Paramecium genome survey: a pilot project.

A consortium of laboratories undertook a pilot sequencing project to gain insight into the genome of Paramecium. Plasmid-end sequencing of DNA fragments from the somatic nucleus together with similarity searches identified 722 potential protein-coding genes. High gene density and uniform small intron size make random sequencing of somatic chromosomes a cost-effective strategy for gene discovery in this organism.

A parafusin-related Toxoplasma protein in Ca2+-regulated secretory organelles.

We cloned a gene, PRPI, of Toxoplasma gondii encoding a 637-amino-acids protein having a calculated mass of 70 kDa. The sequence showed high homology to parafusin, a protein that in Paramecium tetraurelia participates in Ca2+-regulated exocytosis and is a paralog of phosphoglucomutase. We show that Toxoplasma gondii homogenate and an expressed recombinant PRP1 fusion protein cross-react with a specific peptide-derived antibody to parafusin in Western blots. Antibodies to the recombinant PRP1 showed cross-reaction with parafusin and recognized PRP1, as bands at M, 63 x 10(3) and 68 x 10(3), respectively. PRP1 is labeled when Toxoplasma gondii cells are incubated with inorganic 32P and appears as the major band on autoradiograms of SDS-PAGE gels. The localization of PRP1 was examined in secretory organelles of Toxoplasma gondii by deconvolution light microscopy followed by three dimensional reconstruction using pairwise combinations of specific antibodies. PRP1 localized to the apical third of the cell. It co-localized with micronemes, the only secretory organelle the secretion of which is Ca2+ dependent. Quantification of the co-localized stain suggests that only mature micronemes ready for exocytosis have PRP1. These findings suggest that PRP1, parafusin and other members of the phosphoglucomutase superfamily have a conserved role in Ca2+-regulated exocytic processes.

A comparative hybridization analysis of yeast DNA with Paramecium parafusin- and different phosphoglucomutase-specific probes.

Molecular probes designed for the parafusin (PFUS), the Paramecium exocytic-sensitive phosphoglycoprotein, gave distinct hybridization patterns in Saccharomyces cerevisiae genomic DNA when compared with different phosphoglucomutase specific probes. These include two probes identical to segments of yeast phosphoglucomutase (PGM) genes 1 and 2. Neither of the PGM probes revealed the 7.4 and 5.9 kb fragments in Bgl II-cut yeast DNA digest detected with the 1.6 kb cloned PFUS cDNA and oligonucleotide constructed to the PFUS region (insertion 3--I-3) not found in other species. PCR amplification with PFUS-specific primers generated yeast DNA-species of the predicted molecular size which hybridized to the I-3 probe. A search of the yeast genome database produced an unassigned nucleotide sequence that showed 55% identity to parafusin gene and 37% identity to PGM2 (the major isoform of yeast phosphoglucomutase) within the amplified region.

Functional diversity of the phosphoglucomutase superfamily: structural implications.

Three-dimensional structural models of three members of the phosphoglucomutase (PGM) superfamily, parafusin, phosphoglucomutase-related protein and sarcoplasmic reticulum phosphoglucomutase, were constructed by homology modeling based on the known crystal structure of rabbit muscle phosphoglucomutase. Parafusin, phosphoglucomutase-related protein and sarcoplasmic reticulum phosphoglucomutase each have 50% or more identity with rabbit muscle phosphoglucomutase at the amino acid level and all are reported to exhibit no or minor phosphoglucomutase activity. There are four major insertions and two deletions in the parafusin sequence relative to PGM, all of which are located in surface-exposed loops connecting secondary structural elements. The remaining amino acid substitutions are distributed throughout the sequence and are not predicted to alter the polypeptide fold. Parafusin contains a putative protein kinase C site located on a surface loop in domain II that is not present in the homologs. Although the general domain structure and the active site of rabbit muscle phosphoglucomutase are preserved in the model of phosphoglucomutase-related protein, a major structural difference is likely to occur in domain 1 due to the absence of 55 amino acid residues in PGM-RP. This deletion predicts the loss of three alpha-helices and one beta-strand from an anti-parallel beta-sheet in this domain as compared with the rabbit muscle phosphoglucomutase.

POLINA: detection and evaluation of single amino acid substitutions in protein superfamilies.

MOTIVATION AND RESULTS: An algorithm is described for the quick identification and evaluation of amino acid substitutions in multiple protein sequence alignment. The strategy is based on the calculation of a relative conservation index for an amino acid at each position of the alignment. The algorithm is implemented in the computer program POLINA (Protein Oriented LINear Analysis) which provides a summary of analysis in a format suitable for import into a graphing program. AVAILABILITY: A copy of source code is available upon request from the authors or can be downloaded via the WWW at http://www.geocities.com/Athens/4654/POLINA.h tml. CONTACT: slevin@aecom.yu.edu; bsatir@aecom.yu.edu

Parafusin is a membrane and vesicle associated protein that cycles at exocytosis.

