Immunity factors for two related tRNAGln targeting killer toxins distinguish cognate and non-cognate toxic subunits

Abstract

The cytoplasmic virus-like element pWR1A from Debaryomyces robertsiae encodes a toxin (DrT) with similarities to the Pichia acaciae killer toxin PaT, which acts by importing a toxin subunit (PaOrf2) with tRNA anticodon nuclease activity into target cells. As for PaT, loss of the tRNA methyltransferase Trm9 or overexpression of tRNA(Gln) increases DrT resistance and the amount of tRNA(Gln) is reduced upon toxin exposure or upon induced intracellular expression of the toxic DrT subunit gene DrORF3, indicating DrT and PaT to share the same in vivo target. Consistent with a specific tRNase activity of DrOrf3, the protein cleaves tRNA(Gln) but not tRNA(Glu) in vitro. Heterologous cytoplasmic expression identified DrOrf5 as the DrT specific immunity factor; it confers resistance to exogenous DrT as well as to intracellular expression of DrOrf3 and prevents tRNA depletion by the latter. The PaT immunity factor PaOrf4, a homologue of DrOrf5 disables intracellular action of both toxins. However, the DrT protection level mediated by PaOrf4 is reduced compared to DrOrf5, implying a recognition mechanism for the cognate toxic subunit, leading to incomplete toxicity suppression of similar, but non-cognate toxic subunits.

Autoselection of Cytoplasmic Yeast Virus Like Elements Encoding Toxin/Antitoxin Systems Involves a Nuclear Barrier for Immunity Gene Expression

“…For the three known VLE encoded ACNase toxin complexes PaT, zymocin and DrT, immunity functions were proposed to be encoded by PaORF4 , KlORF3 and DrORF5 , respectively [ 16 , 17 , 20 ]. Subsequently, PaORF4 and DrORF5 were functionally expressed from their native promoters in the cytoplasm after integration of the genes into a VLE system (the pGKL1/2 system transferred to S .…”

“…There is hardly any homology among PaORF4 and KlORF3 , and consistent with it, no cross-protection has been observed against zymocin or PaT [ 16 ]. PaT and zymocin are the most thoroughly studied VLE encoded ACNase killer toxins, but there are other systems in yeast [reviewed in 18 ], such as PiT from Pichia inositovora , a ribonuclease inducing specific fragmentation of 25S and 18S rRNAs [ 19 ], or DrT from Debaryomyces robertsiae , an ACNase resembling PaT and cleaving tRNA Gln [ 20 ]. From an evolutionary point of view, toxin and immunity functions implemented in VLEs have to be considered as players of an autoselection system rather than providing advantages for the respective host [ 16 , 21 ], although the latter, clearly benefits from the conferred killer phenotype.…”

DNA damage induced by the anticodon nuclease from aPichia acaciaekiller strain is linked to ribonucleotide reductase depletion

“…Toxins are either chromosomally encoded or associated with extranuclear elements, such as dsRNA containing virus-like particles (VLPs) or dsDNA virus-like elements (VLEs) (for reviews see Magliani et al, 1997;Schmitt and Breinig, 2002;Klassen and Meinhardt, 2009). In case of the VLEs, specific immunity factors are genetically coupled with toxin production, providing a natural autoselection system, which enables the stable, long-term propagation of such extranuclear elements (Paluszynski et al, 2007;Klassen et al, 2014;Kast et al, 2015). With the exception of Pichia inositovora, the latter strategy applies for killer strains of Pichia acaciae, Kluyveromyces lactis and Debaryomyces robertsiae (Gunge et al, 1981;Ligon et al, 1989;Worsham and Bolen, 1990;Cong et al, 1994;Klassen and Meinhardt, 2002;.…”

“…With the exception of Pichia inositovora, the latter strategy applies for killer strains of Pichia acaciae, Kluyveromyces lactis and Debaryomyces robertsiae (Gunge et al, 1981;Ligon et al, 1989;Worsham and Bolen, 1990;Cong et al, 1994;Klassen and Meinhardt, 2002;. In target cells, the VLEencoded toxin of P. inositovora attacks ribosomal RNA (Kast et al, 2014), while killer toxins of K. lactis and P. acaciae target specific transfer RNAs (Lu et al, 2005;Klassen et al, 2008;Satwika et al, 2012). The secreted ribotoxins of P. acaciae and K. lactis, termed PaT and zymocin respectively, are heteromeric glycoproteins.…”

Site-Directed Mutagenesis of the Heterotrimeric Killer Toxin Zymocin Identifies Residues Required for Early Steps in Toxin Action

“…3A and 5A), we wondered if the ␣␤ subunits carry and deliver not only their cognate cargo protein but a heterologous subunit of another known killer system. We utilized the k1⌬ORF4 strain to introduce the toxic ACNase subunit (PaOrf2) from the related Pichia acaciae killer system along with its immunity gene (PaOrf4) (15,36). The last two were expressed cytoplasmically using the in vitro-constructed element TU-PO4PO2-T (see Materials and Methods), and the presence of the linear elements was verified by gel electrophoresis (Fig.…”