title: Site-directed mutagenesis of the heterotrimeric killer toxin zymocin identifies residues required for early steps in toxin action.
abstract: Zymocin is a Kluyveromyces lactis protein toxin composed of αβγ subunits encoded by the cytoplasmic virus-like element k1 and functions by αβ-assisted delivery of the anticodon nuclease (ACNase) γ into target cells. The toxin binds to cells' chitin and exhibits chitinase activity in vitro that might be important during γ import. Saccharomyces cerevisiae strains carrying k1-derived hybrid elements deficient in either αβ (k1ORF2) or γ (k1ORF4) were generated. Loss of either gene abrogates toxicity, and unexpectedly, Orf2 secretion depends on Orf4 cosecretion. Functional zymocin assembly can be restored by nuclear expression of k1ORF2 or k1ORF4, providing an opportunity to conduct site-directed mutagenesis of holozymocin. Complementation required active site residues of α's chitinase domain and the sole cysteine residue of β (Cys250). Since βγ are reportedly disulfide linked, the requirement for the conserved γ C231 was probed. Toxicity of intracellularly expressed γ C231A indicated no major defect in ACNase activity, while complementation of k1ΔORF4 by γ C231A was lost, consistent with a role of β C250 and γ C231 in zymocin assembly. To test the capability of αβ to carry alternative cargos, the heterologous ACNase from Pichia acaciae (P. acaciae Orf2 [PaOrf2]) was expressed, along with its immunity gene, in k1ΔORF4. While efficient secretion of PaOrf2 was detected, suppression of the k1ΔORF4-derived k1Orf2 secretion defect was not observed. Thus, the dependency of k1Orf2 on k1Orf4 cosecretion needs to be overcome prior to studying αβ's capability to deliver other cargo proteins into target cells.
authors.affiliation: Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Münster, Germany.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Wemhoff
authors.affiliation: Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Münster, Germany.
authors.forename: Roland
authors.initials: R
authors.lastname: Klassen
authors.affiliation: Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Münster, Germany meinhar@uni-muenster.de.
authors.forename: Friedhelm
authors.initials: F
authors.lastname: Meinhardt
mesh_terms: D020134:Catalytic Domain; D002686:Chitin / Q000378:metabolism; D002688:Chitinases / Q000378:metabolism; D003545:Cysteine; D005816:Genetic Complementation Test; D055646:Killer Factors, Yeast / Q000235:genetics* / Q000378:metabolism; D007716:Kluyveromyces / Q000235:genetics*; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D021122:Protein Subunits; D012260:Ribonucleases / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics
chemical_list: D055646:Killer Factors, Yeast; D021122:Protein Subunits; C058975:zymocin; D002686:Chitin; D012260:Ribonucleases; C049345:anticodon nuclease; D002688:Chitinases; D003545:Cysteine