A Yeast Killer Toxin Screen Provides Insights into A/B Toxin Entry, Trafficking, and Killing Mechanisms

Abstract

Summary 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.

New Regulators of Clathrin-Mediated Endocytosis Identified in Saccharomyces cerevisiae by Systematic Quantitative Fluorescence Microscopy

“…Thus, a majority of the known endocytic machinery proteins were located high in this ranking (Table S1 and Table S2). Interestingly, all four subunits of the AP-2 complex scored a colocalizing PCC, consistent with recent findings that AP-2 does in fact play a role in yeast CME (Carroll et al 2009;Chapa-Y-Lazo et al 2014). The lower range of the 197 colocalizing proteins was enriched in proteins spanning a variety of functions and containing predicted or known transmembrane domains.…”

“…Furthermore, additional CME machinery components may yet to be uncovered and their functions elucidated. Previous methods for identifying endocytic machinery proteins include knockout, synthetic lethality, cargo based, and drug sensitivity screenings (Burston et al 2009;Carroll et al 2009). These methods may miss proteins for various reasons.…”

Identification and Dynamics of Arabidopsis Adaptor Protein-2 Complex and Its Involvement in Floral Organ Development

“…By contrast, the budding yeast Saccharomyces cerevisiae lacking all three AP b-subunit genes, including the AP-2 homologs, exhibits no deleterious effects on cell growth (Yeung et al, 1999). The only role for the budding yeast AP-2 reported to date is in endocytosis of the killer toxin K28, whereas the internalization of other cargo proteins appears to be independent of AP-2 (Carroll et al, 2009). In the nematode Caenorhabditis elegans, complete loss and C-terminal deletion of the m-subunit have weak impacts on synaptic vesicle recycling, and the mutants are viable (Gu et al, 2008).…”

Endocytic Accessory Factors and Regulation of Clathrin-Mediated Endocytosis

“…In this model, the AP2 complex therefore acts as a coincidence detector directly linking PIP2, receptor cargo, and clathrin. Similarly, in yeast Ede1p (Eps15 homolog), clathrin, the AP2 complex, and Syp1p (FCHo homolog) are recruited early to nascent endocytic sites (Burston et al 2009;Carroll et al 2009;Reider et al 2009). Deleting genes for Syp1, AP2 complex, or Yap1801/2 does not cause defects in endocytic dynamics but these proteins are important for the internalization of certain cargo (Burston et al 2009;Carroll et al 2009;Reider et al 2009).…”

“…Similarly, in yeast Ede1p (Eps15 homolog), clathrin, the AP2 complex, and Syp1p (FCHo homolog) are recruited early to nascent endocytic sites (Burston et al 2009;Carroll et al 2009;Reider et al 2009). Deleting genes for Syp1, AP2 complex, or Yap1801/2 does not cause defects in endocytic dynamics but these proteins are important for the internalization of certain cargo (Burston et al 2009;Carroll et al 2009;Reider et al 2009). The yeast scaffold protein Ede1p (Eps15 homolog) was found necessary for recruitment of the majority of earlyarriving proteins barring the adaptor protein Yap1802p (Carroll et al 2012).…”