In the unicellular eukaryote Paramecium tetraurelia, stimulation of exocytosis leads to Ca2+ activation of an alpha Glc-1-phosphodiesterase that dephosphoglucosylates the phosphoglycoprotein parafusin (PFUS). This process fails in exo mutant nd9 and also in the absence of Ca2+ influx upon stimulation suggesting that PFUS dephosphoglucosylation may be causally related to exocytosis. To further corroborate the hypothesis that PFUS is involved in the molecular events in exocytosis, we used laser confocal scanning microscopy and a PFUS specific peptide antibody to perform localization studies of PFUS in wild type (wt) and mutant Paramecium. In unstimulated wt cells, PFUS was associated both with the exocytic site of the cell membrane and with the membrane of the dense core secretory vesicles. Localization at these two sites was shown to be independent of each other since the exocytosis mutant (exo-) tam8, in which docking of its vesicles is blocked, still showed cell membrane staining. Immunofluorescence and immunoblotting of isolated intact secretory vesicles also revealed PFUS association. Upon stimulation of exocytosis, PFUS dissociated from both the dense core secretory vesicles and the cell membrane in a Ca(2+)-dependent manner. During recovery of exocytic capacity, PFUS reassociated with the newly formed secretory vesicles in the cytoplasm prior to their docking at the exocytic sites. Immunoblot analysis of PFUS during this time showed no changes in levels of the protein. Stimulation of exocytosis in wt in Mg2+ buffer or in the exo- temperature sensitive mutant (nd9) at the non-permissive temperature did not lead to dissociation of the PFUS. We conclude that PFUS is a novel critical component whose cycling probably participates in the molecular exocytic fusion machinery in these cells.

Mammalian homologue of the calcium-sensitive phosphoglycoprotein, parafusin.

Three specific antipeptide antibodies and oligonucleotide probes synthesized to internal sequences of parafusin have been used to search for mammalian counterpart(s) of this protein. Parafusin is an exocytic-sensitive phosphoglycoprotein from a unicellular eukaryote Paramecium that was recently cloned and sequenced (Subramanian et al., Proc. Natl. Acad. Sci. USA 91, 9832-9836 (1994)). Western and Southern blot analyses, polymerase chain reaction (PCR) and reverse transcriptase coupled PCR (RT-PCR) techniques have been used to examine rat liver and pancreas, human pancreas and a murine pancreatic beta-cell line (beta TC3) arising in transgenic mice. The parafusin-specific antibodies showed cross-reaction with a protein at approximately 63 kDa in 4 tissues, whereas a phosphoglucomutase-specific antibody also detected a second band of similar molecular weight in the beta TC3 cells. The presence of two bands shows that parafusin homologue(s) and phosphoglucomutase are separate entities. beta TC3 cells were shown to incorporate [beta 35]UDPGlc into the parafusin homologue in a Ca(++)-sensitive manner characteristic of parafusin. Southern blot analysis revealed that the parafusin-specific probe hybridized with restriction enzyme digests of rat DNA in distinct patterns different from those observed with a phosphoglucomutase-specific probe. Rat genomic DNA and mRNA from the beta TC3 cells were used as the templates for PCR and RT-PCR using internal parafusin primers. In both cases similarly sized products were obtained which hybridized in Southern analysis with a specific parafusion probe located within the amplified region. These results indicate that a parafusin homologue exists in mammalian cells.

Lysozyme acts as a chemorepellent and secretagogue in Paramecium by activating a novel receptor-operated Ca++ conductance.

Using combined intracellular recordings and behavioral bioassays, it was found that lysozyme has two different effects in Paramecium, depending upon the concentrations used. At low concentrations (0.5 mM to 1.0 microM) it acts as an effective chemorepellent that causes reliable electrophysiological changes. Lysozyme-induced somatic depolarizations, isolated by blocking K+ channels with Cs-TEA, showed concentration dependencies that were well correlated with chemorepulsion. Ion dependency experiments showed that these were Ca++ based depolarizations. Addition of either Na+ or Mg++ improves chemorepulsion by providing additional depolarizations. Both the depolarizations and chemorepulsion were blocked by 10 microM neomycin, suggesting that the depolarization is necessary for this chemosensory transduction event. At higher concentrations (100 microM), lysozyme is a secretagogue. A transient inward current, recorded in Ca++ alone solutions with Cs-TEA present, was seen in response to high lysozyme concentrations. The amplitude of this inward current was well correlated with exocytosis. Addition of neomycin (1.0 mM) eliminated both the inward current and exocytosis, suggesting a causal relationship. Neither amiloride or W-7, compounds previously suggested to affect the electrophysiological responses to secretagogues, had any significant effects. The mucopolysaccharide hydrolysis activity of lysozyme was not required for any of these responses. We propose that Paramecium have a high affinity receptor on the body plasma membrane that responds to either lysozyme or a related compound to cause an increase in a novel body Ca++ conductance. This receptor-operated Ca++ conductance causes membrane depolarization and chemorepulsion at low concentrations and triggers a sufficient Ca++ influx at high concentrations to cause exocytosis.

