Immunity to yeast killer toxin K28: interaction of retrograde internalised toxin with the preprotoxin in the cytosol.
Frank Breinig, Tanja Sendzik, Manfred Schmitt
Applied Molecular Biology, Saarland University, Building 2, Saarbruecken, 66123, Germany
Killer toxin K28 is an alpha/beta heterodimeric protein toxin of the A/B toxin family that is secreted by Saccharomyces cerevisiae killer strains infected with the M28 double-stranded RNA virus. The toxin enters the cytosol of a sensitive target cell by travelling the secretion pathway in reverse and inhibits DNA synthesis in the nucleus leading to a cell cycle arrest at the G1/S boundary and final cell death. Here, we show that toxin immunity does not affect uptake of external toxin as the amount of internalised toxin can be increased by the addition of exogenous toxin. Furthermore, both the unprocessed preprotoxin (pptox) and the internalised and retrograde transported K28 dimer are detectable in the cytosol of a killer cell, indicating posttranslational pptox import and conformational stability of the alpha/beta heterodimer. Almost complete immunity is conferred by intracellular expression of the alpha subunit whereas complete protection requires a sequence-unspecific C-terminal extension of alpha. Here, we identified a 'intelligent' strategy of killer cells to protect themselves against their own toxin: immunity takes place within the cytosol and is based on the interaction and complexation of the reinternalised heterodimeric toxin and the unprocessed pptox precursor, yielding a pptox/K28 complex that is rapidly ubiquitinated and degraded by the proteasome. Thus, killer cells can effectively resist their own toxin.
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