Expression of K1 Toxin Derivatives in Saccharomyces cerevisiae Mimics Treatment with Exogenous Toxin and Provides a Useful Tool for Elucidating K1 Mechanisms of Action and Immunity

Abstract

Killer toxin K1 is a heterodimeric protein toxin secreted by Saccharomyces cerevisiae strains infected with the M1 double-stranded RNA ‘killer’ virus. After binding to a primary receptor at the level of the cell wall, K1 interacts with its secondary plasma membrane receptor Kre1p, eventually leading to an ionophoric disruption of membrane function. Although it has been under investigation for decades, neither the particular mechanisms leading to toxicity nor those leading to immunity have been elucidated. In this study, we constructed derivatives of the K1α subunit and expressed them in sensitive yeast cells. We show that these derivatives are able to mimic the action of externally applied K1 toxin in terms of growth inhibition and pore formation within the membrane, leading to a suicidal phenotype that could be abolished by co-expression of the toxin precursor, confirming a mechanistic similarity of external and internal toxin action. The derivatives were successfully used to investigate a null mutant completely resistant to externally applied toxin. They provide a valuable tool for the identification of so far unknown gene products involved in K1 toxin action and/or immunity.

Analysis of Yeast Killer Toxin K1 Precursor Processing via Site-Directed Mutagenesis: Implications for Toxicity and Immunity

“…Furthermore, we analyzed potential effects of the described mutations in the preproregion on the pore-forming abilities of the toxin. Corresponding to previously described lethal constructs consisting only of the toxic ␣ subunit, truncated K1 derivatives containing the different preproregions were obtained via PCR and expressed in the sensitive strain BY4742 (15). The potential impact of the mutations on the growth of the transformants (i.e., the suicidal phenotype) was analyzed via serial dilution assay FIG 1 Effects of mutations within the K1 preproregion on toxicity and the formation of immunity.…”

“…A reduction of the plasma membrane Kre1p population in K1 killer cells was suggested to confer immunity due to complexation of Kre1p and the K1 protoxin in the secretory pathway, leading to vacuolar degradation of this complex (17,18). However, we were recently able to show that the plasma membrane receptor Kre1p is not involved in the immunity mechanism by intracellularly expressing different K1 derivatives in a Δkre1 null mutation background (15). Although a previously conducted comprehensive mutational analysis of the toxin precursor was able to show the importance of the ␥ subunit in K1 immunity, neither the exact mechanism of the toxic effect nor the intrinsic immunity have been completely elucidated to date (19).…”

“…On a molecular level, this mechanism has only been elucidated for K28 and is essentially linked to the posttranslational import of the toxin precursor, which interacts with reinternalized mature toxin molecules in the cytosol, blocking their action (14). Based on the hydrophobicity of the K1 preregion, cotranslational ER import of the precursor molecule is proposed, which would exclude a comparable immunity mechanism; nevertheless, the preprotoxin and its import into the secretory pathway are critically involved in the formation of intrinsic immunity (15,16). A reduction of the plasma membrane Kre1p population in K1 killer cells was suggested to confer immunity due to complexation of Kre1p and the K1 protoxin in the secretory pathway, leading to vacuolar degradation of this complex (17,18).…”

Saccharomyces cerevisiae killer toxins: synthesis, mechanisms of action and practical use

“…Одновременная продукция протоксина приводила к выработке иммунитета у трансформантов против α-субъединицы К1, несмотря на то что α-субъединица не способна транспортироваться в ЭПР и, следовательно, не может взаимодействовать с предшественником токсина. Полученные результаты позволяют говорить о том, что в развитии иммунитета к К1 может принимать участие неидентифицированный белок-эффектор [34].…”

Killer Yeasts And Their Application

“…Prekursor kierowany jest do retikulum endoplazmatycznego, gdzie podlega glikozy-lacji, a następnie jest transportowany do aparatu Golgiego i modyfikowany z udziałem peptydazy. N-końcowy peptyd sygnałowy podlega hydrolizie po stronie karboksylowej Arg 29 . Oddzielnie kodowany jest prekursor podjednostki γ (ORF 4), zawierający N-końcowy peptyd sygnałowy, odcinany przez peptydazę podczas procesu dojrzewania toksyny [51].…”