# The Biology of _Pichia membranifaciens_ Killer Toxins
## 5 Killing Mechanism of Action
### Cellular Responses to Killer Toxins
#### 5.3.2 PMKT2  
The fact that PMKT2 presented a different spectrum of killer activity, as indicated above, soon suggested that the molecular mechanism of action of PMKT2 might be different. In previous works [20], the binding of PMKT2 to manoproteins, its primary receptors, was seen to occur in the first two or three minutes after PMKT2 incorporation and then, cellular viability reduced (80% after 5 h of exposure to PMKT2). Furthermore, none of the cellular events observed, when sensitive cells are intoxated with PMKT, occurred with PMKT2. Leakage of K\({}^{+}\), Na\({}^{+}\) influx and reduction of pH\({}_{\text{i}}\) were not observed during the initial exposure to PMKT2, indicating that the nature of the interaction is not related  
Figure 2: Cellular response to PMKT exposure deduced from the analysis of the transcriptional response of _S. cerevisiae_ to the toxin. Once PMKT has reached the cell wall through an interaction with (1\(\rightarrow\)6)-ß-D-glucans, PMKT interacts with the cytoplasmic membrane through the interaction with cwp2p (not shown in the figure). This interaction, through a mechanism of unknown nature (7), leads to disruption of electrochemical gradients. Cellular receptors are not included in the scheme for simplification. PMKT cause the formation of unregulated channels through which cations and small metabolites (e.g., K\({}^{+}\), H\({}^{+}\), Na\({}^{+}\), glycerol) flow. These changes in pH\({}_{\text{i}}\), turgor or ionic concentrations generates a transcriptional response through the activation of Hog1p. Genes (_CTT1_, _HSP12_, _HSP26_, and _DDR2_), related with a general stress response, and genes (_GPD1_ and _GPP2_), involved in glycerol synthesis, are transcribed to compensate the toxic effects of PMKT. Cations and glycerol, among others, can leak out from sensitive cells generating cell death. Highlighted in a box are the induction levels of the most up-regulated genes. Abbreviations: DHAP (dihydroxyoxyacetone phosphate), G3P (glycerol-3-phosphate), FBP (fructose-1,6-bisphosphate), MG (methylglyoxal), CoQ (coenzyme Q), S-LG (S-D-lactoylglutathione), GSH (glutathione).  
with the formation of unregulated cation-permeable channels. The observed increase of membrane permeability to H\({}^{+}\), K\({}^{+}\), Na\({}^{+}\) after 4-5 h in contact with PMKT2 is considered an indirect effect, since they initiate concomitantly or after cell death, reflecting that they are not primary effects.  
The reason so many dead cells remain non-permeabilized during a long time of exposure to PMKT2 was elucidated when the cell cycle was studied. Flow cytometry studies of sensitive yeast cells exposed to PMKT2 show cells with arrested at an early S-phase with a nascent bud and pre-replicated 1n DNA content (Figure 3). The execution point for lethality was determined with more precision by checking the sensitivity/resistance of cells arrested at different cell cycle stages. Alpha-factor-arrested cells at the G1-phase or G2-phase-arrested cells by treatment with methyl benzimidazole-2-yl-carbamate, are resistant to PMKT2, indicating that sensitive yeasts are not affected in these stages. Furthermore, in both cases, sensitive cells rescued from the arrest are inhibited by the toxin, suggesting, all together, that G2-phase-released cells start to divide, but die in the presence of the toxin after initiating the S-phase, adopting a terminal phenotype. To explain the late ion movements across plasma membrane, we hypothesize that, during the first moments of toxin action, the cell cycle arrest does not cause cytoplasmic membrane permeabilization and then, the arrested cells became converted unviable and plasma membrane permeabilization happens [42].  
In short, the killing of action of PMKT, as well as several other yeast killer toxins, is related to peptide membrane permeation properties [16; 41; 125], whereas the mechanism of PMKT2 is related to cell cycle arrest [42].