Immunolocalization of the exocytosis-sensitive phosphoprotein, PP63/parafusin, in Paramecium cells using antibodies against recombinant protein

Abstract

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 Ca 2+ -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 Ca 2+ homeostasis.& b d y :

Immunolabeling analysis of biosynthetic and degradative pathways of cell surface components (glycocalyx) in Paramecium cells

“…The sizes of markers varied between 14.5 and 94 kOa or 97 and 584 kOa, in low or high molecular weight kits, from Pharmacia (Freiburg, Germany) or from Sigma (Oeisenhofen, Germany), respectively. Gels were stained with silver or alternatively processed for Western blots and probed with anti-nSAg or anti-dSAg antibodies (AB), followed by alkaline phosphatase-tagged anti-rabbit AB (Sigma) as previously described [40].…”

“…These samples were incubated with anti-nSAg or anti-dSAg AB. followed by fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (ICN, Eschwege, Germany) for analysis by confocal laser scanning microscopy (CLSM) as previously described [40]. Controls were treated in the same way, but using preimmune sera instead of the first AB.…”

Molecular aspects of rapid, reversible, Ca2+-dependent de-phosphorylation of pp63/parafusin during stimulated exo-endocytosis in Paramecium cells

“…In the Paramecium cell cortex, any Ca 2 +-release channels would have to be located on the outer part of alveolar sacs, because the part facing the cell center is densely studded with Ca 2 +-pump molecules [56]. In agreement with this postulate, the subplasmalemmal space, including that delineated by alveolar sacs and trichocysts, is the site where pp63/pf [36], as well as the relevant protein phosphatase PP2B/CaN [42] and protein kinases CK-2 and PKG [57,58] are heavily enriched according to immuno-EM studies (see Fig. 1 and below).…”

Comparison of in Vivo and in Vitro Phosphorylation of the Exocytosis-Sensitive Protein PP63/Parafusin by Differential MALDI Mass Spectrometric Peptide Mapping

“…It has been shown that at least a part of the PP63/pf pool in the cell is membrane-associated, i.e., associated with cortical structures (8). This membrane association and additional proteins clustered around the PP63/pf molecule may mask some of the potential phosphorylation sites for CK and PKG, and therefore, these sites cannot be phosphorylated even if they are located on the surface of the molecule.…”

“…This protein is heavily enriched in the cell cortex (8). It is selectively dephosphorylated within 80 ms during stimulated synchronous exocytosis of trichocysts and fully rephosphorylated within 10 s (6,9).…”