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Review
. 2017 Oct 20;9(10):333.
doi: 10.3390/toxins9100333.

Yeast Killer Toxin K28: Biology and Unique Strategy of Host Cell Intoxication and Killing

Björn Becker  1 , Manfred J Schmitt  2
Affiliations
Review

Yeast Killer Toxin K28: Biology and Unique Strategy of Host Cell Intoxication and Killing

Björn Becker et al. Toxins (Basel). .

Erratum in

Abstract

The initial discovery of killer toxin-secreting brewery strains of Saccharomyces cerevisiae (S. cerevisiae) in the mid-sixties of the last century marked the beginning of intensive research in the yeast virology field. So far, four different S. cerevisiae killer toxins (K28, K1, K2, and Klus), encoded by cytoplasmic inherited double-stranded RNA viruses (dsRNA) of the Totiviridae family, have been identified. Among these, K28 represents the unique example of a yeast viral killer toxin that enters a sensitive cell by receptor-mediated endocytosis to reach its intracellular target(s). This review summarizes and discusses the most recent advances and current knowledge on yeast killer toxin K28, with special emphasis on its endocytosis and intracellular trafficking, pointing towards future directions and open questions in this still timely and fascinating field of killer yeast research.

Keywords: A/B toxin; H/KDEL receptor; K28; S. cerevisiae; cell cycle arrest; cell wall receptor; killer toxin; retrograde protein transport; retrotranslocation; toxin immunity.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Genomic organisation of the L-A (+) and M28 (+) strands. The initial GAAAAA sequence at the 5′-end of the M28 (+) ssRNA represents a terminal recognition element (TRE) which is necessary for the initiation of transcription. M28 (+) ssRNA further contains the toxin-encoding K28 open reading frame (ORF), a poly(A)-rich region and potential 3′ elements which are required for in vivo RNA replication (IRE, internal replication enhancer; TRE, 3′ terminal recognition element) and packaging (VBS, viral binding side). Besides the two ORFs on L-A (+) ssRNA, the position of the −1 frameshift region, the encapsidation signal and the replication side for (−) strand synthesis are indicated in brackets. Reproduced and modified from [10,20], 2011 and 2013, American Society for Cell biology.
Figure 2
Figure 2
Replication cycle of L-A helper and M28 killer viruses in S. cerevisiae. The L-A replication cycle starts in vivo, with the transcription of L-A dsRNA into L-A (+) strands, via the transcriptase activity of the Gag-Pol fusion protein. After extrusion into the cytoplasm, the majority of L-A (+) strands are translated into the capsid protein, Gag (grey dot), while only 1–2% are converted into a Gag-Pol fusion protein (green-grey dot) by a −1 ribosomal frameshift event. Viral L-A (+) strands further interact with Gag-Pol, which triggers L-A particle assembly and encapsidation [39]. The protein composition of newly assembled ScV-L-A particles, each of them containing just a single L-A (+) strand, is identical to that of mature virions. In the final step, an L-A (−) strand is synthesized by the replicase activity of Pol, leading to a complete dsRNA genome within each virion. Replication of ScV-M is analogous to that of ScV-L-A. After in vivo M (+) strand synthesis and subsequent extrusion into the cytoplasm, M (+) strands are either translated into the unprocessed K28 toxin precursor or bound by Gag-Pol for subsequent encapsidation into virus particles. ScV-M replication finally finishes with the synthesis of an M (−) strand. Compared to L-A virions, two M-dsRNA copies can be present in a single M virion at the same time, due to their smaller genome size.
Figure 3
Figure 3
Schematic K28 preprotoxin (pptox) structure and processing in the secretory pathway. (a) pptox is completely translated by cytosolic ribosomes and thereafter post-translationally imported into the ER lumen via complex protein import machinery, including Sec61p, Sec71p, Sec72p Sec62p, Ssa1p and Ssa2p [40]; (b) Within the ER, the N-terminal signal peptide (pre-sequence) is removed by signal peptidase (SP) cleavage, the γ-subunit is N-glycosylated ( formula image ) and the connecting disulfide (s-s) between α and β is formed by protein disulfide isomerase (Pdi1p); (c) In the cis-Golgi, the furin-like endopeptidase, Kex2p, removes the pro-sequence and γ-subunit which leads to a disulfide-bonded α/β heterodimer. Although the precise function of the pro-region is still not fully understood, it has been proposed to be important for proper post-translational pptox import into the ER lumen [40]. While the inter-chain disulfide in the heterodimer has clearly been demonstrated to be positioned between the single cysteine in α (Cys56) and Cys333 in β, the exact position of the cysteine residues in β that form the intra-chain disulfide and the single free thiol is still hypothetical [41,42]; (d) In the trans-Golgi, the C-terminus of the α-subunit as well as the C-terminal arginine of the β-subunit are removed by carboxypeptidase Kex1p cleavage, which unmasks the ER retention/targeting motif, HDEL, thereby converting the precursor in its biologically active conformation; (e) K28 is finally secreted as a 21 kDa heterodimer, whose β-C terminus carries a potential ER retention/targeting signal required for toxin uptake in a sensitive target cell [14].
