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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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Genomic structure of PGM1 and Southern and Northern analysis of ΔPGM1 transformants. (A) neo2, which confers  paromomycin resistance in Tetrahymena (Gaertig et al., 1994;  Chilcoat et al., 1996), was used to replace the 5'-most 80% of the  PGM1 gene as shown. (B) Genomic DNA was prepared from  wild-type and ΔPGM1 transformants and digested with XbaI.  Southern blotting using a probe made from a region upstream of  the PGM1 gene (probe A) was performed as described in Materials and Methods. Successful targeting resulted in elimination of a  band at 4.9 kB, concurrent with the appearance of a band at 2.7 kB.  Upon overexposure, the ∼7 kB micronuclear allele could be seen  in all lanes. (C) Northern blotting was performed on total RNA  prepared from wild-type (wt) or ΔPGM1 strains using a PGM1  cDNA probe (probe B), and a probe derived from GRL1 cDNA  (Chilcoat et al., 1996). Ethidium bromide staining of rRNA is  also shown. ΔPGM1 cells contain normal levels of GRL1 transcript and total RNA, but have no detectable PGM1 transcript.
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Figure 6: Genomic structure of PGM1 and Southern and Northern analysis of ΔPGM1 transformants. (A) neo2, which confers paromomycin resistance in Tetrahymena (Gaertig et al., 1994; Chilcoat et al., 1996), was used to replace the 5'-most 80% of the PGM1 gene as shown. (B) Genomic DNA was prepared from wild-type and ΔPGM1 transformants and digested with XbaI. Southern blotting using a probe made from a region upstream of the PGM1 gene (probe A) was performed as described in Materials and Methods. Successful targeting resulted in elimination of a band at 4.9 kB, concurrent with the appearance of a band at 2.7 kB. Upon overexposure, the ∼7 kB micronuclear allele could be seen in all lanes. (C) Northern blotting was performed on total RNA prepared from wild-type (wt) or ΔPGM1 strains using a PGM1 cDNA probe (probe B), and a probe derived from GRL1 cDNA (Chilcoat et al., 1996). Ethidium bromide staining of rRNA is also shown. ΔPGM1 cells contain normal levels of GRL1 transcript and total RNA, but have no detectable PGM1 transcript.

Mentions: To clone the gene encoding the putative homologue as well as any related proteins, we identified protein sequences conserved between Paramecium parafusin, human PGM, and yeast PGM, and designed eight degenerate PCR primers corresponding to those regions. PCR amplification using combinations of these primers, in conjunction with Tetrahymena cDNA or genomic DNA, generated six products encoding a PGM-like protein. Each overlapped with all of the others, and overlapping sequences were identical, indicating that they were derived from a single gene. The largest product hybridized to a 1.7-kB clone from a cDNA library, whose sequence was identical to that of the gene identified by PCR. We extended the search for potential homologues with low stringency Southern blots of genomic DNA, using probes made both from S. cerevisiae PGM (GAL5) as well as the isolated Tetrahymena cDNA clone. Both probes detected a single macronuclear gene (see Fig. 3). Based on these lines of evidence, it appears that Tetrahymena has only a single gene homologous to PGM and parafusin, which we named PGM1 for reasons described below. Northern blots (see Fig. 6 C) were also consistent with a single gene.


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

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

Genomic structure of PGM1 and Southern and Northern analysis of ΔPGM1 transformants. (A) neo2, which confers  paromomycin resistance in Tetrahymena (Gaertig et al., 1994;  Chilcoat et al., 1996), was used to replace the 5'-most 80% of the  PGM1 gene as shown. (B) Genomic DNA was prepared from  wild-type and ΔPGM1 transformants and digested with XbaI.  Southern blotting using a probe made from a region upstream of  the PGM1 gene (probe A) was performed as described in Materials and Methods. Successful targeting resulted in elimination of a  band at 4.9 kB, concurrent with the appearance of a band at 2.7 kB.  Upon overexposure, the ∼7 kB micronuclear allele could be seen  in all lanes. (C) Northern blotting was performed on total RNA  prepared from wild-type (wt) or ΔPGM1 strains using a PGM1  cDNA probe (probe B), and a probe derived from GRL1 cDNA  (Chilcoat et al., 1996). Ethidium bromide staining of rRNA is  also shown. ΔPGM1 cells contain normal levels of GRL1 transcript and total RNA, but have no detectable PGM1 transcript.
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Related In: Results  -  Collection

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Figure 6: Genomic structure of PGM1 and Southern and Northern analysis of ΔPGM1 transformants. (A) neo2, which confers paromomycin resistance in Tetrahymena (Gaertig et al., 1994; Chilcoat et al., 1996), was used to replace the 5'-most 80% of the PGM1 gene as shown. (B) Genomic DNA was prepared from wild-type and ΔPGM1 transformants and digested with XbaI. Southern blotting using a probe made from a region upstream of the PGM1 gene (probe A) was performed as described in Materials and Methods. Successful targeting resulted in elimination of a band at 4.9 kB, concurrent with the appearance of a band at 2.7 kB. Upon overexposure, the ∼7 kB micronuclear allele could be seen in all lanes. (C) Northern blotting was performed on total RNA prepared from wild-type (wt) or ΔPGM1 strains using a PGM1 cDNA probe (probe B), and a probe derived from GRL1 cDNA (Chilcoat et al., 1996). Ethidium bromide staining of rRNA is also shown. ΔPGM1 cells contain normal levels of GRL1 transcript and total RNA, but have no detectable PGM1 transcript.
Mentions: To clone the gene encoding the putative homologue as well as any related proteins, we identified protein sequences conserved between Paramecium parafusin, human PGM, and yeast PGM, and designed eight degenerate PCR primers corresponding to those regions. PCR amplification using combinations of these primers, in conjunction with Tetrahymena cDNA or genomic DNA, generated six products encoding a PGM-like protein. Each overlapped with all of the others, and overlapping sequences were identical, indicating that they were derived from a single gene. The largest product hybridized to a 1.7-kB clone from a cDNA library, whose sequence was identical to that of the gene identified by PCR. We extended the search for potential homologues with low stringency Southern blots of genomic DNA, using probes made both from S. cerevisiae PGM (GAL5) as well as the isolated Tetrahymena cDNA clone. Both probes detected a single macronuclear gene (see Fig. 3). Based on these lines of evidence, it appears that Tetrahymena has only a single gene homologous to PGM and parafusin, which we named PGM1 for reasons described below. Northern blots (see Fig. 6 C) were also consistent with a single gene.

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