The K28 toxin of S. cerevisiae is a secreted alpha/beta
heterodimeric protein that kills sensitive cells in a
receptor-mediated fashion by blocking DNA synthesis. In vivo
processing of the toxin precursor results in a protein whose beta
C-terminus carries the ER retention signal HDEL. Yeast
end3/end4 mutants as well as cells lacking the HDEL-receptor
Erd2p or mutants defective in Golgi-to-ER protein recycling are
toxin-resistant since the toxin can no longer enter and/or retrograde
pass the cell. Site-directed mutagenesis of the preprotoxin gene
indicated that the beta-HDEL-motif ensures retrograde transport,
although in a toxin-secreting yeast the beta C-terminus is initially
masked by an R residue until Kex1p cleavage uncovers the toxin's
targeting signal in a late Golgi compartment. Prevention of Kex1p
cleavage results in high level secretion of an inactive protein
uncapable to re-enter the yeast secretion pathway. We present evidence
that once the toxin has reached the ER lumen, retranslocation into the
cytosol is mediated by Kar2p (BiP), Cne1p (calnexin), and the ER
translocon Sec61p. Within the cytosol, the toxin is interacting with
so far unknown proteins, thereby transmitting its toxic signal into
the nucleus. Results of two-hybrid screens identified TBP and PRP42 as
target proteins which interact with the cytotoxic alpha-subunit. This
work was kindly supported by grants from the Deutsche
Forschungsgemeinschaft (Schm 541/3-3 and SFB399, project B10).
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