The β-phosphoro[35S]thioate analogue of UDP-Glc is efficiently utilized by the glucose phosphotransferase and is relatively resistant to hydrolytic degradation

Rb, Marchase; Saunders, Aleister J.; Aa, Rivera; Jm, Cook

doi:10.1016/0167-4838(87)90103-8

Cited by 14 publications

(6 citation statements)

References 12 publications

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“…A band of the expected mobility for PRP1 (M r 68 kDa) was labelled in lysates from isolated tachyzoites. Since the presence of the breakdown product [α‐ 35 S]‐glucose‐1‐phosphate from [β‐ 35 S]‐UDP‐glucose could artifactually label PRP1 (Marchase et al ., 1987; Satir et al ., 1990), a chase was performed to determine if labelling was due to a potential labeling of an active PGM site of PRP1. Therefore, high excess levels of cold glucose‐1‐phosphate or UDP‐glucose were used in order to chase the labelled species (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…To determine if PRP1 would serve as an acceptor for a glucose phosphotransferase ( Fig. 2A (Marchase et al ., 1987;Satir et al ., 1990), a chase was performed to determine if labelling was due to a potential labeling of an active PGM site of PRP1. Therefore, high excess levels of cold glucose-1-phosphate or UDPglucose were used in order to chase the labelled species ( Fig.…”

Section: Prp1 Becomes Phosphorylated In Vitro With Udp-glucosementioning

confidence: 99%

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Role of the parafusin orthologue, PRP1, in microneme exocytosis and cell invasion inToxoplasma gondii

Matthiesen

Shenoy

Kim

et al. 2003

Cellular Microbiology

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SummaryThe association of PRP1, a Paramecium parafusin orthologue, with Toxoplasma gondii micronemes, now confirmed by immunoelectron microscopy, has here been studied in relation to exocytosis and cell invasion. PRP1 becomes labelled in vivo by inorganic 32 P and is dephosphorylated when ethanol is used to stimulate Ca 2+ + + + -dependent exocytosis of the micronemes. The ethanol Ca 2+ + + + -stimulated exocytosis is accompanied by translocation of PRP1 and microneme content protein (MIC3) from the apical end of the parasite. Immunoblotting showed that PRP1 is redistributed inside the parasite, while microneme content is secreted. To study whether similar changes occur during cell invasion, quantitative microscopy was performed during secretion, invasion and exit (egress) from the host cell. Time-course experiments showed that fluorescence intensities of PRP1 and MIC3 immediately after invasion were reduced 10-fold compared to preinvasion levels, indicating that PRP1 translocation and microneme secretion accompanies invasion. MIC3 regained fluorescence intensity and apical distribution after 15 min, while PRP1 recovered after 1 h. Intensity of both proteins then increased throughout the parasite division period until host cell lysis, suggesting the need to secrete microneme proteins to egress. These studies suggest that PRP1 associated with the secretory vesicle scaffold serves an important role in Ca 2+ + + + -regulated exocytosis and cell invasion.

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Section: Resultsmentioning

confidence: 99%

Section: Prp1 Becomes Phosphorylated In Vitro With Udp-glucosementioning

confidence: 99%

Role of the parafusin orthologue, PRP1, in microneme exocytosis and cell invasion inToxoplasma gondii

Matthiesen

Shenoy

Kim

et al. 2003

Cellular Microbiology

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“…Glc-phosphotransferase assays, SDS-PAGE, and autoradiography were performed as described previously (Srisomsap et ai., 1988). [f132p]UDP-Glc (700-900 Ci/mmol) was synthesized and purified as described by Marchase et al (1987). The 35S-labeled phosphorothioate analogue of UDP-GIc [(/~35S)UDP-Glc] (1,100-1,300 Ci/mmol), which had been determined by Marchase et al (1987) to be used with high efficiency by the Glc-pbosphotransferase, was obtained from Dupont-NEN Products (Wilmington, DE) and used in some of the experiments reported here.…”

Section: Glc-phosphotransferase Assaysmentioning

confidence: 99%

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

Satir

Srisomsap

Reichman

et al. 1990

The Journal of Cell Biology

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Abstract. 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 otGlc-l-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.

