This study seeks to identify phosphoproteins in axonemes from Paramecium tetraurelia whose phosphorylation responses to adenosine 3', 5'-cyclic monophosphate (cAMP) and Ca2+ parallel responses induced by these agents in ciliary behavior in this cell. In purified axonemes, over 15 bands ranging from Mr greater than 300 kDa to 19 kDa on SDS-PAGE incorporate 32P from adenosine 5'-gamma-[32P]triphosphate (gamma-32P-ATP) at pCa 7 in the absence of cAMP. A major band whose label turns over rapidly was identified at Mr 43 kDa. In the presence of 5 microM cAMP, more than eight bands, but not the Mr 43 kDa band, were labeled additionally or enhanced their labeling. These phosphoproteins and their kinases are structural components of the axoneme. Overall, some of the same major bands are labeled in the presence of cAMP in Triton X-100-permeabilized paramecia that retain their behavioral responses and in axonemes mechanically isolated from these cells. In particular, two major bands have been identified whose phosphorylation is greatly enhanced by cAMP at low concentrations: 1) a 29 kDa polypeptide whose cAMP-dependent phosphorylation is diminished at pCa 4 compared with pCa 7 and 2) a 65 kDa polypeptide whose phosphorylation is pCa insensitive. These polypeptides meet minimal criteria for signal-sensitive regulators of motility parameters in the Paramecium axoneme.
A cytosolic phosphoprotein that appears to function in membrane fusion during exocytosis of secretory products has previously been isolated from Paramecium tetraurelia. This phosphoprotein, parafusin, with Mr 63,000, is rapidly dephosphorylated via a Ca2-dependent process when secretagogues induce exocytosis in competent cells. Dephosphorylation does not occur in exocytosis-incompetent cells. Polyclonal antibodies against purified parafusin have now been used to show that this protein is present in unicellular organisms and cells of metazoan groups of wide evolutionary divergence, such as yeast, insects, and mammals, including humans. These results suggest that parafusin was present early in the history of eukaryotes and that it is of functional importance in the general mechanism of exocytosis and membrane fusion.
This paper deals with the detailed mechanisms of signal transduction that lead to exocytosis during regulative secretion induced by specific secretagogues in a eukaryotic cell, Paramecium tetraurelia. There are at least three cellular compartments involved in the process: I) the plasma membrane, which contains secretagogue receptors and other transmembrane proteins, II) the cytoplasms, particularly in the region between the cell and secretory vesicle membranes, where molecules may influence interactions of the membranes, and III) the secretory vesicle itself. The ciliated protozoan Paramecium tetraurelia is very well suited for the study of signal transduction events associated with exocytosis because this eukaryotic cell contains thousands of docked secretory vesicles (trichocysts) below the cell membrane which can be induced to release synchronously when triggered with secretagogue. This ensures a high signal-to-noise ratio for events associated with this process. Upon release the trichocyst membrane fuses with the cell membrane and the trichocyst content undergoes a Ca2+-dependent irreversible expansion. Secretory mutants are available which are blocked at different points in the signal transduction pathway. Aspects of the three components mentioned above that will be discussed here include a) the properties of the vesicle content, its pH, and its membrane; b) the role of phosphorylation/dephosphorylation of a cytosolic 63-kilodalton (kDa)Mr protein in membrane fusion; and c) how influx of extracellular Ca2+ required for exocytosis may take place via exocytic Ca2+ channels which may be associated with specific membrane microdomains (fusion rosettes).
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