Yeasts respond to changes in extracellular pH in a complex manner (Arino 2010; Serra-Cardona et al. 2015) Basically, this response affects the functioning of the main signaling pathways. Until now, there was no data on the effect of extracellular pH on the process of yeast cell death. However, such data have been obtained on metazoan cells. It has been shown that acidification of the extracellular pH changes the manner of cell death of cancer cells from an apoptotic to a necrotic mode (Meurette et al. 2005; Lan et al. 2007) The initial impetus for the present study was a neglect of the dual nature of neutralized ascorbate – which is a scavenger of ROS on one hand, and a pH-buffering agent with pH7.0 on the other. The incubation of S.cerevisiae SEY6210 with glucose in 50 mM HEPES buffer pH 7.0 led to a complete suppression of SICD (Fig. 2), which is a case of primary necrosis. Decreasing of the external pH leads to a gradual resumption of SICD. This observation seems to link necrosis with the degree of the extracellular acidification. A similar effect was observed in the induction of apoptosis by valproic acid in the yeast Schizosaccharomyces pombe (Mutoh et al. 2011). We also observed similar mitigating effects of external pH on another, recently discovered mode of death, termed division-associated necrosis (Alexandrov A.I. et al., manuscript in preparation), however death was mitigated only in some of the tested mutations.
As mentioned above, alkalization of the external environment leads to a complex response from the yeast cell. We examined the involvement of some of the signaling pathways and individual genes in the suppression of the necrosis by the neutral pH. In Saccharomyces cerevisiae, the Rim101 pathway senses external alkalization and alteration in plasma membrane lipid asymmetry through a complex consisted of Rim8, Rim9 and Rim21 at the plasma membrane. It is known that Rim21p, acts as a sensor of extracellular pH (Obara et al. 2012; Nishino et al. 2015). It was logical to assume that deletion of the rim21 gene could lead to the restoration of SICD in a neutral environment. However, the Δrim21 strain showed the same suppression of SICD by neutral pH as the parental strain BY4741 (Table 2). We conclude that the Rim101/PacC signaling pathway is not involved in SICD.
The cAMP/protein kinase A (PKA) pathway is one of the major glucose-signaling pathways of budding yeast. Activation of the PKA pathway cause sensitivity to alkaline pH (Casado et al. 2011). Conversely, deactivation of this system increases resistance to alkaline stress. However, activation of the PKA pathway by deleting the ira2 gene (upstream member of cAMP/PKA pathway) did not affect the development of SICD nor its suppression at neutral pH. Thus, the development of SICD and its suppression by neutral pH turned out to be insensitive to the cAMP level.
It should be noted that the study of the regulation of Rim101 and PKA signaling systems by extracellular pH was carried out at pH 8 (Casado et al. 2011; Obara et al. 2012). We tested the involvement of these systems in the suppression of SICD at pH 7. Perhaps this explains the insensitivity of SICD to the functional state of these signaling systems.
Caspases are a family of protease enzymes playing essential roles in programmed cell death. Inhibition of caspases promote alternative cell death pathways (Vandenabeele et al. 2006). For example, inhibition of caspases induced a switch from apoptosis to necrosis in B lymphocytes (Lemaire et al. 1998). It has also been shown that inhibition of caspases which occurs at acidic pH leads to a change in cell death from apoptosis to necrosis (Lan et al. 2007). Only one metacaspase, Yca1, is present in yeast (Madeo et al. 2002). It is involved in the regulation of apoptosis-like cell death caused by exposure to various treatments such as H2O2, acetic acid, salt- and osmotic stress, valproic acid and some metals (Madeo et al. 2009). We reasoned as follows. If metacaspase (by analogy with metazoan caspases), after inhibition in an acidic medium, leads to the switching of apoptosis to necrosis, then in Δyca1 strain, we either should not observe SICD in an acidic medium, or SICD will also be observed at neutral pH. However, the Δyca1 mutant did not show any of the expected responses (Table 2). Therefore, yca1 is not involved in SICD. This is in agreement with published data that only about half of the cell death scenarios are caspase-dependent (Madeo et al. 2009).
