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 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.

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Francisella tularensis (FT) is a gram‐negative, highly infectious, facultative intracellular pathogen that causes tularemia, and is a potential bioterrorism agent. Macrophages represent the main site of initial FT replication but the mechanism of survival and bacteriostasis upon activation remains poorly characterized. We used genome‐wide genetic screen to identify host genes responsible for survival of infection. We used macrophages (RAW 264.7) transduced with an EST‐lentiviral "knock‐down" library targeting 40,000 sequences. The macrophages were infected with attenuated Live Vaccine Strain (LVS) FT expressing GFP. We screened for EST interference resulting in a phenotype that allows survival of infection and intracellular bacterial proliferation. Flow cytometric sorting of cells with highest FT load (i.e. top 1% by green fluorescence) was performed. The EST inserts from sorted cells were amplified from genomic DNA by PCR, and corresponding EST inserts were identified by cloning and DNA sequencing. Initial screening has analyzed 1400 clones from 6 different sorts and has identified 7 genes of interest based on repeated detection. Validation of potential targets is underway to confirm gene expression and to assess effects of directed lentiviral knockdown of candidate genes. This approach may prove useful to identify mechanisms in macrophage innate immunity against intracellular pathogens.