Previous work carried out in our laboratory (Burlini, N., Lamponi S., Radrizzani, M., Monti, E. and Tortora P. (1987) Biochim. Biophys. Acta 930, 220-229) led to the immunological identification of a yeast 65-kDa phosphoprotein as a modified form of phosphoenolpyruvate carboxykinase; moreover the appearance of this phospho form was proven to be independent of cAMP, whereas the glucose-induced inactivation of the native enzyme is cAMP-dependent. Here, we report further investigations on the mechanism of the glucose-triggered degradation of the enzyme which led to the following results: (a) the aforementioned phospho form displayed a binding pattern to 5 AMP-Sepharose 4B quite similar to that of native enzyme, although it did not retain its oligomeric structure, nor was it catalytically active; (b) its phosphate content was of about two residues per monomer; (c) its isoelectric point was slightly higher than that of native enzyme, this shows that the enzyme undergoes additional modifications besides phosphorylation; (d) it represented about 4% of the native enzyme in glucose-depressed cells; (e) other forms immunologically cross-reactive with the native enzyme were also isolated, whose molecular mass was in the range of 60-62 kDa, and they are probable candidates as degradation products of the phospho form; (f) time courses of the native and phospho forms in the presence and the absence of glucose provided data consistent with a kinetic model involving a strong stimulation of the decay of both forms effected by the sugar; (g) in the mutant ABYS1 (Achstetter, T., Emter, O., Ehmann, C. and Wolf, D.H. (1984) J. Biol. Chem. 259, 13334-13343) which is devoid of the four major vacuolar proteinases, the decay pattern was essentially the same as in wild-type; (h) effectors lowering intracellular ATP also retarded the first step of enzyme degradation; this points to an ATP-dependence of this step. Based on these results we propose a degradation mechanism consisting of an initial cAMP- and ATP-dependent modification of the enzyme, followed by a cAMP-independent phosphorylation, which leads to the appearance of the aforementioned monomeric phospho form; this in turn seems to undergo limited proteolysis. These data strongly suggest the occurrence of an intermediate form arising from the native one and whose phosphorylation gives rise to the 65-kDa phosphoprotein described here.
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Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Phenotype | Experiment Type | Experiment Type Category | Mutant Information | Strain Background | Chemical | Details | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Regulator | Regulator Systematic Name | Target | Target Systematic Name | Direction | Regulation of | Happens During | Regulator Type | Direction | Regulation Of | Happens During | Method | Evidence | Strain Background | Reference |
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Site | Modification | Modifier | Source | Reference |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Assay | Annotation | Action | Modification | Source | Reference | Note |
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Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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