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In vivo analysis of the major exocytosis-sensitive phosphoprotein in Tetrahymena.

Chilcoat ND, Turkewitz AP - J. Cell Biol. (1997)

Bottom Line: Comparison of deduced protein sequences, taking advantage of the known atomic structure of rabbit muscle PGM, suggests that both ciliate enzymes and all other PGM-like proteins have PGM activity.Surprisingly, DeltaPGM1 cells displayed no detectable defect in exocytosis, but showed a dramatic decrease in PGM activity.Both our results, and reinterpretation of previous data, suggest that any potential role for PGM-like proteins in regulated exocytosis is unlikely to precede membrane fusion.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, Illinois 60637, USA.

ABSTRACT
Phosphoglucomutase (PGM) is a ubiquitous highly conserved enzyme involved in carbohydrate metabolism. A number of recently discovered PGM-like proteins in a variety of organisms have been proposed to function in processes other than metabolism. In addition, sequence analysis suggests that several of these may lack PGM enzymatic activity. The best studied PGM-like protein is parafusin, a major phosphoprotein in the ciliate Paramecium tetraurelia that undergoes rapid and massive dephosphorylation when cells undergo synchronous exocytosis of their dense-core secretory granules. Indirect genetic and biochemical evidence also supports a role in regulated exocytotic membrane fusion. To examine this matter directly, we have identified and cloned the parafusin homologue in Tetrahymena thermophila, a ciliate in which protein function can be studied in vivo. The unique T. thermophila gene, called PGM1, encodes a protein that is closely related to parafusin by sequence and by characteristic post-translational modifications. Comparison of deduced protein sequences, taking advantage of the known atomic structure of rabbit muscle PGM, suggests that both ciliate enzymes and all other PGM-like proteins have PGM activity. We evaluated the activity and function of PGM1 through gene disruption. Surprisingly, DeltaPGM1 cells displayed no detectable defect in exocytosis, but showed a dramatic decrease in PGM activity. Both our results, and reinterpretation of previous data, suggest that any potential role for PGM-like proteins in regulated exocytosis is unlikely to precede membrane fusion.

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In vitro and in vivo labeling of  Pgm1p. (A) Labeling of wild-type and  ΔPGM1 cell lysates with [β-35S]UDP-Glc was conducted as described in Materials and Methods. The predominantly  labeled protein in wild-type (wt) lysates  (marked by *) has the mobility predicted  for Pgm1p. It is absent in labeled lysates  of ΔPGM1 cells. All other bands appear  to be breakdown products of Pgm1p as  they vary between experiments, have  faster electrophoretic mobilities than Pgm1p, and are absent in  ΔPGM1 lysates. (B) Excess unlabeled UDP-Glc specifically reduces labeling by [β-35S]UDP-Glc. Wild-type lysates were labeled with [β-35S]UDP-Glc alone (lane 1) or with unlabeled  UDP-Glc (lane 2), or unlabeled Glc-1-P (lane 3). (C) In vivo labeling of wild-type (wt) and ΔPGM1 cells with [32P]orthophosphate. A major phosphoprotein (marked by *) in wild-type cells,  of the mobility predicted for Pgm1p, is absent in ΔPGM1 cells. A  minor phosphoprotein of slightly lower mobility is observed in  both. The generally lower background in the ΔPGM1 cells was  reproducible, and may be due to a role of PGM metabolites in  macromolecular synthesis.
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Figure 7: In vitro and in vivo labeling of Pgm1p. (A) Labeling of wild-type and ΔPGM1 cell lysates with [β-35S]UDP-Glc was conducted as described in Materials and Methods. The predominantly labeled protein in wild-type (wt) lysates (marked by *) has the mobility predicted for Pgm1p. It is absent in labeled lysates of ΔPGM1 cells. All other bands appear to be breakdown products of Pgm1p as they vary between experiments, have faster electrophoretic mobilities than Pgm1p, and are absent in ΔPGM1 lysates. (B) Excess unlabeled UDP-Glc specifically reduces labeling by [β-35S]UDP-Glc. Wild-type lysates were labeled with [β-35S]UDP-Glc alone (lane 1) or with unlabeled UDP-Glc (lane 2), or unlabeled Glc-1-P (lane 3). (C) In vivo labeling of wild-type (wt) and ΔPGM1 cells with [32P]orthophosphate. A major phosphoprotein (marked by *) in wild-type cells, of the mobility predicted for Pgm1p, is absent in ΔPGM1 cells. A minor phosphoprotein of slightly lower mobility is observed in both. The generally lower background in the ΔPGM1 cells was reproducible, and may be due to a role of PGM metabolites in macromolecular synthesis.

Mentions: A well-characterized biochemical property of parafusin in Paramecium lysates is that it serves as the acceptor of a glucose phosphotransferase (Satir et al., 1990). To determine whether this property was shared by Tetrahymena Pgm1p, we prepared cell lysates and incubated them with the donor molecule for this reaction, [β-35S]UDP-glucose. A single band with the mobility predicted for Pgm1p was labeled in lysates prepared from wild-type cells (Fig. 7 A). In contrast, no band was labeled in parallel experiments done with ΔPGM1 lysates.