Cloning and sequencing of parafusin, a calcium-dependent exocytosis-related phosphoglycoprotein.

A cDNA for parafusin, an evolutionarily conserved phosphoglycoprotein involved in exocytosis, has been cloned and sequenced from a unicellular eukaryote, Paramecium tetraurelia. A Paramecium cDNA library was screened with an oligonucleotide probe synthesized to an internal amino acid sequence of isolated parafusin. The insert was 3 kb long with an open reading frame of 1.75 kb. Data base searches of the deduced amino acid sequence showed that Paramecium parafusin had a 50.7% sequence identity to rabbit muscle phosphoglucomutase, although no detectable phosphoglucomutase activity has been detected in isolated parafusin. The deduced parafusin amino acid sequence had four inserts and two deletions, which might confer on the protein specific functions in signal transduction events related to exocytosis. Furthermore, searches for potential phosphorylation sites showed the presence of a protein kinase C site (KDFSFR) specific to parafusin. Southern blot analysis with probes specific for parafusin and phosphoglucomutase suggested that these proteins were products of different genes. We propose that parafusin and phosphoglucomutase are members of a superfamily that conserve homologies important for the tertiary structure of the molecules.

The activity of parafusin is distinct from that of phosphoglucomutase in the unicellular eukaryote Paramecium.

In this paper we identified the presence of a Paramecium phosphoglucomutase enzymatic activity which is clearly distinct from that of parafusin-the exocytosis-related phosphoglycoprotein. Since the recently cloned parafusin showed homology to rabbit muscle phosphoglucomutase, we have designed a specific peptide parafusin antibody-generated to a region not present in any known sequenced phosphoglucomutases-to distinguish parafusin from the Paramecium phosphoglucomutase. Separation of these two proteins was obtained using liquid chromatography, enzymatic activity assay and immunoblotting analysis with the specific parafusin peptide antibody. Parafusin fractions incorporated [B35S]UDP-Glc but not [35S]Glc-1-P whereas Paramecium phosphoglucomutase fractions incorporated [35S]Glc-1-P but not [B35S]UDP-Glc. This indicates that these two proteins are separate entities exhibiting different properties and most likely distinct functions in the cell.

Carbohydrate cycling in signal transduction: parafusin, a phosphoglycoprotein and possible Ca(2+)-dependent transducer molecule in exocytosis in Paramecium.

Parafusin, a cytosolic phosphoglycoprotein of M(r) 63,000, is dephosphorylated and rephosphorylated rapidly in a Ca(2+)-dependent manner upon stimulation of exocytosis in vivo in wild-type (wt) Paramecium. In contrast, the temperature-sensitive exocytosis mutant nd9, grown at the nonpermissive temperature (27 degrees C), does not exocytose or dephosphorylate parafusin upon stimulation in the presence of Ca2+; grown at the permissive temperature (18 degrees C), nd9 cells show a wt phenotype. Parafusin contains two types of phosphorylation sites: one where glucose 1-phosphate is added by an alpha-glucose-1-phosphate phosphotransferase and removed by a phosphodiesterase and one where phosphate from ATP is added directly to a serine residue by a protein kinase and removed by a phosphatase. We show here that, in cell fractions from wt Paramecium, both reactions can be carried out in vitro by using uridine(5'-[beta-[35S]thio])diphospho(1)-glucose (UDP[beta 35S]-Glc) and [gamma-32P]ATP, respectively. The characteristics of these pathways are different. Specifically, in the presence of Ca2+, the amount of UDP[beta 35S]-Glc label in parafusin is reduced. In contrast, identical labeling experiments with [gamma-32P]ATP show that Ca2+ enhances labeling of parafusin. Mg2+ had no appreciable effect on either labeling. Removal of the UDP[beta 35S]-Glc label on parafusin in the presence of Ca2+ correlates with the in vivo dephosphorylation seen upon exocytosis. Incubations with UDP[beta 35S]-Glc were then performed with homogenates and nd9 cell fractions grown at 27 degrees C under the ionic conditions used for wt cells. These labelings were not affected by Ca2+, in contrast to results from wt cells but in accord with those obtained earlier with nd9-27 mutant cells in vivo. Factors responsible for both dephosphorylation and Ca2+ sensitivity were found in the high-speed pellet (P2) in wt cells, suggesting that the putative phosphodiesterase is in this fraction and that the defect in the mutant nd9-27 residues in the Ca2+ activation of the phosphodiesterase. We conclude that the in vivo dephosphorylation of parafusin that occurs upon exocytosis is a dephosphoglucosylation due to removal of the alpha-glucose 1-phosphate and more generally that carbohydrates on cytoplasmic glycoproteins may be cyclically added and/or removed in response to extracellular stimuli.

