Microdomain arrangement of the SERCA-type Ca2+ Pump (Ca2+-ATPase) in subplasmalemmal calcium stores of Paramecium cells
1999, Plattner, Helmut, Flötenmeyer, Matthias, Kissmehl, Roland, Pavlovic, Nada, Hauser, Karin, Momayezi, Massoud, Braun, N., Tack, Jens, Bachmann, Luis
We localized SERCA pumps to the inner region of alveolar sac membranes, facing the cell interior, by combining ultrastructural and biochemical methods. Immunogold labeling largely predominated in the inner alveolar sac region which displayed aggregates of intramembrane particles (IMPs). On image analysis, these represented oligomeric arrangements of ~8-nm large IMP subunits, suggesting formation of SERCA aggregates (as known from sarcoplasmic reticulum). We found not only monomers of typical molecular size (~106 kD) but also oligomeric forms on Western blots (using anti-SERCA antibodies, also against endogenous SERCA from alveolar sacs) and on electrophoresis gelautoradiographs of 32P-labeled phosphoenzyme intermediates. Selective enrichment of SERCA-pump molecules in the inner alveolar sac membrane region may eliminate Ca2+ after centripetal spread observed during exocytosis activation, while the plasmalemmal Ca2+ pump may maintain or reestablish [Ca2+] in the narrow subplasmalemmal space between the outer alveolar sac membrane region and the cell membrane. We show for the first time the microzonal arrangement of SERCA molecules in a Ca2+ store of a secretory system, an intensely discussed issue in stimulus secretion coupling research.
Subplasmalemmal Ca-stores in Paramecium tetraurelia : identification and characterisation of a sarco(endo)plasmic reticulum-like Ca2+-ATPase by phosphoenzyme intermediate formation and its inhibition by caffeine
1998, Kissmehl, Roland, Huber, Sabine, Kottwitz, Brunhilde, Hauser, Karin, Plattner, Helmut
Considering increasing interest in calcium stores in protozoa, including parasitic forms, and specifically in subplasmalemmal stores in higher eukaryotes, we have isolated subplasmalemmal calcium stores (alveolar sacs) from the ciliated protozoan, Paramecium tetraurelia. Using antibodies against established sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs) we detected in Western blots of subcellular fractions a band of 106 kDa size selectively in alveolar sacs but not, for example, in plasma membranes and concomitant restriction of immunofluorecence labelling to the cell cortex of permeabilised cells. These results are the same as with ABs against a peptide derived from a cloned SERCA-like gene from Paramecium [Hauser K., Pavlovic N., Kissmehl R., Plattner H. Molecular characterization of a sarco(endo)plasmic reticulum Ca2+-ATPase gene from Paramecium tetraurelia and localisation of its gene product to subplasmalemmal calcium stores. Biochem J 1998; 334: 31 38]. When such isolated alveolar sacs were now tested for phosphoenzyme intermediate (EP) formation, a phosphoprotein of the same apparent molecular mass (106 kDa) as in blots could be identified in gel autoradiograms. This EP corresponds to that formed in the reaction cycle of different SERCA-types, with dependency on Ca2+ and Mg2+, sensitivity to La3+ or insensitivity towards calmodulin, calmodulin antagonists and vanadate. However, EP formation in alveolar sacs is not inhibited by established SERCA inhibitors (e.g. thapsigargi[ci]n tested up to 100 μM). Surprisingly, caffeine, which is frequently used to mobilise Ca2+ from intracellular stores, strongly inhibits EP formation. In parallel experiments, we did not find any similar effect with sarcoplasmic reticulum isolated from skeletal muscle. We conclude that the 106 kDa protein of alveolar sacs in Paramecium may represent a SERCA-like Ca2+-ATPase with some unorthodox features, which might be relevant also for some other protozoan systems. In this case, the established Ca2+-mobilizing effect of caffeine may be amplified by inhibiting store refilling.