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“…Glc-phosphotransferase assays were carried out in the presence of 50 mM Bis-Tris (pH 6.0); 105 mM NaCl; 5 mM CaCl 2 ; 1 mM phenylmethylsulfonyl fluoride; 200 M Glc-1-P; 1 mM NaH 2 PO 4 ; 5 Ci (ϳ350 Ci/mmol) of the 35 S-labeled ␤-phosphorothioate analog of UDP-Glc, ␤-35 S-UDP-Glc; and 1 g of yeast lysate per l (30 g total). It was previously shown that this phosphorothioate analog of UDP-Glc is utilized in the same manner as the native compound in the Glc-phosphotransferase assay (20). Reactions were carried out for 30 min at room temperature and were terminated by the addition of sodium dodecyl sulfate (SDS) sample buffer and boiling.…”

Section: Methodsmentioning

confidence: 99%

The posttranslational modification of phosphoglucomutase is regulated by galactose induction and glucose repression in Saccharomyces cerevisiae

Bounelis

Dey

et al. 1995

J Bacteriol

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The enzyme phosphoglucomutase functions at a key point in carbohydrate metabolism. In this paper, we show that the synthesis of the major isoform of yeast phosphoglucomutase, encoded by the GAL5 (PGM2) gene, is regulated in a manner that is distinct from that previously described for other enzymes involved in galactose metabolism in the yeast Saccharomyces cerevisiae. Accumulation of this isoform increased four-to sixfold when the culture experienced either glucose depletion or heat shock. However, heat shock induction did not occur unless the cells were under glucose repression. This nonadditive increase in expression suggests that the regulatory mechanisms controlling the heat shock induction and glucose repression of the GAL5 gene are functionally related. We previously demonstrated that phosphoglucomutase is modified by a posttranslational Glc-phosphorylation reaction. We now show that this posttranslational modification, like phosphoglucomutase expression itself, is also regulated by galactose induction and glucose repression. Finally, no evidence was found to indicate that the Glc-phosphorylation of phosphoglucomutase alters its enzymatic activity under the conditions examined.The interconversion of Glc-1-P and Glc-6-P, a key metabolic trafficking point in all cells, is carried out by the enzyme phosphoglucomutase (EC 2.7.5.1). In Saccharomyces cerevisiae cells grown on galactose as the primary carbon source, the Glc-1-P formed as a result of galactose metabolism is converted to Glc-6-P by phosphoglucomutase as a required step in providing cells with carbon-containing precursors. When yeast cells are grown on other carbon sources, phosphoglucomutase functions primarily to convert Glc-6-P to Glc-1-P. Glc-1-P is required for the synthesis of the sugar nucleotide UDP-Glc, which acts as a precursor for the synthesis of oligosaccharides and trehalose. Phosphoglucomutase consists of two isoforms in S. cerevisiae (31). The major isoform is encoded by the GAL5 (PGM2) gene (8), while the gene encoding the minor isoform is called PGM1 (3).The synthesis of most enzymes involved in the galactose utilization (Leloir) pathway is subject to tight control by overlapping mechanisms of glucose repression and galactose induction, leading to as much as a 1,000-fold induction of enzyme levels when a yeast culture is shifted from glucose to galactose medium. In contrast, phosphoglucomutase was regarded as a constitutively expressed enzyme for many years (3, 12). Since phosphoglucomutase function is also required for growth on media containing glucose as the carbon source, this conclusion was consistent with its metabolic function. However, phosphoglucomutase synthesis is also regulated by glucose repression and galactose induction, although the magnitude of this regulation is considerably weaker than that of other genes involved in galactose metabolism (23). The galactose regulation of the GAL5 gene, like other genes encoding proteins in the Leloir pathway, is mediated by the Gal4p-Gal80p regulatory system. Gal4p acts as a posi...

show abstract

The β-phosphoro[35S]thioate analogue of UDP-Glc is efficiently utilized by the glucose phosphotransferase and is relatively resistant to hydrolytic degradation

Cited by 14 publications

References 12 publications

Role of the parafusin orthologue, PRP1, in microneme exocytosis and cell invasion inToxoplasma gondii

Role of the parafusin orthologue, PRP1, in microneme exocytosis and cell invasion inToxoplasma gondii

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

The posttranslational modification of phosphoglucomutase is regulated by galactose induction and glucose repression in Saccharomyces cerevisiae

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