As noted earlier, SICD occurs when the ROS content is high (Valiakhmetov et al. 2019). Two major sources of ROS are known in the yeast cell - the mitochondrial respiratory chain and NADPH oxidase (Rinnerthaler et al. 2012) of the ER. We found no data on regulation of ROS production by the ambient pH. However, if such regulation existed, it would explain the fact that SICD was suppressed by neutral pH. To test this assumption, we used two deletion mutants: Δafo1(Heeren et al. 2009) with reduced mitochondrial ROS generation due to the lack of a large subunit of the mitochondrial ribosome and Δyno1 in which there is no NADPH oxidase (Rinnerthaler et al. 2012). Surprisingly, both strains continued to exhibit generation of ROS when cells were incubated with glucose in an unbuffered medium. The percentage of cells with ROS (and with SICD) roughly coincided with the percentage of such cells in the parental strain BY4741 (Table 2.). And just as the parent strain, both strains showed a pH dependence of the SICD. Incubation with glucose at pH 7 resulted in the suppression of the number of cells with ROS (and with SICD) by 90%. The data obtained indicate that the generation of ROS by the respiratory chain of mitochondria or NADPH oxidase of ER is not regulated by the extracellular pH.
However, the level of ROS in the cell can be indirectly altered by actin dynamics. It was previously shown that a decrease in actin dynamics leads to depolarization of the mitochondrial membrane and an increase in ROS generation (Gourlay et al. 2004; Gourlay and Ayscough 2005a). To test the involvement of genes regulating actin dynamics, we used three strains: Δend3, Δscp1 and Δsla1. Scp1p is the small actin-binding protein related to mammalian SM22/transgelin (Goodman et al. 2003; Winder et al. 2003). Sla1p is the endocytic adaptor protein that interacts with ubiquitin and involved in endocytosis and actin cortical patch assembly (Warren et al. 2002). End3p is the EH domain-containing protein involved in endocytosis, actin cytoskeletal organization and cell wall morphogenesis and forms a complex with Sla1p (Tang et al. 1997). In wild-type cells, Scp1p decreases actin dynamics and thus increases the amount of cellular ROS. The Δscp1 cells, respectively, have a reduced amount of ROS. Conversely, in wild cells, Sla1p and End3p increase actin dynamics, which leads to a decrease in the amount of ROS. In strains Δsla1 and Δend3, respectively, the ROS level is increased. Incubation of the Δend3, Δscp1 and Δsla1strains with glucose in water showed that cells with deleted genes generate ROS and undergo SICD at approximately the same efficiency as the parent strain. Incubation of these strains with glucose at pH 7.0 resulted in a complete absence of ROS and SICD (Table 2). Such results are to be expected, since End3p, Scp1p and Sla1p alter the ROS level indirectly by modulating the membrane potential of mitochondria, which, as can be seen from experiments with Δafo1, are not involved in the development of SICD.
