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

Abstract

Calf signalling governs stimulated exocytosis and exocytosis-coupled endocytosis also in Paramecium cells. Upon stimulation, the ::s10 3 dense-core exocytotic organelles (trichocysts) can be synchronously (80 ms) released, followed by endocytotic membrane resealing (350 ms) and retrieval. Paramecium is the most synchronous dense-core exocytotic system known, allowing to dissect rapidly reversible Calf -dependent phenomena. This holds for the reversible de-/re-phosphorylation cycle of a 63 kD phosphoprotein, pp63/parafusin (pf), which we have cloned, immuno-Iocalised, and characterised as phosphoglucomutase. the enzyme funneling glucose into the glycolytic pathway. It was isolated ex vivo. followed by MALDl analysis, while X-ray structure analysis was performed after heterologous expression. We found multiple phosphorylation of superficial SerlThr residues. Although present also in exo-mutants. pp63/pf is selectively de-phosphorylated only in exo+ strains during synchronous exocytosis (80 ms) and re-phosphorylated within~20 s, Le., the time required to re-establish [Calf] homeostasis. We have isolated relevant protein phosphatases and kinases and probed their activity on pp63/pf in vitro. We consider CalfIcalmodulin-activated PP2B (calcineurin, whose subunits have been cloned) relevant for de-phosphorylation. Re-phosphorylation can be achieved by two protein kinases that also have been cloned. One is activated by cGMP (PKG) which in turn is formed by Calf-activated guanylate cyclase. Another kinase, casein kinase 2. is inhibited by Calf and. hence, activated with some delay in parallel to decreasing [Calf] after exocytosis. In total, several Calf -sensitive cycles cooperate whose protein components have been localised to the cell cortex. Regulation of the phosphorylation degree of pp63/pf may affect structure binding on a microscale and/or its enzymatic activity. All this may serve fueling substrate into glycolysis with increased ATP re-formation (compromised in exo-mutants) and NADH formation. with effects on Calf signalling including mobilisation from cortical stores (alveolar sacs) and overall effects on ATP and Ca 2 + dynamics during synchronous exo-and endocytosis.

Signalling in ciliates: long- and short-range signals and molecular determinants for cellular dynamics

“…This serves for the rephosphorylation of ADP to ATP, the substrate of the dynein ATPase that drives ciliary beating, and involves phosphoarginine provided by a ciliary arginine kinase which in Tetrahymena pyriformis is 40 kDa in size (Michibata et al, ). Generally, phosphagenes help to maintain the ATP level in narrow spaces, as discussed previously (Plattner & Kissmehl, ). In muscle, compensation mechanisms include phosphocreatine (in mammals) or phosphoarginine in crustaceans as a store of sufficient free enthalpy.…”

“…During AED‐induced synchronous trichocyst exocytosis in P. tetraurelia , ATP decays significantly by ∼20% (averaged from different strains), from between 1.1 and 1.25 mM (Matt, Bilinski & Plattner, ; Lumpert, Kersken & Plattner, ), within ∼5 s. ATP content recovers, depending on the strain, during the following ∼20–30 s (Vilmart‐Seuwen et al, ; Plattner & Kissmehl, ). Considering a turnover time of ATP of 1–2 min in mammalian cells under steady‐state conditions (Skog, Tribukait & Sundius, ; Jauker, Lades & Nowack, ), the decay of [ATP] by ∼20% within 5 s is difficult to explain.…”

Protein Phosphatase 2B (PP2B, Calcineurin) in Paramecium: Partial Characterization Reveals That Two Members of the Unusually Large Catalytic Subunit Family Have Distinct Roles in Calcium-Dependent Processes

“…In Paramecium, CaN has been shown to specifically dephosphorylate the 63-kDa phosphoprotein, pp63 (45,46), a 63-kDa protein with multiple Ser/Thr phosphorylation sites on its surface (67), in strict correlation with synchronous (80 ms) exocytosis (35). In a recent survey, pp63 could be reasonably connected with several aspects of Ca 2ϩ signaling, specifically in subplasmalemmal domains (73) where pp63 (42) colocalizes with CaN (62). This may include a role for CaN in Ca 2ϩ store activation (10,87).…”

Multigene Family Encoding 3′,5′-Cyclic-GMP-Dependent Protein Kinases in Paramecium tetraurelia Cells

“…CELL although the precise PKG form(s) contributing remains to be identified. A putative role of this phosphorylation has already been discussed recently (63), including regulating contact with substrate proteins (7). In summary, considering exo-endocytosis-regulated processes, the potential role of PKG involvement is not less intriguing than, for instance, the level of discussion concerning neuronal (14,85) and a variety of other systems (20,22).…”