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. 2009 Oct;17(4):552-60.
doi: 10.1016/j.devcel.2009.08.006.

A yeast killer toxin screen provides insights into a/b toxin entry, trafficking, and killing mechanisms

Susheela Y Carroll  1 , Peter C Stirling, Helen E M Stimpson, Esther Giesselmann, Manfred J Schmitt, David G Drubin
Affiliations

A yeast killer toxin screen provides insights into a/b toxin entry, trafficking, and killing mechanisms

Susheela Y Carroll et al. Dev Cell. 2009 Oct.

Abstract

Like Ricin, Shiga, and Cholera toxins, yeast K28 is an A/B toxin that depends on endocytosis and retrograde trafficking for toxicity. Knowledge of the specific proteins, lipids, and mechanisms required for trafficking and killing by these toxins remains incomplete. Since K28 is a model for clinically relevant toxins, we screened over 5000 yeast mutants, identifying 365 that affect K28 sensitivity. Hypersensitive mutants revealed cytoprotective pathways, including stress-activated signaling and protein degradation. Resistant mutants clustered to endocytic, lipid organization, and cell wall biogenesis pathways. Furthermore, GPI anchors and transcriptional regulation are important for K28-cell binding. Strikingly, the AP2 complex, which in metazoans links endocytic cargo to the clathrin coat, but had no assigned function in yeast, was critical for K28 toxicity. Yeast AP2 localizes to endocytic sites and has a cargo-specific function in K28 uptake. This comprehensive genetic analysis identified conserved processes important for A/B toxin trafficking and killing.

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Figures

Figure 1
Figure 1
A genome-wide screen for K28 resistance or hypersensitivity. (A) Examples of killer assay results for BY4742 (WT), hypersensitive 192.2d and resistant sla2Δ strains. Arrows represent halo measurements. Scale bar, 5 mm. (B and C) Manually annotated groupings of K28 resistant (B) and hypersensitive (C) mutants. (D and E) Statistically enriched gene ontology (GO) terms among K28 resistant (D) and hypersensitive (E) mutations.
Figure 2
Figure 2
Genes from selected complexes and pathways implicated in K28 resistance or hypersensitivity. Osprey generated network diagrams of genes related to (A) cell wall and lipid biogenesis, (B) vesicular trafficking, and (C) the regulation of gene expression. Yellow and orange circles represent K28 resistant deletions or ts-alleles, respectively, and light blue and dark blue circles represent K28 hypersensitive deletions or ts-alleles, respectively. Grey lines depict the K28 phenotype while red lines depict published physical, functional or genetic interactions within a subgroup of genes.
Figure 3
Figure 3
K28 binding defects underlie a proportion of toxin resistant mutations. Relative toxin activity remaining after depletion of K28 containing culture supernatant with wild-type or mutant cells. Shown are the mean values of at least three experiments for mutants that deplete toxin activity significantly more poorly than wild-type (p <0.01). Error bars indicate standard error of the mean.
Figure 4
Figure 4
Yeast AP2 subunits are endocytic coat proteins. (A) Killer assays of BY4742 (WT), a control endocytic mutant, and AP2 subunit deletions. (B) Yeast expressing Apl1-GFP or Apm4-GFP. Kymographs of the patches indicated by the arrows from movies of cells expressing the GFP-tagged proteins. (C) Yeast expressing Apl1-GFP and Abp1-mRFP or Sla1-mCherry. Kymographs and montages of single patches indicated by the arrows from two-color movies of cells expressing the indicated proteins. (D) Wild-type (WT) or apl1Δ yeast expressing Apm4-GFP. (E) Network diagram of the endocytic mutants identified in our screen. Grey lines depict the K28 phenotype and red lines depict published interactions within a subgroup of genes. (F) Subcellular fractionation for the indicated strains treated with K28. Fractions were probed with the indicated antibodies by Western blotting. P13 = 13,000 g pellet; P100 = 100,000 g pellet; S100 = 100,000 g supernatant. (G) Western blots of K28 remaining in cell-free culture supernatant after incubation of spheroplasts with K28 over time. Scale bars, 4 μm.
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References

    1. Amberg DC, Amberg DC, Burke D, Strathern JN, Burke D, Cold Spring Harbor Laboratory . Methods in yeast genetics: a Cold Spring Harbor Laboratory course manual, 2005. Cold Spring Harbor Laboratory Press; Cold Spring Harbor, N.Y.: 2005.
    1. Beh CT, Rine J. A role for yeast oxysterol-binding protein homologs in endocytosis and in the maintenance of intracellular sterol-lipid distribution. J Cell Sci. 2004;117:2983–2996. - PubMed
    1. Ben-Aroya S, Coombes C, Kwok T, O'Donnell KA, Boeke JD, Hieter P. Toward a comprehensive temperature-sensitive mutant repository of the essential genes of Saccharomyces cerevisiae. Mol Cell. 2008;30:248–258. - PMC - PubMed
    1. Boyle EI, Weng S, Gollub J, Jin H, Botstein D, Cherry JM, Sherlock G. GO::TermFinder--open source software for accessing Gene Ontology information and finding significantly enriched Gene Ontology terms associated with a list of genes. Bioinformatics. 2004;20:3710–3715. - PMC - PubMed
    1. Breinig F, Sendzik T, Eisfeld K, Schmitt MJ. Dissecting toxin immunity in virus-infected killer yeast uncovers an intrinsic strategy of self-protection. Proc Natl Acad Sci U S A. 2006;103:3810–3815. - PMC - PubMed

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