In vivo analysis of the major exocytosis-sensitive phosphoprotein in Tetrahymena.

Chilcoat ND, Turkewitz AP - J. Cell Biol. (1997)

In vitro and in vivo labeling of  Pgm1p. (A) Labeling of wild-type and  ΔPGM1 cell lysates with [β-35S]UDP-Glc was conducted as described in Materials and Methods. The predominantly  labeled protein in wild-type (wt) lysates  (marked by *) has the mobility predicted  for Pgm1p. It is absent in labeled lysates  of ΔPGM1 cells. All other bands appear  to be breakdown products of Pgm1p as  they vary between experiments, have  faster electrophoretic mobilities than Pgm1p, and are absent in  ΔPGM1 lysates. (B) Excess unlabeled UDP-Glc specifically reduces labeling by [β-35S]UDP-Glc. Wild-type lysates were labeled with [β-35S]UDP-Glc alone (lane 1) or with unlabeled  UDP-Glc (lane 2), or unlabeled Glc-1-P (lane 3). (C) In vivo labeling of wild-type (wt) and ΔPGM1 cells with [32P]orthophosphate. A major phosphoprotein (marked by *) in wild-type cells,  of the mobility predicted for Pgm1p, is absent in ΔPGM1 cells. A  minor phosphoprotein of slightly lower mobility is observed in  both. The generally lower background in the ΔPGM1 cells was  reproducible, and may be due to a role of PGM metabolites in  macromolecular synthesis.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2140215&req=5

Figure 7: In vitro and in vivo labeling of Pgm1p. (A) Labeling of wild-type and ΔPGM1 cell lysates with [β-35S]UDP-Glc was conducted as described in Materials and Methods. The predominantly labeled protein in wild-type (wt) lysates (marked by *) has the mobility predicted for Pgm1p. It is absent in labeled lysates of ΔPGM1 cells. All other bands appear to be breakdown products of Pgm1p as they vary between experiments, have faster electrophoretic mobilities than Pgm1p, and are absent in ΔPGM1 lysates. (B) Excess unlabeled UDP-Glc specifically reduces labeling by [β-35S]UDP-Glc. Wild-type lysates were labeled with [β-35S]UDP-Glc alone (lane 1) or with unlabeled UDP-Glc (lane 2), or unlabeled Glc-1-P (lane 3). (C) In vivo labeling of wild-type (wt) and ΔPGM1 cells with [32P]orthophosphate. A major phosphoprotein (marked by *) in wild-type cells, of the mobility predicted for Pgm1p, is absent in ΔPGM1 cells. A minor phosphoprotein of slightly lower mobility is observed in both. The generally lower background in the ΔPGM1 cells was reproducible, and may be due to a role of PGM metabolites in macromolecular synthesis.
Mentions: A well-characterized biochemical property of parafusin in Paramecium lysates is that it serves as the acceptor of a glucose phosphotransferase (Satir et al., 1990). To determine whether this property was shared by Tetrahymena Pgm1p, we prepared cell lysates and incubated them with the donor molecule for this reaction, [β-35S]UDP-glucose. A single band with the mobility predicted for Pgm1p was labeled in lysates prepared from wild-type cells (Fig. 7 A). In contrast, no band was labeled in parallel experiments done with ΔPGM1 lysates.

Bottom Line: Comparison of deduced protein sequences, taking advantage of the known atomic structure of rabbit muscle PGM, suggests that both ciliate enzymes and all other PGM-like proteins have PGM activity.Surprisingly, DeltaPGM1 cells displayed no detectable defect in exocytosis, but showed a dramatic decrease in PGM activity.Both our results, and reinterpretation of previous data, suggest that any potential role for PGM-like proteins in regulated exocytosis is unlikely to precede membrane fusion.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, Illinois 60637, USA.

ABSTRACT
Phosphoglucomutase (PGM) is a ubiquitous highly conserved enzyme involved in carbohydrate metabolism. A number of recently discovered PGM-like proteins in a variety of organisms have been proposed to function in processes other than metabolism. In addition, sequence analysis suggests that several of these may lack PGM enzymatic activity. The best studied PGM-like protein is parafusin, a major phosphoprotein in the ciliate Paramecium tetraurelia that undergoes rapid and massive dephosphorylation when cells undergo synchronous exocytosis of their dense-core secretory granules. Indirect genetic and biochemical evidence also supports a role in regulated exocytotic membrane fusion. To examine this matter directly, we have identified and cloned the parafusin homologue in Tetrahymena thermophila, a ciliate in which protein function can be studied in vivo. The unique T. thermophila gene, called PGM1, encodes a protein that is closely related to parafusin by sequence and by characteristic post-translational modifications. Comparison of deduced protein sequences, taking advantage of the known atomic structure of rabbit muscle PGM, suggests that both ciliate enzymes and all other PGM-like proteins have PGM activity. We evaluated the activity and function of PGM1 through gene disruption. Surprisingly, DeltaPGM1 cells displayed no detectable defect in exocytosis, but showed a dramatic decrease in PGM activity. Both our results, and reinterpretation of previous data, suggest that any potential role for PGM-like proteins in regulated exocytosis is unlikely to precede membrane fusion.

Show MeSH
Related in: MedlinePlus