A Chemical Genomic Screen in Saccharomyces cerevisiae Reveals a Role for Diphthamidation of Translation Elongation Factor 2 in Inhibition of Protein Synthesis by Sordarin

Abstract

Sordarin and its derivatives are antifungal compounds of potential clinical interest. Despite the highly conserved nature of the fungal and mammalian protein synthesis machineries, sordarin is a selective inhibitor of protein synthesis in fungal organisms. In cells sensitive to sordarin, its mode of action is through preventing the release of translation elongation factor 2 (eEF2) during the translocation step, thus blocking protein synthesis. To further investigate the cellular components required for the effects of sordarin in fungal cells, we have used the haploid deletion collection of Saccharomyces cerevisiae to systematically identify genes whose deletion confers sensitivity or resistance to the compound. Our results indicate that genes in a number of cellular pathways previously unknown to play a role in sordarin response are involved in its growth effects on fungal cells and reveal a specific requirement for the diphthamidation pathway of cells in causing eEF2 to be sensitive to the effects of sordarin on protein synthesis. Our results underscore the importance of the powerful genomic tools developed in yeast (Saccharomyces cerevisiae) to more comprehensively understanding the cellular mechanisms involved in the response to therapeutic agents.

Diphtheria Disease and Genes Involved in Formation of Diphthamide, Key Effector of the Diphtheria Toxin

“…In an effort to further study diphthamide function and the interrelation between components of the diphthamide pathway, we found by co-immune precipitation and tandem affinity purification protocols that the Dph1, Dph2 and Dph3 factors form a protein complex, assembly of which is crucial for EF2 ADP-riboslyation by DT (Fichtner et al, 2003;Bär et al, 2008). Moreover, we and others discovered that the Dph1-Dph5 proteins are all required for the cytotoxic activity of sordarin Botet et al, 2008), another EF2-related antifungal and translation inhibitor (Justice et al, 1998). When using biotin-labeled NAD + as donor for the in vitro ADP ribosylation assay, wild-type strains display EF2 ADP-ribosylation acceptor activity (indicated by the arrow) whereas diphthamide mutans dph1 and dph5 fail to do so.…”

“…In addition, possible effector roles of YBR246w and YLR143w for EF2 specific antifungals including sordarin are becoming evident: when deleted, these new loci not only affect the communication between Dph5 and EF2 but also phenocopy traits (including sordarin resistance) that are typical of dph1, dph2, dph3, dph4 and dph5 mutants from yeast Botet et al, 2008;Carette et al, 2009). Although being aware that the sordarin phenotype may also be ascribable to defects in EF2-unrelated genes that are required for binding and/or import of the deadly antifungal (Botet et al, 2008), we consider these ORFs to be candidate diphthamide biosynthesis genes. In support of this notion, preliminary data based on in vitro EF2 modification assays demonstrate that inactivation of YBR246w and YLR143w eliminates the ADP-ribosylation acceptor activity of EF2 in the presence of DT.…”

“…8) but are also all resistant to growth inhibition by sordarin (Fig. 8) Botet et al, 2008). The latter is an ascomycetous glycoside (Hauser & Sigg, 1971) whose antifungal activity obviously depends on diphthamide, but that operates by selectively blocking the EF2-ribosome complex rather than inhibiting EF2 by ADP-ribosylation (Dominguez et al, 1999).…”

“…In a search for new diphthamide-related factors, two novel and uncharacterized open reading frames (ORFs), YBR246w and YLR143w, have been identified recently as new potential components for diphthamide biosynthesis using genetic screens in human and yeast cells (Botet et al, 2008;Carette et al, 2009). Based on synthetic genetic interaction data deposited at the genetic interaction database (GID; University of Toronto, Canada) (Baryshnikova et al, 2010) and the significance of the phenotypic correlation scores, both budding yeast ORFs are predicted to have EF2-related functions (Fig.…”

Functional analysis of the uL11 protein impact on translational machinery

“…42,43 In contrast, the eEF2 defect might affect the sensitivity toward another translation inhibitor, namely sordarin. 44 We showed that the uL11 protein is involved in the reading frame maintenance and translation fidelity. The lack of the uL11 protein on the ribosome induces both C1 and -1 PRF.…”