Since we did not observe the sensitivity of SICD to neutral pH to depend on the functioning of several tested cellular systems, we hypothesized that ΔµH+ might be involved in this effect. During incubation with glucose, S.cerevisiae cells decreases the pH of the medium to pH 3.7 after 5 minutes (Fig. 1). This creates a large ΔpH on the plasma membrane, which is absent when cells are incubated at 50 mM HEPES pH 7. Hence dissipation of ΔpH by the protonophore should lead to the suppression of SICD. Figure 3 shows that DNP suppresses SICD by more than 80% already at a concentration of 0.5 mM. In this case, the extracellular pH was 4.7 (Fig. 1), which is significantly higher than the value for glucose metabolism in the absence of DNP (pH 3.5). It is noteworthy that the ROS level also dropped by more than 80%. The role of the second component of the membrane potential - ΔΨ - was verified in experiments with exogenous KCl. The ROS generation and SICD was suppressed by 80% when S.cerevisiae SEY6210 is incubated with glucose in the presence of 150 mM KCl (Fig. 4). Interestingly, the same data were obtained in experiments with yeast in the stationary growth phase, though at lower KCl concentrations (Hoeberichts et al. 2010). In order to make sure that the exogenous KCl really affected ΔΨ rather than non-specifically inhibits SICD we used DiOC2(3) dye to monitor changes in plasma membrane potential (Fig. 5). Since the dye enters cells in a voltage-dependent manner, it was important to exclude the contribution of the mitochondrial membrane potential to the observed changes in ΔΨ. Strain SEY6210 rho0 showed the same SICD development as the wild type (Table 2). Therefore, we used this strain to record changes in ΔΨ on the plasma membrane. As can be seen from Fig. 5, the incubation of cells in an aqueous solution of glucose leads to a significant increase in ΔΨ. Incubation of cells with glucose in a buffer with pH7.0 or in the presence of 150 mM KCl completely suppresses this increase of ΔΨ. It is important to note that the acidification of the medium in the presence of 150 mM KCl even slightly exceeded acidification during incubation with glucose only (Fig. 1). It is known that extracellular K+ is a regulator of the membrane potential on the plasma membrane of yeast(Madrid et al. 1998) and causes additional acidification of the medium (Martinez-Munoz and Kane 2008). An 80% inhibition of the ROS generation and SICD development under these conditions suggests that ΔΨ, rather than ΔpH, is responsible for ROS generation and subsequent development of SICD. Whether a hyperpolarized membrane can generate ROS on its own, or some abnormal cellular process is triggered via this hyperpolarization, is currently unclear. However, since we found a direct correlation between the ROS generation and the magnitude of ΔΨ on the plasma membrane, we do not exclude the possibility of ROS production by some membrane-bound complexes at a high ΔΨ value. This is supported by the fact that the removal of any of the two main producers of ROS - Afo1p or Yno1p - had no effect on ROS generation in our conditions. It should also be emphasized that an important issue is the selection of a suitable buffer to study the effect of extracellular pH on the development of primary necrosis. The presence of high concentrations of inorganic ions in buffers with pH 3.5, such as K+, Na+, citrate, phthalate and tartrate, distorts the real picture, because monovalent ions significantly change ΔµH +. Therefore, to test the SICD at an initially low pH of 3.5, we used water acidified with HCl. Notably, dissipating the ΔΨ on the plasma membrane also had profound effects on nearly all of the tested mutations causing rapid division-associated necrosis.
Summarizing the obtained data, we can state that both SICD and rapid division associated necrosis in some mutants are suppressed 1) upon dissipation of ΔpH at neutral pH (or treatment with DNP for SICD) and 2) upon dissipation of ΔΨ in the presence of 150 mM extracellular KCl. Thus, our current understanding of SICD, as well as, possibly, a subset of necrotic death associated with some mutations, is summarized in Fig. 7. ATP, synthesized during glucose metabolism, is consumed by the plasma membrane H-ATPase to create and maintain membrane potential through the extrusion of protons into the extracellular environment. When yeast grows on a rich medium, the transport of K+ into the cell prevents membrane hyperpolarization. The conditions of SICD (i.e. incubation in deionized water in the presence of glucose) do not provide sufficient ions to counterbalance the hyperpolarization, thus triggering some yet uncharacterized process resulting in necrosis. This notion is supported by the recent observations that deletion of K+ transporters of the Trk-family results in exacerbated SICD (Dušková et al. 2021), and coincides with the previously stated hypothesis about the involvement of the membrane potential in SICD observed in cells in the stationary growth phase (Hoeberichts et al. 2010). Notably, addition of KCl and stabilization of alkaline pH have been reported to be highly efficient methods of increasing yeast tolerance to ethanol, which also perturbs membrane structure and may thus cause necrotic cell death (Lam et al. 2014). Overall, our observations and the data in the literature suggest that deciphering the interplay between membrane potential and cell death may have wide-ranging ramifications for the understanding of cell death mechanisms in yeast.