Photochemical release of ATP from "Caged ATP" studied by time-resolved infrared spectroscopy
1995, Barth, Andreas, Hauser, Karin, Maentele, Werner, Corrie, John E. T., Trentham, David R.
Rapid scan Fourier transform infrared (FTIR) spectroscopy and time-resolved single wavelength infrared (IR) spectroscopy have been used to follow the photochemical release of adenosine 5’-triphosphate (ATP) from P3-( 1 -(2-nitrophenyl)ethyl) adenosine 5’-triphosphate (caged ATP). Vibrational difference spectra for the formation first of the aci-nitro anion intermediate and subsequently of ATP and the byproduct(s) were obtained by rapid scan FTIR spectroscopy in the millisecond-to-second time domain. Vibrational modes of the phosphate groups of ATP and caged ATP in the range 1250-900 cm-I could be assigned on the basis of triple and single l8O labeling in caged ATP at the terminal phosphate group and at the bridging oxygen between the terminal phosphate and the 1-(2-nitrophenyl)ethyl group, respectively. The rapid formation and subsequent decay of the aci-nitro anion intermediate were monitored by single-wavelength time-resolved IR spectroscopy at 125 1 cm- ’ (predominantly a PO2- mode of caged ATP and the aci-nitro intermediate). The appearance of the free y-phosphate group of ATP was monitored at 1119 cm-I (P03’- mode of ATP). Decay of the aci-nitro anion intermediate and formation of ATP were well fitted by single exponentials to give a mean rate constant of 218 & 33 s-’ at pH 7 and 22 OC.
Comparison of in vivo and in vitro phosphorylation of the exocytosis-sensitive protein PP63/Parafusin by differential MALDI mass spectrometric peptide mapping
1999, Kussmann, Martin, Hauser, Karin, Kissmehl, Roland, Breed, Jason, Plattner, Helmut, Roepstorff, Peter
PP63 (parafusin) is a 63 kDa phosphoprotein, which exists in at least two different isoforms. It is very rapidly (80 ms) dephosphorylated during triggered trichocyst exocytosis. This occurs selectively in exocytosis-competent Paramecium tetraurelia strains. At least two protein kinases isolated from Paramecium, casein kinase type II kinase and cGMP-dependent kinase, are able to phosphorylate the two recombinant PP63/parafusin isoforms, both with phosphoglucomutase activity, in vitro. By performing mass spectrometric peptide mapping, we have investigated in vitro phosphorylation of recombinant PP63/ parafusin by these kinases in comparison to in vivo phosphorylation of native PP63/parafusin isolated from Paramecium homogenates. Low picomolar quantities of proteolytic digests of recombinant and native PP63/parafusin, prior to and following alkaline phosphatase treatment, were directly analyzed by MALDI mass spectrometry. In native PP63-1/parafusin-1, six of 64 serine and threonine residues (S-196, T-205, T-280, T-371, T-373, and T-469) were found definitely, 27 were found possibly phosphorylated, 28 were identified as nonphosphorylated, and three were not covered by mapping. Three of the six certainly phosphorylated amino acids represent consensus phosphorylation sites for casein kinase II or cGMPdependent protein kinase. In vitro phosphorylation studies of recombinant PP63/parafusin confirm that some of the sites found were used in vivo; however, also significant differences with respect to in vivo phosphorylation of native PP63/parafusin were observed. The two Paramecium protein kinases that were used do not preferably phosphorylate expected consensus sites in vitro. Homology structure modeling of PP63/parafusin with rabbit phosphoglucomutase revealed that the majority of residues found phosphorylated is located on the surface of the molecule.
