K28 killer strains of Saccharomyces cerevisiae are permanently infected with a cytoplasmic persisting dsRNA virus encoding a secreted alpha/beta heterodimeric protein toxin that kills sensitive cells by cell-cycle arrest and inhibition of DNA synthesis. In vivo processing of the 345 aa toxin precursor (preprotoxin; pptox) involves multiple internal and carboxy-terminal cleavage events by the prohormone convertases Kex2p and Kex1p. By site-directed mutagenesis of the preprotoxin gene and phenotypic analysis of its in vivo effects it is now demonstrated that secretion of a biological active virus toxin requires signal peptidase cleavage after Gly(36) and Kex2p-mediated processing at the alpha subunit N terminus (after Glu-Arg(49)), the alpha subunit C terminus (after Ser-Arg(149)) and at the beta subunit N terminus (after Lys-Arg(245)). The mature C terminus of the beta subunit is trimmed by Kex1p, which removes the terminal Arg(345) residue, thus uncovering the toxin's endoplasmic reticulum targeting signal (HDEL) which--in a sensitive target cell--is essential for retrograde toxin transport. Interestingly, both toxin subunits are covalently linked by a single disulfide bond between alpha-Cys(56) and beta-Cys(340), and expression of a mutant toxin in which beta-Cys(340) had been replaced by Ser(340) resulted in the secretion of a non-toxic alpha/beta heterodimer that is blocked in retrograde transport and incapable of entering the yeast cell cytosol, indicating that one important in vivo function of beta-Cys(340) might be to ensure accessibility of the toxin's beta subunit C terminus to the HDEL receptor of the target cell.
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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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