A potential mucus precursor in Tetrahymena wild type and mutant cells.

By using an antibody to a specific mucus polypeptide (34 kDa) to study whole cell extracts of both a secretory mutant (SB281) and wild type (wt) Tetrahymena, we demonstrate that a 57-kDa polypeptide is a probable precursor to the 34-kDa secretory polypeptide. We postulate that the precursor accumulates in the mutant cells because it cannot be cleaved. This mutant contains no recognizable mature secretory granules (mucocysts). By immunoelectron microscopy, the 34-kDa polypeptide was localized in wt cells specifically to the mature mucocysts and to their released products. Localization in mutant cells occurred in two different types of cytoplasmic vesicles: small electron dense vesicles (0.3-0.5 microns in diameter) and large electron lucent vacuoles (1.2-3.5 microns in diameter). Immunoblot analyses of homogenates of mutant and wt cells with the anti-34-kDa serum revealed a dominant band in the mutant at Mr 57 kDa whereas the wt showed a dominant band only at Mr 34 kDa. Furthermore, the 57-kDa polypeptide is immunoprecipitated with anti-34-kDa serum from the mutant cell. Further evidence for a precursor relation of the 57-kDa polypeptide in mutant cells to the 34-kDa mucus polypeptide of wt cells was obtained by the use of drugs (monensin, chloroquine, NH4Cl) that block secretory product processing in wt cells. Extracts of drug-treated wt cells showed the presence of a 57-kDa cross reacting band even after 18 h of incubation in growth medium whereas untreated control cells contained the 34-kDa mature protein almost exclusively. These results indicate that processing of the precursor to the 34-kDa polypeptide occurs in an acidic compartment(s) possibly in either the trans Golgi network, or condensing vacuoles or both.

Chromosomal localization of an exocytosis mutant in Tetrahymena thermophila.

Exocytosis mutants of Tetrahymena thermophila are deficient in mucus release. Experiments to chromosomally locate two of these mutants are described, using the technique of deletion mapping with nullisomic strains. One exo locus has been assigned to chromosome 5.

Parafusin, an exocytic-sensitive phosphoprotein, is the primary acceptor for the glucosylphosphotransferase in Paramecium tetraurelia and rat liver.

Parafusin, the major protein in Paramecium tetraurelia to undergo dephosphorylation in response to secretory stimuli, appears to be the primary acceptor for the glucosylphosphotransferase in this species based on five independent criteria: identical molecular size of 63 kD; identical isoelectric points in the phosphorylated state of pH 5.8 and 6.2; identical behavior in reverse-phase chromatography; immunological cross-reactivity with an affinity-purified anti-parafusin antibody; the presence of a phosphorylated sugar after acid hydrolysis. It appears likely that the dephosphorylation observed with secretion reflects the removal of alpha Glc-1-P from parafusin's oligosaccharides and is consistent, therefore, with a regulatory role for this cytoplasmic glycosylation event. The glucosylphosphotransferase acceptor in rat liver is also immunoprecipitated by the anti-parafusin antibody and is very similar in physical characteristics to the paramecium protein. This conservation suggests a role for parafusin in mammalian exocytosis as well, at a step common to both the regulated and constitutive secretory pathways.

Species distribution of a phosphoprotein (parafusin) involved in exocytosis.

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.

The secretory vesicle in living Paramecium is acidic.

In Paramecium, secretory proteins are packaged within membrane-bounded vesicles in a condensed form. This form expands when the proteins are released. We have now determined that a proton gradient is present in the secretory vesicles of living Paramecium. Acridine Orange, used as an in vivo indicator of acidic compartments, stained the secretory vesicles in both wild-type and mutant cells. Addition of the two agents that dissipate proton gradients (protonophores), namely, 2,4-dinitrophenol (DNP) and carbonylcyanide m-chlorophenylhydrazone (CCCP), eliminated this staining. Washed cells re-established their intravesicular acidity. Effects of sodium azide on vesicular acidity suggest that proton transport in these vesicles involves an ATP-dependent mechanism.

In vitro phosphorylation of Paramecium axonemes and permeabilized cells.

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.

Aspects of signal transduction in stimulus exocytosis-coupling in Paramecium.

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).