Parafusin: Molecular cloning and studies on its two pathways of phosphorylation

Abstract

Parafusin, a cytosolic phosphoglycoprotein of M{dollar}\sb{lcub}\rm r{rcub}{dollar} 63,000, has been shown to dephosphorylate rapidly in a Ca{dollar}\sp{lcub}2+{rcub}{dollar}-dependent manner upon stimulation of exocytosis in Paramecium cells and thereafter to become rephosphorylated. Dephosphorylation does not occur in exo- mutants (nd9-27) or in wild type cells inhibited to exocytose in the absence of extracellular calcium. Parafusin contains two types of phosphorylation sites: one is a {dollar}\alpha{dollar} glucose phosphate added to a short oligosaccharide chain, O-linked to a serine and the other is a serine phosphate. To study the characteristics of the two pathways, both phosphorylations were carried out in vitro using the labels {dollar}\beta{dollar}({dollar}\sp{lcub}35{rcub}{dollar}S) UDPGlc and {dollar}\tau{dollar}({dollar}\sp{lcub}32{rcub}{dollar}P)ATP. Results showed that the presence of Ca{dollar}\sp{lcub}2+{rcub}{dollar} decreased the amount of {dollar}\beta{dollar}({dollar}\sp{lcub}35{rcub}{dollar}S)UDPGlc radioactivity incorporated into parafusin. In contrast, results from identical labeling experiments performed with {dollar}\tau{dollar}({dollar}\sp{lcub}32{rcub}{dollar}P)ATP showed that Ca{dollar}\sp{lcub}2+{rcub}{dollar} enhances incorporation of label into parafusin. In order to determine whether the removal of the {dollar}\beta{dollar}({dollar}\sp{lcub}35{rcub}{dollar}S)UDPGlc label from parafusin by calcium correlated with the in vivo dephosphorylation of parafusin, incubations with {dollar}\beta{dollar}({dollar}\sp{lcub}35{rcub}{dollar}S)UDPGlc were performed using cell fractions of the mutant nd9 grown at the non-permissive temperature (27{dollar}\sp\circ{dollar}C). In contrast to wt cells, the high speed pellet (P2) fraction did not dephosphoglucosylate parafusin in the presence of calcium suggesting that the putative phosphodiesterase was defective in the mutant. We conclude that the in vivo dephosphorylation of parafusin that occurs upon exocytosis is due to removal of the {dollar}\alpha{dollar}Glc-1-P from its oligosaccharide. More generally carbohydrates on cytoplasmic glycoproteins may be cyclically added and/or removed in response to external stimuli.;In addition parafusin cDNA was cloned and sequenced. The amino acid sequence deduced from the cDNA showed a high homology to a glycolytic enzyme PGM (phosphoglucomutase) which catalyzes the interconversion of Glc-1-P and Glc-6-P. It seems probable to infer that parafusin and PGM belong to the same family of genes such that the homologies pertain to areas important for the secondary structure of the molecules which enable them to interact with the Glc-1-P moiety. However, the parafusin sequence has certain specific regions which seem to confer a unique function of the protein in signal transduction related to exocytosis.