Pkh1p-Ypk1p and Pkh1p-Sch9p Pathways Are Activated by Acetic Acid to Induce a Mitochondrial-Dependent Regulated Cell Death

Abstract

The yeast Saccharomyces cerevisiae undergoes a mitochondrial-dependent regulated cell death (RCD) exhibiting typical markers of mammalian apoptosis. We have previously shown that ceramide production contributes to RCD induced by acetic acid and is involved in mitochondrial outer membrane permeabilization and cytochrome c release, especially through hydrolysis of complex sphingolipids catalyzed by Isc1p. Recently, we also showed that Sch9p regulates the translocation of Isc1p from the endoplasmic reticulum into mitochondria, perturbing sphingolipid balance and determining cell fate. In this study, we addressed the role of other signaling proteins in acetic acid-induced RCD. We found that single deletion of PKH1 or YPK1, as shown for SCH9 and ISC1, leads to an increase in cell survival in response to acetic acid and that Pkh1/2p-dependent phosphorylation of Ypk1p and Sch9p increases under these conditions. These results indicate that Pkh1p regulates acetic acid-induced RCD through Ypk1p and Sch9p. In addition, our results suggest that Pkh1p-Ypk1p is necessary for isc1Δ resistance to acetic acid-induced RCD. Moreover, double deletion of ISC1 and PKH1 has a drastic effect on cell survival associated with increased ROS accumulation and release of cytochrome c, which is counteracted by overexpression of the PKA pathway negative regulator PDE2. Overall, our results suggest that Pkh1p-Ypk1p and Pkh1p-Sch9p pathways contribute to RCD induced by acetic acid.

Regulation of Cell Death Induced by Acetic Acid in Yeasts

“…Acetic acid-induced mitochondrial degradation was demonstrated by several authors ( Fannjiang et al, 2004 ; Pereira et al, 2010 ; Rego et al, 2012 ; Pereira et al, 2013 ; Dong et al, 2017 ; Martins et al, 2019 ) and it was shown that the normal tubular mitochondrial network becomes fragmented and deformed, acquiring a punctate pattern ( Fannjiang et al, 2004 ; Pereira et al, 2010 ; Rego et al, 2012 , 2018 ; Longo et al, 2015 ; Trindade et al, 2016 ), or even forming clusters in some mutant cells ( Pereira et al, 2010 ; Rego et al, 2012 ) in an actin-dependent process ( Pereira et al, 2010 ). Mitochondrial outer membrane permeabilization (MOMP) associated with cytochrome c release, a central event in mammalian intrinsic apoptosis, was also characterized: this hallmark in AA-RCD has been studied by assessing permeabilization of the outer mitochondrial membrane and maintenance of IMM integrity by in vitro enzymatic assays ( Pereira et al, 2007 ; Rego et al, 2012 , 2014a ), and cytochrome c release by western blot and/or spectra analysis ( Ludovico et al, 2002 ; Pereira et al, 2007 ; Giannattasio et al, 2008 ; Guaragnella et al, 2010a , b , 2011 , 2013 ; Rego et al, 2014a , 2018 , 2020 ; Guerreiro et al, 2016 ; Trindade et al, 2016 ; Martins et al, 2019 ). Besides mitochondria, a functional vacuole also seems to be crucial in AA-RCD ( Schauer et al, 2009 ), involving vacuolar membrane permeabilization and subsequent release of Pep4p, the yeast ortholog of mammalian cathepsin D (CatD), which acts on mitochondrial degradation ( Pereira et al, 2010 ).…”

“…These include the serine/threonine kinase Pkh1p, ortholog of the mammalian 3-phosphoinositide-dependent kinase PDK1 and an important regulator of the yeast sphingolipid biosynthetic pathway, which controls sphingolipid balance through Sch9p [yeast ortholog of mammalian protein kinase B (Akt/PKB)] and Ypk1p [yeast ortholog of mammalian serum- and glucocorticoid-inducible protein kinase (SGK)]. Acetic acid exposure was shown to trigger Pkh1/2p-dependent phosphorylation of both Sch9p and Ypk1p, demonstrating that Pkh1p-Ypk1p and Pkh1p-Sch9p pathways are activated during AA-RCD ( Rego et al, 2020 ). The contribution of these signaling pathways and how sphingolipid signaling regulates AA-RCD, mainly with Isc1p as a downstream target, was then addressed in more detail.…”

“…These results suggest that the Sch9p pathway modulates AA-RCD through the regulation of Isc1p cellular distribution, thus affecting the sphingolipid balance that regulates cell fate. On the other hand, the Pkh1p-Ypk1p pathway seems necessary for the resistance to acetic acid displayed by isc1 Δ cells ( Rego et al, 2020 ). Indeed, in contrast with individual mutations, which caused resistance, absence of both ISC1 and PKH1 drastically reduced survival of yeast cells to acetic acid, accompanied by increased ROS accumulation and release of cytochrome c ( Rego et al, 2020 ).…”

“…On the other hand, the Pkh1p-Ypk1p pathway seems necessary for the resistance to acetic acid displayed by isc1 Δ cells ( Rego et al, 2020 ). Indeed, in contrast with individual mutations, which caused resistance, absence of both ISC1 and PKH1 drastically reduced survival of yeast cells to acetic acid, accompanied by increased ROS accumulation and release of cytochrome c ( Rego et al, 2020 ). However, this was likely associated with hyperactivation of the cAMP/PKA pathway and not due to a more specific role of the proteins in sphingolipid signaling (see next section).…”

“…Apart from a possible role in mitochondria, Ras activation may promote AA-RCD by increasing cAMP levels. Overexpression of PDE2 , which encodes a high-affinity cAMP phosphodiesterase that inhibits PKA by hydrolyzing cAMP, increased the resistance of yeast cells to acetic acid ( Rego et al, 2020 ). On the other hand, excessive levels of cAMP seem to result in sensitivity to acetic acid.…”