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

Abstract

A cDNA for parafusin, an evolutionarily conserved phosphoglycoprotein involved in exocytosis, has been cloned and sequenced from a uniceilular 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 phosphoglucomutate 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) specifi 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.Previously we discovered a cytosolic phosphoprotein, parafusin, that plays a role in regulated exocytosis in the unicellular eukaryote Paramecium (1, 2) and that is evolutionarily conserved (3). Parafusih has been shown to be phosphorylated via a Ca2+-dependent protein kinase (4). Surprisingly, parafusin is also a phosphoglycoprotein in which a short chain ofmannose residues is 0-linked to serine. This chain is phosphoglucosylated by a glucose-1-phosphate phosphotransferase that uses UDP glucose (5). We have recently demonstrated that dephosphoglucosylation is catalyzed by a Ca2+-activated phosphodiesterase. Cells in which parafusin is normal but that are unable to release the content of their dense core secretory vesicles upon stimulation show inactive phosphodiesterase, suggesting that dephosphoglucosylation is a critical event in the pathway to exocytosis (4).Tryptic digests of parafusin purified as described earlier (6)

Effect of wortmannin and phorbol ester on Paramecium fluid-phase uptake in the presence of transferrin

“…This suggests that the PMA influence on fluid phase endocytosis is probably not mediated by PKC signalling but may be resulting from its effect on the other factors -discussed abovereported by Aballay et al (1999). Paramecium was a subject of studies on endo-and exocytic processes (Allen and Fok 1980, 2000, Ziessenis and Plattner 1985, Wyroba 1987, 1991, Subramanian et al 1994, but both the regulation of endocytic routes and a relationship between pinocytosis and RME uptake requires further investigation.…”

Dynamin-association with agonist-mediated sequestration of beta-adrenergic receptor in single-cell eukaryoteParamecium

“…Polymerase chain reaction (PCR) was performed on the genomic DNA isolated from Paramecium (Subramanian et al, 1994). PCR settings were: denaturation at 94°C (30·s), annealing at 52°C (30·s) and extension at 72°C (1·min) for 29 cycles using the PTC-200 DNA Engine from MJ Research.…”

[No Title Available]

“…Compared to PGM, PFUS has several specific insertions and deletions in the primary sequence (Subramanian et al 1994). These regions have been shown to be located in surface loops of the molecule, thus conserving the overall three-dimensional structure of PGM.…”