A novel, calcium-inhibitable casein kinase in Paramecium cells
1997, Kissmehl, Roland, Treptau, Tilman, Hauser, Karin, Plattner, Helmut
This is the first identification of a Ca2+-inhibitable casein kinase (CPK) which we have isolated from the 100 000×g supernatant of Paramecium cell homogenates. The 1000-fold enriched CPK activity depends on millimolar Mg2+ and is inhibited by low concentrations of heparin or by ≥100 μM Ca2+. Enzyme activity is stimulated by polylysine or polyarginine with either casein or with specific casein kinase-2 (CK-2) peptide substrates (RRRDDDSDDD and RREEETEEE). The enzymic properties are similar with GTP instead of ATP. CPK does not undergo autophosphorylation. In gel kinase assays, enzyme activity is associated with a 36 kDa band. Calmodulin as another characteristic substrate for mammalian CK-2 has not been phosphorylated by this protein kinase. Besides casein, CPK phosphorylates in vitro the catalytic subunit of bovine brain calcineurin (CaN), a typical substrate of type 1 mammalian casein kinase (CK-1) in vitro. Again this phosphorylation is significantly reduced by Ca2+. Thus, CPK combines aspects of different casein kinases, but it is clearly different from any type known by its Ca2+ inhibition. Since CPK also phosphorylates the exocytosis-sensitive phosphoprotein, PP63, in Paramecium, which is known to be dephosphorylated by CaN, an antagonistic Ca2+-effect during phosphorylation/dephosphorylation cycles may be relevant for exocytosis regulation.
Immunolocalization of the exocytosis-sensitive phosphoprotein, PP63/parafusin, in Paramecium cells using antibodies against recombinant protein
1998, Kissmehl, Roland, Hauser, Karin, Gössringer, Markus, Momayezi, Massoud, Klauke, Norbert, Plattner, Helmut
We have localized a structure-bound fraction of the exocytosis-sensitive phosphoprotein, PP63/parafusin (PP63/pf), in Paramecium cells by widely different methods. We combined cell fractionation, western blots, as well as light and electron microscopy (pre- and postembedding immunolabeling), applying antibodies against the recombinant protein. PP63/pf is considerably enriched in certain cortical structures, notably the outlines of regular surface fields (kinetids), docking sites of secretory organelles (trichocysts) and the membranes of subplasmalemmal Ca2+-stores (alveolar sacs). From our localization studies we tentatively derive several potential functions for PP63/pf, including cell surface structuring, assembly of exocytosis sites, and/or Ca2+ homeostasis.
Identification of isoforms of the exocytosis-sensitive phosphoprotein PP63/parafusin in Paramecium tetraurelia and demonstration of phosphoglucomutase activity
1997, Hauser, Karin, Kissmehl, Roland, Linder, Jürgen, Schultz, Joachim E., Lottspeich, Friedrich, Plattner, Helmut
PP63 (parafusin) is a 63 kDa phosphoprotein which is very rapidly (within 80 ms) dephosphorylated (to P63) during triggered trichocyst exocytosis; this occurs selectively in exocytosis-competent Paramecium tetraurelia strains. In the present work, two cDNAs coding for PP63}parafusin have been isolated, one of which is a new isoform. These isoforms are 99.6% identical and are derived from two different genes. Similarity searches revealed 43±51% identity of the deduced
amino acid sequences with known phosphoglucomutases from yeast and mammals. The sequences of two proteolytic peptides obtained from PP63}parafusin isolated from Paramecium are identical to parts of the amino acid sequence deduced from the major cDNA. The major cDNA was mutated from the macronuclear ciliate genetic code into the universal genetic code and expressed in Escherichia coli. The recombinant protein shows the same biochemical and immunological characteristics as the (P)P63}parafusin originally isolated from Paramecium. It has the same specifc phosphoglucomutase activity as phosphoglucomutase from chicken muscle. We also show that recombinant P63-1}parafusin 1 is a substrate of an endogenous casein kinase from Paramecium, as is the originally isolated P63}parafusin.
Polyclonal antibodies against recombinant P63-1}parafusin 1 were raised which recognized phosphoglucomutases from different sources. Thus we show that PP63}parafusin and phosphoglucomutase in Paramecium are identical.