuwenhoek 52:467–482
     [Google Scholar]
  43. Pitt D., Poole P.C. 1981; Calcium-induced conidiation in Penicillium notatum in submerged culture. Transactions of the British Mycological Society 76:219–230
     [Google Scholar]
  44. Pitt D., Stewart P. 1981; Isolation and characterization of components of the lytic compartment of several plant tissues including separations with a zonal rotor. Annals of Botany 48:665–692
     [Google Scholar]
  45. Pitt D., Barnes J.C., Ugalde U.O. 1988; Differential uptake of calcium by strains of Penicillium notatum and relationships to calcium-induced conidiation. Transactions of the British Mycological Society 91:489–199
     [Google Scholar]
  46. Prentki M., Janic D., Wolheim C.B. 1983; The regulation of extra-mitochondrial steady state free Ca2+ concentration by rat insulinoma mitochondria. Journal of Biological Chemistry 258:7597–7602
     [Google Scholar]
  47. Rink T.J., Pozzan T. 1985; Using quin-2 in cell suspensions. Cell Calcium 6:133–144
     [Google Scholar]
  48. Robson G.D., Trinci A.P.J., Wiebe M.G., Best L.C. 1991; Phosphatidylinositol 4,5-biphosphate (PIP2) is present in Fusarium graminearum. Mycological Research 95:1082–1084
     [Google Scholar]
  49. Roncal T., Ugalde U.O., Barnes J.C., Pitt D. 1991; Production of protoplasts of Penicillium cyclopium with improved viability and functional properties. Journal of General Microbiology 137:1647–1651
     [Google Scholar]
  50. Roos W., Lückner M. 1984; Relationships between proton extrusion and fluxes of ammonium ions and organic acids in Penicillium cyclopium. Journal of General Microbiology 130:1007–1014
     [Google Scholar]
  51. Slavik J. 1982; Intracellular pH of yeast cells measured with fluorescent probes. FEBS Letters 140:22–26
     [Google Scholar]
  52. Stleger R.J., Roberts D.W., Staples R.C. 1989; Calcium and calmodulin-mediated protein synthesis and protein phosphorylation during germination, growth and protein production by Metarhizium anisopliae. Journal of General Microbiology 135:2141–2154
     [Google Scholar]
  53. Streb H., Irvine R.F., Berridge M.J., Schulz I. 1983; Release of Ca2+ from a non-mitochondrial intracellular store in pancreatic acinar cells by inositol-1,4,5-trisphosphate. Nature; London: 30667–69
     [Google Scholar]
  54. Tsien R.Y., Pozzan T., Rink T.J. 1982; Calcium homeostasis in intact lymphocytes: cytoplasmic free calcium monitored with a new, intracellularly trapped fluorescent indicator. Journal of Cell Biology 94:325–334
     [Google Scholar]
  55. Ugalde U.O. 1991; Preparation of highly purified plasma membrane vesicles of controlled orientation from Penicillium cyclopium. Mycological Research 95:1303–1307
     [Google Scholar]
  56. Ugalde U.O., Pitt D. 1983; Morphology and calcium-induced conidiation of Penicillium cyclopium in submerged culture. Transactions of the British Mycological Society 80:319–325
     [Google Scholar]
  57. Ugalde U.O., Pitt D. 1984; Subcellular sites of calcium accumulation and relationships with conidiation in Penicillium cyclopium. Transactions of the British Mycological Society 83:547–555
     [Google Scholar]
  58. Ugalde U.O., Pitt D. 1986; Calcium uptake kinetics in relation to submerged cultures of Penicillium cyclopium. Transactions of the British Mycological Society 87:199–203
     [Google Scholar]
  59. Ugalde U.O., Virto M.E., Pitt D. 1990; Calcium binding and induction of conidiation in protoplasts of Penicillium cyclopium. Antonie van Leeuwenhoek 57:43–49
     [Google Scholar]
  60. Ugalde U.O., Henandez A., Galindo I., Pitt D., Barnes J.C., Wakley G. 1992; Preparation of right-side-out plasma membrane vesicles from Penicillium cyclopium: critical assessment of markers. Journal of General Microbiology 138:2205–2212
     [Google Scholar]
  61. Weete J.D. 1974 Fungal Lipid Biochemistry London: Plenum;
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-139-12-3053
Loading
Calcium homeostasis, signalling and protein phosphorylation during calcium-induced conidiation in Penicillium notatum
Microbiology 139, 3053 (1993); https://doi.org/10.1099/00221287-139-12-3053
/content/journal/micro/10.1099/00221287-139-12-3053
/content/journal/micro/10.1099/00221287-139-12-3053
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error