Figure 4
Figure 4
Schematic outline of K28 cell surface binding and internalization. Initially, the β-subunit of the mature K28 toxin rapidly binds in an energy-independent process to primary mannoprotein receptors in the yeast cell wall. In a second energy-dependent step, K28 penetrates the cell wall and subsequently interacts with the plasma membrane (PM)-localized pool of the yeast H/KDEL receptor Erd2p [14]. This interaction is mediated by the C-terminal HDEL motif of the toxin’s β-subunit, which finally triggers receptor/toxin complex internalization by clathrin-mediated endocytosis.
Figure 5
Figure 5
Schematic overview of retrograde toxin trafficking routes in a K28 sensitive target cell. (a) Biologically active K28 toxin (α-subunit shown in orange, β-subunit in green) initially binds to primary receptors in the yeast cell wall and then interacts with a secondary receptor, the yeast H/KDEL receptor, Erd2p, at the level of the plasma membrane; (b) After clathrin-mediated endocytosis, a minor fraction of internalized toxin/receptor complexes is targeted to the vacuole and degraded (c), while toxin/receptor complexes that escaped degradation are transported in a retrograde manner to the pH-neutral ER lumen, where the receptor/cargo complexes dissociate and release K28 (d + e). The natural pH gradient between the extracellular killer yeast environment (pH 4.7) and the intracellular compartment of the ER (pH 7.2) presumably prevents premature toxin dissociation from its receptor (Erd2p) and likewise prevents spontaneous formation of inactive K28 oligomers (see also Chapter 7 below); (f) In contrast to, for example, cholera toxin, ER exit of K28 occurs in its oxidized and α/β heterodimeric conformation and is likely gated by the major protein import/export channel in the ER membrane, Sec61p [56,58]; (g) Within the cytosol, the heterodimeric toxin dissociates into its subunits: while the β-subunit is ubiquitylated and proteasomally degraded; (h) the α-subunit enters the nucleus and finally kills, by causing an irreversible G1/S cell cycle arrest and inhibiting DNA synthesis.
Figure 6
Figure 6
Model of pH-driven and Pdi1p-dependent thiol rearrangements in the K28 heterodimer during its retrograde transport to the cytosol. In the natural habitat and extracellular environment of a K28 killer yeast, the mildly acidic pH of 4.7 stabilizes the biologically active K28 toxin and keeps it in a heterodimeric conformation. During host cell intoxication, the α/β heterodimer faces a continuous increase in intra-compartmental pH. The neutral pH leads to fast deprotonation of free sulfhydryls in the β-subunit and subsequently causes the formation of inactive K28 trimers, tetramers and oligomers. Due to the presence of the chaperone, Pdi1p, in the ER-lumen, these disulfide bond rearrangements are efficiently prevented, in vivo and in vitro, ensuring ER exit of the heterodimeric toxin. At the neutral pH of the yeast cell cytosol, the intra-chain disulfide is cleaved through nucleophilic attack of a reactive thiol in β, which finally releases the monomeric α-subunit. This model is reproduced and modified from [42], 2017, American Society for Cell biology.
Figure 7
Figure 7
Model of the protecting toxin immunity mechanism in a K28 killer yeast. After toxin internalization and retrotranslocation of mature K28 from the ER into the cytosol, a cytosolic immunity complex is formed between the re-internalized α/β toxin and the unprocessed toxin precursor (pptox) which is encoded by the M28 killer virus and post-translationally imported into the ER lumen. Within each K28/pptox complex, the β-subunit of pptox and mature α/β toxin is poly-ubiquitylated (●) and degraded by the proteasome, thereby protecting a K28 killer cell against the lethal action of K28. In addition, free cytosolic and non-ubiquitylated pptox can enter the secretory pathway for enzymatic processing and toxin maturation, resulting in the secretion of biologically active K28.
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