Role for Sit4p‐dependent mitochondrial dysfunction in mediating the shortened chronological lifespan and oxidative stress sensitivity of Isc1p‐deficient cells

Abstract

Summary Saccharomyces cerevisiae cells lacking Isc1p, an orthologue of mammalian neutral sphingomyelinase 2, display a shortened lifespan and an increased sensitivity to oxidative stress. A lipidomic analysis revealed specific changes in sphingolipids that accompanied the premature aging of Isc1p deficient cells under severe calorie restriction conditions, including a decrease of dihydrosphingosine levels and an increase of dihydro-C26-ceramide and phyto-C26-ceramide levels, the latter raising the possibility of activation of ceramide-dependent protein phosphatases. Consequently, deletion of the SIT4 gene, which encodes for the catalytic subunit of type 2A ceramide-activated protein phosphatase in yeast, abolished the premature aging and hydrogen peroxide sensitivity of isc1Δ cells. SIT4 deletion also abolished the respiratory defects and catalase A deficiency exhibited by isc1Δ mutants. These results are consistent with catabolic derepression associated with the loss of Sit4p. The overall results show that Isc1p is an upstream regulator of Sit4p and implicate Sit4p activation in mitochondrial dysfunction leading to the shortened chronological lifespan and oxidative stress sensitivity of isc1Δ mutants.

Ceramide signalling impinges on Sit4p and Hog1p to promote mitochondrial fission and mitophagy in Isc1p-deficient cells

“…Consistently, the deletion of TOR1, SCH9, SIT4 or HOG1 suppresses mitochondrial fragmentation [26-28; this study], which in turn is associated with improvement of the organelle function, as shown by our lab [26][27][28], and lowers the induction of mitophagy in isc1Δ cells under respiratory conditions (in lactate medium, this study). Since mitochondrial fragmentation is a critical step for the sequestration of dysfunctional mitochondrial by autophagosomes [38] and oxidative stress is a signal for mitophagy induction [39], it seems that the suppression of mitochondrial fragmentation and reduction of ROS levels [26-28; this study] in isc1Δtor1Δ, isc1 Δsch9Δ, isc1Δsit4Δ and isc1Δhog1Δ cells lead to the attenuation of mitophagy.…”

“…Importantly, this was correlated with loss of cell viability in isc1Δ cells (Fig. 1C), as previously described [26]. We also analysed total levels of Por1p, a mitochondrial outer membrane protein, as a means to monitor mitochondrial turnover during ageing.…”

“…Our previous observations that isc1Δ cells present mitochondrial dysfunction associated with mitochondrial fragmentation [23,26,28] led us to hypothesize that Isc1p and ceramide signalling are involved in the modulation of mitochondrial dynamics and mitophagy. We first monitored mitophagy in cells grown to post-log phase by incubating them in lactate medium for 48 h (Fig.…”

“…7A). In addition, we showed earlier that the ceramide-activated protein phosphatase Sit4p and the MAPK Hog1p, which responds to ceramide signalling via Sch9p, also mediate mitochondrial loss-of-function in Isc1p-deficient cells [26][27][28]. To assess if ceramide signalling is involved in the regulation of mitochondrial dynamics and mitophagy in yeast, we monitored the mitochondrial morphology in isc1Δsit4Δ and isc1Δhog1Δ cells.…”

“…Consistently with a role of Isc1p in the regulation of mitochondrial function, isc1Δ cells fail to upregulate genes required for the transition to respiratory metabolism, exhibit oxidative stress sensitivity associated with iron overload and shortened chronological lifespan (CLS) [23,24]. We previously reported that the ceramide-activated protein phosphatase Sit4p, related to the type 2A family of protein phosphatases and with high homology to human protein phosphatase 6 [25], is activated in cells lacking Isc1p in response to very long chain ceramide species during ageing and that deletion of SIT4 suppresses mitochondrial dysfunction and other phenotypes of isc1Δ cells [26]. More recently, we demonstrated that the activation of the TORC1-Sch9p pathway and of the Hog1p kinase, the latter in response to ceramide signalling by Sch9p-dependent mechanisms, also contributes to the phenotypes of Isc1p-deficient cells [27,28].…”