# The Biology of _Pichia membranifaciens_ Killer Toxins
## 5 Killing Mechanism of Action
### The Dual Mechanism of Action of PMKT and PMKT2  
It has been described how the killing of _S. cerevisiae_ sensitive cells by _P. membranifaciens_ killer toxins may be expressed as a combination of two mechanisms depending on toxin dosage. High doses  
Figure 3: Mechanisms of the killing activity of PMKT2 depending on toxin dosage. (**A**) High concentrations of PMKT2 caused cell cycle arrest at G1/S. Flow cytometry analyses of DNA content of _S. cerevisiae_ revealed that cells treated with high doses of PMKT2 were arrested at an early S phase with a nascent bud. (**B**) Asynchronously growing cells exposed to inactivated toxin remained fully viable. (**C**) Cells treated with low concentrations of PMKT2 lead to the typical markers of apoptosis. Figure adapted and reproduced with permission ([PERSON] et al. 2013) [42]. (between 1250 and 2000 AU/mL) of PMKT and PMKT2 are observed to induce the aforementioned effects on sensitive yeast cells. By contrast, low doses (200 AU/mL) of the toxins lead to a cell death process in _S. cerevisiae_ accompanied by cytological and biochemical indicators of apoptotic cell death (PCD, Programmed Cell Death). The observed cell death process is characterized by the presence of an initial apoptotic state that rapidly evolves to a necrotic state. The rate at which this process occurs is proportional to the amount of killer toxin used in the study. Low PMKT concentrations (up to 200 AU/mL) lead to a higher number of apoptotic cells, whereas higher concentrations (from 300 to 3000 AU/mL) cause both apoptosis and necrosis. Necrotic cells are usually characterized by a permeabilized plasma membrane (usually detected by propidium iodide stain), whereas the plasma membrane that have initiated a PCD phenotype is usually intact. As a result of these investigations, it can be determined that cells appear to start the process of death with a PCD response, die as a consequence of apoptosis, and then, as a late consequence, become necrotic [74]. In these experimental conditions, cell death is characterized by cytological, genetic and biochemical indicators of apoptosis, namely, an increment of reactive oxygen species (ROS), DNA strand breaks, externalization of phosphatidylinositolserine, metacaspase activation and cytochrome \(c\) release, confirming the hypothesis of PCD induction. It has been also demonstrated that the secondary receptors in the sensitive cells have the same function in both experimental conditions. _CWP2\(\Delta\)_ mutants are resistant to pro-apoptotic, as well as for pro-ionophoric amounts of PMKT, also in agreement with the observations obtained for Kre1p, the plasma membrane secondary receptor involved in the K1 mechanism of action [76; 135], indicating that Cwp2p is always involved in the killing action [76; 135]. In the comparison studies between the two experimental conditions, it has been determined whether the transcriptional response of the cells is coincident. Therefore, using DNA microarrays, global gene expression has been also evaluated in the presence of pro-apoptotic concentrations of PMKT. The results obtained from DNA microarrays indicate that genes related with an oxidative stress response are induced, showing that the transcriptional response is not coincident with that obtained for pro-ionophoric doses of PMKT. The most highly induced genes are, in order, _DCS2_, _FLR1_, _GSH1_, _APH1_, _VPS13_, _ TSA2_, _GLR1_, _ECM4_, _AAD14_ and _GT03_, most of which are observed to be induced by different oxidative compounds. Several genes encoding for ROS detoxifying enzymes, such as _CTT1_, _PRX1_, _TsA1_, and _SOD1_, are also observed to be up-regulated (activations comprised between +16.1 and +11.3), indicating an oxidative stress that can be observed by analyzing the ROS production with dihydroethidium in the presence of PMKT (Figure 4). Taking into account the previous research on PMKT, we raised the possibility that PMKT2 also presented the aforementioned duality in its mode of action.