The carboxyl-terminal sequences of the $ alpha$ and $ beta$ subunits of the secreted yeast K1 toxin have been determined by protein sequencing and amino acid analysis of peptide fragments generated from the purified toxin. It revealed that the $ alpha$ and $ beta$ subunits consist of amino acid residues 45-147 and 236-316 from the preprotoxin, respectively. The preprotoxin configuration can be represented as: prepropeptide-ArgPro-$ alpha$-ArgArg-$ gamma$-LysArg-$ beta$. This structure for the preprotoxin defines a specific processing pathway in yeast involving a dibasic endoprotease, encoded by the KEX2 gene and a carboxypeptidase B like enzyme which is probably encoded by the KEX1 gene. / By using the patch-clamp technique, it is shown both in vivo with sensitive yeast spheroplasts and in vitro with asolectin liposomes that the toxin forms ion permeable channels. The toxin induced ion channels are voltage independent with a unit conductance of 118pS, often appearing in pairs and prefer monovalent cations. / K1 toxin has a much wider killing spectrum at the spheroplast level than at the whole cell level as demonstrated by the fact that the toxin kills spheroplasts from the genera Candida, Kluyveromyces, and Schwanniomyces, whose cells are toxin insensitive. A toxin binding study shows that the wall receptor can define toxin specificity and is necessary but not sufficient for toxin action on intact cells. / Using various mutagenesis techniques, a set of mutations throughout regions encoding the $ alpha$ and $ beta$ subunits that allow secretion of mutant toxins were generated. By analyzing the phenotypes of these mutant toxins, the ion channel forming domain is assigned exclusively to the hydrophobic $ alpha$ subunit and the cell wall receptor binding domain is localized to both the $ alpha$ and $ beta$ subunits.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.74623 |
| Date | January 1990 |
| Creators | Zhu, Hong, 1963- |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Biology.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001234359, proquestno: AAINN67664, Theses scanned by UMI/ProQuest. |
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