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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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Amino acid sequence alignment of PGMs. Residues differing from the consensus are lightly boxed. Residues located in the active site of the three-dimensional structure, which are predicted to be necessary for enzymatic function, are heavily boxed. Residues that  form a magnesium binding pocket in the active site are shaded. The active site phosphoserine is heavily boxed and shaded. Thick bars  mark four insertions and two deletions in parafusin, relative to mammalian PGMs, which were previously identified (Subramanian et al.,  1994). Sequences were aligned using Lasergene software (DNAStar, Madison, WI) and the clustal method with the PAM250 residue  weight table. Genes and Genbank accession codes are T. thermophila (PGM1, AF020726), P. tetraurelia (Parafusin, L12471), S. cerevisiae (GAL5, U09499), H. sapiens PGM1 (M83088), H. sapiens PGM5 (L40933), O. cuniculus PGM1 (M97663), and O. cuniculus PGM2  (M97664).
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Figure 4: Amino acid sequence alignment of PGMs. Residues differing from the consensus are lightly boxed. Residues located in the active site of the three-dimensional structure, which are predicted to be necessary for enzymatic function, are heavily boxed. Residues that form a magnesium binding pocket in the active site are shaded. The active site phosphoserine is heavily boxed and shaded. Thick bars mark four insertions and two deletions in parafusin, relative to mammalian PGMs, which were previously identified (Subramanian et al., 1994). Sequences were aligned using Lasergene software (DNAStar, Madison, WI) and the clustal method with the PAM250 residue weight table. Genes and Genbank accession codes are T. thermophila (PGM1, AF020726), P. tetraurelia (Parafusin, L12471), S. cerevisiae (GAL5, U09499), H. sapiens PGM1 (M83088), H. sapiens PGM5 (L40933), O. cuniculus PGM1 (M97663), and O. cuniculus PGM2 (M97664).

Mentions: Based on known codon usage in Tetrahymena thermophila (Martindale, 1989), degenerate primers (Integrated DNA Technologies, Coralville, IA) were designed to amplify parafusin homologues via the polymerase chain reaction. Primers were designed to correspond to regions conserved between GAL5 (Saccharomyces cerevisiae), PGM1 (Homo sapiens), and parafusin (P. tetraurelia). A total of four forward and four reverse primers were designed and used in all 16 possible combinations. The primers correspond to residues 126-133, 142-149, 321-327, 339-344, 388-384, 369-362, 410-403, and 427-421 of the deduced Pgm1p protein sequence (see Fig. 4). PCR was performed on a Tetrahymena thermophila λgt10 cDNA library generously provided by Tohru Takemasa (University of Tsukuba, Japan), or on Tetrahymena thermophila strain CU428.1 genomic DNA. The 50-μl reactions contained 108 plaque-forming units or 10 ng genomic DNA, 2.5 U Taq polymerase (Promega Corp., Madison, WI), 10 mM Tris-HCl pH 8.3, 50 mM KCl, 2.5 mM MgCl2, 250 μM each dNTP, and 1 μM each primer. 35 cycles of 92°C, 0.5 min; 40, 45, 50, or 55°C, 1 min; 72°C, 0.5 min, were performed. A total of 12 bands of the expected sizes were cloned into pCRII (Invitrogen, San Diego, CA) and sequenced; 5 products were unrelated to PGM, the other 6 (3 amplified from genomic DNA, 3 from cDNA, using 6 of the 8 primers in all) were related to various PGMs. These were determined to be identical in overlapping regions and derived from a single gene.


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

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

Amino acid sequence alignment of PGMs. Residues differing from the consensus are lightly boxed. Residues located in the active site of the three-dimensional structure, which are predicted to be necessary for enzymatic function, are heavily boxed. Residues that  form a magnesium binding pocket in the active site are shaded. The active site phosphoserine is heavily boxed and shaded. Thick bars  mark four insertions and two deletions in parafusin, relative to mammalian PGMs, which were previously identified (Subramanian et al.,  1994). Sequences were aligned using Lasergene software (DNAStar, Madison, WI) and the clustal method with the PAM250 residue  weight table. Genes and Genbank accession codes are T. thermophila (PGM1, AF020726), P. tetraurelia (Parafusin, L12471), S. cerevisiae (GAL5, U09499), H. sapiens PGM1 (M83088), H. sapiens PGM5 (L40933), O. cuniculus PGM1 (M97663), and O. cuniculus PGM2  (M97664).
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Related In: Results  -  Collection

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Figure 4: Amino acid sequence alignment of PGMs. Residues differing from the consensus are lightly boxed. Residues located in the active site of the three-dimensional structure, which are predicted to be necessary for enzymatic function, are heavily boxed. Residues that form a magnesium binding pocket in the active site are shaded. The active site phosphoserine is heavily boxed and shaded. Thick bars mark four insertions and two deletions in parafusin, relative to mammalian PGMs, which were previously identified (Subramanian et al., 1994). Sequences were aligned using Lasergene software (DNAStar, Madison, WI) and the clustal method with the PAM250 residue weight table. Genes and Genbank accession codes are T. thermophila (PGM1, AF020726), P. tetraurelia (Parafusin, L12471), S. cerevisiae (GAL5, U09499), H. sapiens PGM1 (M83088), H. sapiens PGM5 (L40933), O. cuniculus PGM1 (M97663), and O. cuniculus PGM2 (M97664).
Mentions: Based on known codon usage in Tetrahymena thermophila (Martindale, 1989), degenerate primers (Integrated DNA Technologies, Coralville, IA) were designed to amplify parafusin homologues via the polymerase chain reaction. Primers were designed to correspond to regions conserved between GAL5 (Saccharomyces cerevisiae), PGM1 (Homo sapiens), and parafusin (P. tetraurelia). A total of four forward and four reverse primers were designed and used in all 16 possible combinations. The primers correspond to residues 126-133, 142-149, 321-327, 339-344, 388-384, 369-362, 410-403, and 427-421 of the deduced Pgm1p protein sequence (see Fig. 4). PCR was performed on a Tetrahymena thermophila λgt10 cDNA library generously provided by Tohru Takemasa (University of Tsukuba, Japan), or on Tetrahymena thermophila strain CU428.1 genomic DNA. The 50-μl reactions contained 108 plaque-forming units or 10 ng genomic DNA, 2.5 U Taq polymerase (Promega Corp., Madison, WI), 10 mM Tris-HCl pH 8.3, 50 mM KCl, 2.5 mM MgCl2, 250 μM each dNTP, and 1 μM each primer. 35 cycles of 92°C, 0.5 min; 40, 45, 50, or 55°C, 1 min; 72°C, 0.5 min, were performed. A total of 12 bands of the expected sizes were cloned into pCRII (Invitrogen, San Diego, CA) and sequenced; 5 products were unrelated to PGM, the other 6 (3 amplified from genomic DNA, 3 from cDNA, using 6 of the 8 primers in all) were related to various PGMs. These were determined to be identical in overlapping regions and derived from 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