Killer yeasts

Introduction

Some yeast strains kill competitors of the own but also of different species by secreting protein toxins into the growth medium (Fig. 1).

Fig. 1: A killer yeast prevents the growth of sensitive cells by secreting a toxin.

The toxin encoding genes can be located on the yeast chromosomes, but also on dsRNA virus-like particles (VLPs) or on the dsDNA of virus-like elements (VLEs, Fig. 2). Our focus is on the toxins of the latter group, in which four killer strains are known (Tab. 1).

Fig. 2: Genomic organization of the VLEs pPac1-1 and pPac1-2 of Pichia acaciae. ORFs are depicted as arrows. Black triangles: terminal inverted repeat (TIR), Black circles: terminal protein (TP), SSB: single-strand binding protein, TRF: terminal recognition factor, (Fig. modified from Klassen & Meinhardt, 2007).

Table 1: Yeast VLE mediated killer phenotypes.

Killer strain	Toxin	VLEs	Size	References
Kluyveromyces lactis	Zymocin	pGKL1 pGKL2	8,9 kb 13,5 kb	Gunge et al., 1981 Lu et al., 2005
Pichia acaciae	PaT	pPac1-2 pPac1-1	6,8 kb 13,6 kb	Worsham & Bolen, 1990 Klassen et al., 2004 Klassen et al., 2008 Jeske & Meinhardt, 2006
Debaryomyces robertsiae	DrT	pWR1A pWR1B	8,0 kb 14,6 kb	Cong et al., 1994 Klassen & Meinhardt, 2002
Pichia inositovora	PiT	pPin1-3 pPin1-2 pPin1-1	10 kb 13 kb 18 kb	Hayman & Bolen, 1991 Klassen & Meinhardt, 2003 Kast et al., 2014

The structure of VLEs

VLEs (originally called linear plasmids) are located in the cytoplasm. Since the replication and transcription machinery is located in the nucleus, VLEs have to ensure extranuclear autonomous replication and transcription. Indeed, the larger elements are highly conserved in the respective strains and encode all the functions necessary for autonomous cytoplasmic replication and maintenance, such as a DNA polymerase, an RNA polymerase, a helicase, and single-strand binding proteins (Fig. 2). Some proteins as well as the replication mechanism display similarities to adenoviruses thereby pointing at the viral origin of these DNA elements. The smaller VLEs depend on the larger ones; their sequence greatly varies in different yeasts. They encode the toxin and the appropriate immunity. The immunity factor rescues the cells from their own toxin; the immunity mechanism still remains obscure. Together with the toxins, the immunity proteins assure the autoselection of the VLEs. Once a cell loses its VLEs and concomitantly the immunity, it will be killed by the toxin secreted by neighbour cells (Satwika et al., 2012).

The toxin structure

The VLE encoded toxins are heteromeric protein complexes. One of the subunits is highly conserved among all the toxins. It recognizes the chitin of target cells and - together with a highly hydrophobic subunit - manages uptake of the actual toxic subunit into target cells.

The intracellular toxic subunit of zymocin from K. lactis shows only faint sequence similarity to PiT from P. inositovora and but none to PaT from P. acaciae. The protein of D. robertsiae displays a remarkable homology to PaT, but not to zymocin.

The molecular mechanism of toxicity

The toxic mechanism is well known for zymocin and PaT. After binding to the chitin of the target cell, which - as for zymocin - serves as the primary toxin receptor, a hydrophobic domain assists to translocate the actual toxic subunit into the target cell. While zymocin causes a G1 cell cycle arrest, PaT provokes an arrest in the S-phase (Schaffrath & Meinhardt, 2005; Stark et al., 1990).

For identifying the molecular target of zymocin, the toxic subunit was expressed intracellularly from an inducible promoter in transposon mutants revealing zymocin resistant Elongator (elp3) mutants. As Elp3 plays an important role in tRNA modification, tRNAs got into the focus as possible molecular targets. Indeed, the overexpression of tRNA^E protected from zymocin action and heterologous expression of the toxin γ-subunit led to the decrease of the tRNA^E level. In vitro experiments identified the cleavage position of tRNA^E between nucleotides 34 and 35 within the anticodon loop (Lu et al., 2005; Jablonowski et al., 2006).

tRNA^Q was identified as the molecular target of PaT. In contrast to zymocin PaT cleaves twice within the anticodon loop, i.e. at the position between 34 and 35 and once more two nucleotides 5`-upstream between nucleotides 32 and 33 (Klassen et al., 2008) (Fig. 3).

Fig. 3: The secondary structure of tRNA^Gln. Triangles show the cleavage sites (Δ) and (▲) of PaT within the anticodon loop.

Recently, we have disclosed the molecular mode of action of the killer toxin PiT from P. inositovora (Kast et al., 2014). PiT induces fragmentation of the 25S and 18S rRNA (Fig. 4A), the latter being cut at least three times at the positions ~130 nt, ~700 nt und ~1100 nt. Mutational analysis confirmed that amino acid residues E9 and H214, conserved in all VLE-encoded toxins and identified to be catalytically active in zymocin and PaT (Keppetipola et al., 2009; Jain et al., 2011; Meineke et al., 2012), are also essential for the in vivo toxicity of PiT too and are crucial for rRNA fragmentation. Interestingly, the cleavage sites of the ~130 nt fragment of the 18S rRNA are located in a loop displaying a similar nucleotide sequence as for the anticodon loop of tRNA^Glu, the target of zymocin (Fig. 4B). Although the toxic subunits of PiT and zymocin hardly show any sequence allliance, there are similarities to PaT and to zymocin, e.g. the genetic localization on VLEs as well as the heteromeric protein consisting of a variable, intracellularly active toxic and a conserved chitin binding and transport subunit, suggesting a common evolutionary origin for all hitherto known VLE-encoded toxins (Kast et al., 2014).

Fig. 4: A. Fragmentation of rRNA after intracellular expression of PiT (PiOrf4) in Saccharomyces cerevisiae. B. 18S rRNA region with PiT cleavage sites at ~130 nt (▲). For comparison: anticodon loop of tRNA^Glu_(UUC) with indicated cleavage site (▲) of zymocin.

Our current research topics

Our current experimental efforts focus on the only poorly understood toxin DrT and on the VLE encoded immunity. Surprisingly, DNA repair mechanisms were found to protect against the translation-inhibitory PaT, enabling us to address the hitherto largely unknown link between translational integrity and genome surveillance.