We demonstrate that a virally encoded yeast ‘killer’ toxin is entering its eukaryotic target cell by endocytosis, subsequently travelling the yeast secretory pathway in reverse to exhibit its lethal effect. The K28 killer toxin is a secreted α/β heterodimer that kills sensitive yeasts in a receptor‐mediated fashion by blocking DNA synthesis in the nucleus. In vivo processing of the toxin precursor results in a protein whose β‐C‐terminus carries the endoplasmic reticulum (ER) retention signal HDEL, which, as we show here, is essential for retrograde toxin transport. Yeast end3/4 mutants as well as cells lacking the HDEL receptor (Δerd2) or mutants defective in Golgi‐to‐ER protein recycling (erd1) are toxin resistant because the toxin can no longer enter and/or retrograde pass the cell. Site‐directed mutagenesis further indicated that the toxin's β‐HDEL motif ensures retrograde transport, although in a toxin‐secreting yeast the β‐C‐terminus is initially masked by an R residue (β‐HDELR) until Kex1p cleavage uncovers the toxin's targeting signal in a late Golgi compartment. Prevention of Kex1p processing results in high‐level secretion of a biologically inactive protein incapable of re‐entering the secretory pathway. Finally, we present evidence that ER‐to‐cytosol toxin export is mediated by the Sec61p translocon and requires functional copies of the lumenal ER chaperones Kar2p and Cne1p.

Toxin-secreting ''killer'' yeasts were initially identified >40 years ago in Saccharomyces cerevisiae strains infected with a doublestranded RNA ''killer'' virus. Despite extensive research conducted on yeast killer toxins, the mechanism of protecting immunity by which toxin-producing cells evade the lethal activities of these proteins has remained elusive. Here, we identify the mechanism leading to protecting immunity in a killer yeast secreting a viral ␣͞␤ protein toxin (K28) that enters susceptible cells by receptor-mediated endocytosis and, after retrograde transport into the cytosol, blocks DNA synthesis, resulting in both cell-cycle arrest and caspase-mediated apoptosis. We demonstrate that toxin immunity is effected within the cytosol of a toxin-secreting yeast and occurs via the formation of complexes between reinternalized toxin and unprocessed precursor moieties that are subsequently ubiquitinated and proteasomally degraded, eliminating the active form of the toxin. Interference with cellular ubiquitin homeostasis, either through overexpression of mutated ubiquitin (Ub-RR 48͞63 ) or by blocking deubiquitination, prevents ubiquitination of toxin and results in an impaired immunity and the expression of a suicidal phenotype. The results presented here reveal the uniquely elegant and efficient strategy that killer cells have developed to circumvent the lethal effects of the toxin they produce.␣͞␤ protein toxin ͉ preprotoxin ͉ ubiquitination T he killer phenomenon in yeast, originally discovered in certain killer strains of Saccharomyces cerevisiae in 1963, is associated with the secretion of a protein toxin that kills sensitive target cells in a receptor-mediated fashion without direct cell-to-cell contact (1, 2). In baker's yeast, the killer phenotype depends on the presence of cytoplasmic dsRNA killer viruses encoding the unprocessed toxin precursor protein (3). Until now, three killer toxins have been identified, of which K1 and K28 have been studied most extensively. Each killer toxin is translated as a preprotoxin (pptox) precursor that is imported into the yeast secretory pathway where it is processed to the mature and cytotoxic ␣͞␤ heterodimer, which is then released from the cell and secreted into the medium (4-6).Although the two ''prototypes'' of yeast killer toxins K1 and K28 exert their lethal effect in a receptor-mediated fashion, the mechanism of toxin-induced lethality is significantly different. K1 disrupts cytoplasmic membrane function, whereas K28 enters its target cell by receptor-mediated endocytosis and blocks DNA synthesis, leading to both G1͞S cell-cycle arrest and caspasemediated apoptosis (7,8). Both toxins initially bind to a primary receptor within the yeast cell wall and are then translocated to the plasma membrane, where they interact with a secondary toxin receptor (9, 10). The glycosylphosphatidylinositol-anchored cellsurface protein Kre1p has recently been identified as the plasma membrane receptor for K1 toxin, whereas the membrane receptor for K28 remains unknown (11). Af...

K28 killer strains of

SUMMARY Iron is an essential element for the growth of nearly all organisms. In order to overcome the problem of its low bioavailability, microorganisms (including fungi) secrete siderophores, high-affinity iron chelators. As the acquisition of iron is also a key step in infection processes, siderophores have been considered as potential virulence factors in several host-pathogen interactions. Most fungi produce siderophores of the hydroxamate-type, which are synthesized by non-ribosomal peptide synthetases (NRPSs). Magnaporthe grisea, the causal agent of rice blast disease, produces ferricrocin as intracellular storage siderophore and excretes coprogens. In the M. grisea genome we identified SSM1, an NRPS gene, and a gene encoding an l-ornithine N5-monooxygenase (OMO1) that is clustered with SSM1 and responsible for catalysing the first step in siderophore biosynthesis, the N(5) hydroxylation of ornithine. Disruption of SSM1 confirmed that the gene encodes ferricrocin synthetase. Pathogenicity of these mutants towards rice was reduced, suggesting a role of this siderophore in pathogenicity of M. grisea.

K28 is a viral A/B toxin that traverses eukaryotic cells by endocytosis and retrograde transport through the secretory pathway. Here we show that toxin retrotranslocation from the endoplasmic reticulum (ER) requires Kar2p/ BiP, Pdi1p, Scj1p, Jem1p, and proper maintenance of Ca 2 þ homeostasis. Neither cytosolic chaperones nor Cdc48p/ Ufd1p/Npl4p complex components or proteasome activity are required for ER exit, indicating that K28 retrotranslocation is mechanistically different from classical ER-associated protein degradation (ERAD). We demonstrate that K28 exits the ER in a heterodimeric but unfolded conformation and dissociates into its subunits as it emerges into the cytosol where b is ubiquitinated and degraded. ER export and in vivo toxicity were not affected in a lysinefree K28 variant nor under conditions when ubiquitination and proteasome activity was blocked. In contrast, toxin uptake from the plasma membrane required Ubc4p (E2) and Rsp5p (E3) and intoxicated ubc4 and rsp5 mutants accumulate K28 at the cell surface incapable of toxin internalization. We propose a model in which ubiquitination is involved in the endocytic pathway of the toxin, while ER-to-cytosol retrotranslocation is independent of ubiquitination, ERAD and proteasome activity.