The Activity of Parafusin Is Distinct from That of Phosphoglucomutase in the Unicellular Eukaryote Paramecium

Andersen, Anders P.; Wyroba, Elżbieta; Reichman, Milaniya; Zhao, Hong; Satir, Birgit H.

doi:10.1006/bbrc.1994.1600

Cited by 19 publications

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“…The sequence and structural information presented here suggest that the region near the active site of PGM is altered in the pPFUS molecule, which is consistent with the observation that isolated parafusin does not have detectable PGM activity (21). We do not know what role the other structural alterations in the PGM motifs play, but the specific disruption of four Ca2+/calmodulin kinase sites is quite striking and probably highly significant.…”

supporting

confidence: 77%

“…4 (4,5). This is in contrast to PGM, which incorporates glucose 1-32P and does not incorporate UDP glucose (21). Therefore, we hypothesize that parafusin and PGM are related molecules but with different functions in the cell.…”

mentioning

confidence: 86%

“…The second, rabbit muscle PGM type 2, is identical to a Mr 60,000 Ca2+/calmodulindependent phosphoprotein in the sarcoplasmic reticulum that is believed to potentially regulate Ca2+ release via its phosphorylation/dephosphorylation. The presence of another PGM-related protein has been reported from smooth muscle cells and fibroblasts; similarly to parafusin (21), this protein has no PGM activity (22). Several other examples of homologous proteins with varied functions are currently being reported (23 Fig.…”

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confidence: 94%

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

Subramanian

Wyroba

Andersen

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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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)

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confidence: 86%

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confidence: 94%

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

Subramanian

Wyroba

Andersen

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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“…Paramecium has been proved to be an excellent model in the studies on the process of endo-and exocytosis (Allen and Fok, 1980;Gilligan and Satir, 1983;Cohen et al, 1984;Plattner et al, 1985;Wyroba, 1989a;Andersen et al, 1994;Subramanian et al, 1994;Gautier et al, 1996;Allen and Fok, 2000;Wiejak et al, 2001;Froissard et al, 2002) including internalization, distribution and retention of the photosensitizers such as hematoporphyrin derivative applied in photodynamic therapy of tumours (Croce et al, 1990).…”

Section: The Relevance Of the Studies On The Unicellular Eukaryotementioning

confidence: 99%

Evolutionary conservancy of the endocytic and trafficking machinery in the unicellular eukaryote Paramecium

Surmacz

Wiejak

Wyroba

2003

Biology of the Cell

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Molecular search for the homologues of the mammalian proteins in the unicellular eukaryote Paramecium involved in endocytosis and membrane trafficking is discussed. We cloned and sequenced the gene fragments encoding the following components participating in endosome formation, sorting and maturation of the proprotein precursors, respectively, dynamin 2, Rab7 and furin. There is a proof that all these genes are expressed in this unicellular organism. The function of the identified immunoanalogues of the above described components of Paramecium endocytic machinery as well as a high degree of sequence homology to the respective human counterparts points to the evolutionary conservancy of these pathways.

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“…The coincidence of exocytosis competence and pp63/pf N de-phosphorylation led to the proposal that this protein may govern membrane fusion, from which the name was derived [30][31][32]. Cloning of its homologue in Tetrahymena and disruption of its single pp63/pf gene suggested this not to be the case [33].…”

Section: Introductionmentioning

confidence: 99%

Molecular aspects of rapid, reversible, Ca2+-dependent de-phosphorylation of pp63/parafusin during stimulated exo-endocytosis in Paramecium cells

Plattner

Kissmehl

2005

Cell Calcium

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Calf signalling governs stimulated exocytosis and exocytosis-coupled endocytosis also in Paramecium cells. Upon stimulation, the ::s10 3 dense-core exocytotic organelles (trichocysts) can be synchronously (80 ms) released, followed by endocytotic membrane resealing (350 ms) and retrieval. Paramecium is the most synchronous dense-core exocytotic system known, allowing to dissect rapidly reversible Calf -dependent phenomena. This holds for the reversible de-/re-phosphorylation cycle of a 63 kD phosphoprotein, pp63/parafusin (pf), which we have cloned, immuno-Iocalised, and characterised as phosphoglucomutase. the enzyme funneling glucose into the glycolytic pathway. It was isolated ex vivo. followed by MALDl analysis, while X-ray structure analysis was performed after heterologous expression. We found multiple phosphorylation of superficial SerlThr residues. Although present also in exo-mutants. pp63/pf is selectively de-phosphorylated only in exo+ strains during synchronous exocytosis (80 ms) and re-phosphorylated within~20 s, Le., the time required to re-establish [Calf] homeostasis. We have isolated relevant protein phosphatases and kinases and probed their activity on pp63/pf in vitro. We consider CalfIcalmodulin-activated PP2B (calcineurin, whose subunits have been cloned) relevant for de-phosphorylation. Re-phosphorylation can be achieved by two protein kinases that also have been cloned. One is activated by cGMP (PKG) which in turn is formed by Calf-activated guanylate cyclase. Another kinase, casein kinase 2. is inhibited by Calf and. hence, activated with some delay in parallel to decreasing [Calf] after exocytosis. In total, several Calf -sensitive cycles cooperate whose protein components have been localised to the cell cortex. Regulation of the phosphorylation degree of pp63/pf may affect structure binding on a microscale and/or its enzymatic activity. All this may serve fueling substrate into glycolysis with increased ATP re-formation (compromised in exo-mutants) and NADH formation. with effects on Calf signalling including mobilisation from cortical stores (alveolar sacs) and overall effects on ATP and Ca 2 + dynamics during synchronous exo-and endocytosis.

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The Activity of Parafusin Is Distinct from That of Phosphoglucomutase in the Unicellular Eukaryote Paramecium

Cited by 19 publications

References 0 publications

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

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

Evolutionary conservancy of the endocytic and trafficking machinery in the unicellular eukaryote Paramecium

Molecular aspects of rapid, reversible, Ca2+-dependent de-phosphorylation of pp63/parafusin during stimulated exo-endocytosis in Paramecium cells

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