# Effects of Pulsed Electric Fields on Yeast with Prions and the Structure of Amyloid Fibrils
## 4 Discussion  
The biological meaning and functions of prions are still under intensive discussion in the scientific community. [PSI+] yeast prion is a perfect model system to analyze the behavior and functions of the amyloid inside the cell. [PSI+] prion has mild pathogenicity, so the cell can survive longer and divide without lethal disturbances. Native Sup35 protein is a translation termination factor and [PSI+] prion helps to overcome STOP codon and prevents translation termination [35; 36; 37; 38]. Strong and weak [PSI+] variants have different abilities to overcome translation termination. It is proven that strong prions form smaller aggregates as compared to weak ones, therefore giving more polymerization ends. Strong prions are more stable and better transmitted in the cell population [6; 39]. In this research, we tried to answer three main scientific questions. 1. Do [PSI+] prions increase PEF resistance in _S. cerevisiae_ cells and how do different variants change this resistance? Our results show that _S. cerevisiae_ cells containing [PSI+] prions exhibited extremely increased resistance to PEF treatment even in the case of weak prion variants (Figure 3). There is also a significant difference between survivability after PEF between strong and weak variants. Almost 10% viability was observed in the case of strong prions, even after applying 22 kV/cm, 1000 \(\upmu\)s pulse duration for 5 pulses. Prions provided cells the ability to survive in such harsh treatment. Other authors agree that prions are related to stress resistance and improve cell viability in chronological aging [40; 41]. Prions in the amyloid conformation are resistant to proteases, heat shock, and other inactivation methods [14]. [PERSON] et al. showed that [PSI+] prion can regulate the phase separation of native Sup35 protein during stress [15]. In cell growth, Sup35 acts as a translation terminator. In  
Figure 4: Atomic force microscopy images of Sup35 NM amyloid fibrils before (**A**) and after exposure to a pulsed electric field (PEF) using different parameters: (**B**) 10 kV/cm, 50 \(\upmu\)s pulse duration, 10 pulses; (**C**) 10 kV/cm, 1000 \(\upmu\)s pulse duration, 10 pulses; (**D**) 20 kV/cm, 1000 \(\upmu\)s pulse duration, 10 pulses. The adjacent color scale indicates the height of the fibrils found in the samples. the stress conditions, the prion and sensor domains act together to promote phase separation and to establish protective and reversible biomolecular condensates. 2. Can PEF induce the appearance of prions in the cell population _de novo_ or change the type of prion from weak to strong by producing free amyloid ends for protein polymerization? There is a hypothesis that prions in the cell population can appear _de novo_ in cells experiencing stress conditions [41; 42] and prion variants can modulate prion-dependent proteomes in response to environmental stress [43], producing a facilitated adaptation of the cells by enhancing survival in rapidly changing environments [44; 45; 46]. We hypothesized that PEF could act as a factor capable of disrupting cell homeostasis and lead to prion induction. In the case of the cells already containing prions, it could serve as an agent that generates free amyloid ends for the more effective intake of the Sup35 to the amyloids. Analysis of the [psi\(-\)] and weak [PSI\(+\)] colonies after PEF treatment showed no signs of showing new prions or shift from weak to strong variants. This is the possibility that the PEF itself is not capable of inducing prionization in yeast cells, which makes PEF safe for the biocontrol of yeasts without the opportunity to induce prionization. 3. Is it possible to disintegrate [PSI\(+\)] amyloids by applying PEF to the purified proteins? There is no covalent bond in the formation of the secondary and tertiary structure between the fibril assembling prion proteins. Stabilizing factors are electrostatic interactions, such as salt bridges and self-energy [28]. Therefore, we hypothesized that those interactions can easily be broken by applying PEF.