In vivo analysis of the major exocytosis-sensitive phosphoprotein in Tetrahymena.
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.
Chilcoat
N D
ND
Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, Illinois 60637, USA.
Turkewitz
A P
AP
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018076:DNA, Complementary / Q000235:genetics; D005089:Exocytosis; D005455:Fluorescent Antibody Technique; D017125:Genes, Protozoan; D018503:Genome, Protozoan; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010733:Phosphoglucomutase / Q000235:genetics* / Q000302:isolation & purification; D010750:Phosphoproteins / Q000235:genetics* / Q000302:isolation & purification; D015800:Protozoan Proteins / Q000235:genetics* / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D016808:Tetrahymena thermophila / Q000235:genetics*
D018076:DNA, Complementary; D010750:Phosphoproteins; D015800:Protozoan Proteins; D010733:Phosphoglucomutase