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

Abstract

Parafusin, a cytosolic phosphoglycoprotein of The background data that tie parafusin to the process of membrane fusion and exocytosis come from studies using the ciliates Paramecium tetraurelia and Tetrahymena thermophila. Paramecium tetraurelia in axenic cultures take up 32p, and phosphorylate a number of polypeptides. The most heavily labeled polypeptide is parafusin, a minor component of Mr 63,000 that shows serine phosphorylation (1, 2). Gilligan and Satir (3,4) showed that, when in vivo-prelabeled wild-type (wt) cells were stimulated to secrete the content of their docked dense-core secretory vesicles (trichocysts) with trinitrophenol, the labeled parafusin was dramatically dephosphorylated. Dephosphorylation was correlated with the massive exocytosis of secretory vesicles in two types of experiments: one testing ionic requirements for exocytosis (Ca2+ vs. Mg2+) and the other using exocytosis mutants.Exocytosis and dephosphorylation required the presence of Ca2+ in the medium in that 5 mM MgCl2, in the absence of added Ca2+, inhibited both processes. Both membrane fusion and changes within the secretory vesicle content are Ca2+-dependent processes, the former requiring an increase of cytosolic Ca2+, probably via receptor-gated Ca2+ channels in the membrane (5). Further, the temperature-sensitive secretory mutant nd9 grown at the restrictive temperature (270C) and then stimulated with trinitrophenol in the presence of 5 mM Ca2+ neither exocytosed nor dephosphorylated parafusin (3). Dephosphorylation and exocytosis, very rapid events, are normally followed by rephosphorylation within 10 sec (6).Parafusin was isolated and purified to apparent homogeneity. An affinity-purified polyclonal antibody was made that recognizes both the phosphorylated and dephosphorylated forms. Two slightly acidic (pI = 5.8 and 6.2) phosphorylated forms of parafusin are found in a cytosolic fraction. The affinity-purified antibody recognizes a third isoelectric form at pI 6.3 that appears unlabeled and is probably membrane bound (1, 2). Parafusin shows immunological cross-reactivity with proteins from a variety ofunicellular organisms and cells of metazoan groups of wide evolutionary divergence, as well as with rat tissues, including brain, heart, pituitary, and kidney. These results suggest that parafusin was present early in the history of eukaryotes and may be of functional importance in the general mechanism of exocytosis and membrane fusion (7,8).A Mr 62,000 phosphoglycoprotein in liver that possessed an a-glucose-l-phosphate (aGlc-l-P) moiety linked to a short chain of mannoses that was O-linked to a senine residue of the protein was independently discovered (9). The bond between glucose and mannose in this protein was a phosphodiester linkage formed by the action of a unique aGlc-1-P phosphotransferase (9). By incorporation of label from a thiophos-

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

“…Anti-peptide antibodies made against the derived amino acid sequence of the pPFUS I-4 (see Fig. 4 (4,5). This is in contrast to PGM, which incorporates glucose 1-32P and does not incorporate UDP glucose (21).…”

“…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.…”

“…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).…”

Structures and Functions of Nuclear and Cytoplasmic Glycoproteins

“…In this case it is thought that the mannose remains associated with the protein while the glucose l-phosphate cycles on and off. Changes in the glycosylation state of PGM and parafusin have been demonstrated to occur in several biological systems [79,80,861, demonstrating that such a cycle is indeed functional in cells. Changes in cytoplasmic calcium levels appear to alter the glucosylation state of both PGM (in the pheochromocytoma PC-12 cell line) [79] and parafusin (in Parumeciunz) [SO].…”

[No Title Available]

“…II, Nov. 1998 7 A series of in vitro experiments later suggested that it was this modification, Glc-1-P that was removed upon exocytosis by a Ca2+-dependent phosphodiesterase, which was defective in the exo-mutant nd9. It was further hypothesized that PFUS might represent a new type of signal transduction molecule involving carbohydrate cycling (Subramanian & Satir 1992).PFUS has been cloned and sequenced and found to have 51% identity to rabbit muscle phosphoglucomutase (PGM), a glycolytic enzyme, although PFUS showed no or minor PGM activity , Levin et al 1998). Compared to PGM, PFUS has several specific insertions and deletions in the primary sequence (Subramanian et al 1994).…”