Abstract
K28 is a viral A/B protein toxin that intoxicates yeast and fungal cells by endocytosis and retrograde transport to the endoplasmic reticulum (ER). Although toxin translocation into the cytosol occurs on the oxidized α/β heterodimer, the precise mechanism of how the toxin crosses the ER membrane is unknown. Here we identify pH-triggered, toxin-intrinsic thiol rearrangements that crucially control toxin conformation and host cell killing. In the natural habitat and low-pH environment of toxin-secreting killer yeasts, K28 is structurally stable and biologically active as a disulfide-bonded heterodimer, whereas it forms inactive disulfide-bonded oligomers at neutral pH that are caused by activation and thiol deprotonation of β-subunit cysteines. Because such pH increase reflects the pH gradient during compartmental transport within target cells, potential K28 oligomerization in the ER lumen is prevented by protein disulfide isomerase. In addition, we show that pH-triggered thiol rearrangements in K28 can cause the release of cytotoxic α monomers, suggesting a toxin-intrinsic mechanism of disulfide bond reduction and α/β heterodimer dissociation in the cytosol.