Tobias Wilms

Molecular mechanisms linking the evolutionary conserved TORC1–Sch9 nutrient signalling branch to lifespan regulation in Saccharomyces cerevisiae

The knowledge on the molecular aspects regulating ageing in eukaryotic organisms has benefitted greatly from studies using the budding yeast Saccharomyces cerevisiae. Indeed, many aspects involved in the control of lifespan appear to be well conserved among species. Of these, the lifespan-extending effects of calorie restriction (CR) and downregulation of nutrient signalling through the target of rapamycin (TOR) pathway are prime examples. Here, we present an overview on the molecular mechanisms by which these interventions mediate lifespan extension in yeast. Several models have been proposed in the literature, which should be seen as complementary, instead of contradictory. Results indicate that CR mediates a large amount of its effect by downregulating signalling through the TORC1-Sch9 branch. In addition, we note that Sch9 is more than solely a downstream effector of TORC1, and documented connections with sphingolipid metabolism may be particularly interesting for future research on ageing mechanisms. As Sch9 comprises the yeast orthologue of the mammalian PKB/Akt and S6K1 kinases, future studies in yeast may continue to serve as an attractive model to elucidate conserved mechanisms involved in ageing and age-related diseases in humans.

The protein kinase Sch9 is a key regulator of sphingolipid metabolism in Saccharomyces cerevisiae

Sphingolipids play crucial roles in the determination of growth and survival of eukaryotic cells. The budding yeast protein kinase Sch9 is not only an effector, but also a regulator of sphingolipid metabolism. This new function provides a crucial link between nutrient and sphingolipid signaling.

The yeast protein kinase Sch9 adjusts V-ATPase assembly/disassembly to control pH homeostasis and longevity in response to glucose availability

The conserved protein kinase Sch9 is a central player in the nutrient-induced signaling network in yeast, although only few of its direct substrates are known. We now provide evidence that Sch9 controls the vacuolar proton pump (V-ATPase) to maintain cellular pH homeostasis and ageing. A synthetic sick phenotype arises when deletion of SCH9 is combined with a dysfunctional V-ATPase, and the lack of Sch9 has a significant impact on cytosolic pH (pHc) homeostasis. Sch9 physically interacts with, and influences glucose-dependent assembly/disassembly of the V-ATPase, thereby integrating input from TORC1. Moreover, we show that the role of Sch9 in regulating ageing is tightly connected with V-ATPase activity and vacuolar acidity. As both Sch9 and the V-ATPase are highly conserved in higher eukaryotes, it will be interesting to further clarify their cooperative action on the cellular processes that influence growth and ageing.

pH homeostasis in yeast; the phosphate perspective

Recent research further clarified the molecular mechanisms that link nutrient signaling and pH homeostasis with the regulation of growth and survival of the budding yeast Saccharomyces cerevisiae. The central nutrient signaling kinases PKA, TORC1, and Sch9 are intimately associated to pH homeostasis, presumably allowing them to concert far-reaching phenotypical repercussions of nutritional cues. To exemplify such repercussions, we briefly describe consequences for phosphate uptake and signaling and outline interactions between phosphate homeostasis and the players involved in intra- and extracellular pH control. Inorganic phosphate uptake, its subcellular distribution, and its conversion into polyphosphates are dependent on the proton gradients created over different membranes. Conversely, polyphosphate metabolism appears to contribute in determining the intracellular pH. Additionally, inositol pyrophosphates are emerging as potent determinants of growth potential, in this way providing feedback from phosphate metabolism onto the central nutrient signaling kinases. All these data point towards the importance of phosphate metabolism in the reciprocal regulation of nutrient signaling and pH homeostasis.

pH homeostasis links the nutrient sensing PKA/TORC1/Sch9 ménage-à-trois to stress tolerance and longevity

The plasma membrane H+-ATPase Pma1 and the vacuolar V-ATPase act in close harmony to tightly control pH homeostasis, which is essential for a vast number of physiological processes. As these main two regulators of pH are responsive to the nutritional status of the cell, it seems evident that pH homeostasis acts in conjunction with nutrient-induced signalling pathways. Indeed, both PKA and the TORC1-Sch9 axis influence the proton pumping activity of the V-ATPase and possibly also of Pma1. In addition, it recently became clear that the proton acts as a second messenger to signal glucose availability via the V-ATPase to PKA and TORC1-Sch9. Given the prominent role of nutrient signalling in longevity, it is not surprising that pH homeostasis has been linked to ageing and longevity as well. A first indication is provided by acetic acid, whose uptake by the cell induces toxicity and affects longevity. Secondly, vacuolar acidity has been linked to autophagic processes, including mitophagy. In agreement with this, a decline in vacuolar acidity was shown to induce mitochondrial dysfunction and shorten lifespan. In addition, the asymmetric inheritance of Pma1 has been associated with replicative ageing and this again links to repercussions on vacuolar pH. Taken together, accumulating evidence indicates that pH homeostasis plays a prominent role in the determination of ageing and longevity, thereby providing new perspectives and avenues to explore the underlying molecular mechanisms.

The TORC1-Sch9 pathway as a crucial mediator of chronological lifespan in the yeast Saccharomyces cerevisiae

The concept of ageing is one that has intrigued mankind since the beginning of time and is now more important than ever as the incidence of age-related disorders is increasing in our ageing population. Over the past decades, extensive research has been performed using various model organisms. As such, it has become apparent that many fundamental aspects of biological ageing are highly conserved across large evolutionary distances. In this review, we illustrate that the unicellular eukaryotic organism Saccharomyces cerevisiae has proven to be a valuable tool to gain fundamental insights into the molecular mechanisms of cellular ageing in multicellular eukaryotes. In addition, we outline the current knowledge on how downregulation of nutrient signaling through the target of rapamycin (TOR)-Sch9 pathway or reducing calorie intake attenuates many detrimental effects associated with ageing and leads to the extension of yeast chronological lifespan. Given that both TOR Complex 1 (TORC1) and Sch9 have mammalian orthologues that have been implicated in various age-related disorders, unraveling the connections of TORC1 and Sch9 with yeast ageing may provide additional clues on how their mammalian orthologues contribute to the mechanisms underpinning human ageing and health.

Hexokinase 2; Tangled between sphingolipid and sugar metabolism

Sphingolipids are important constituents of all eukaryotic cell membranes. In addition to a mere structural role, sphingolipids are being identified as second messengers in a growing number of pathways with impact on diverse biological processes. Similar as for other lipids, the disturbance of sphingolipid homeostasis is associated to the pathobiology of several diseases. Sphingolipid metabolism is tightly controlled and evolutionary conserved. Biosynthesis starts in the endoplasmic reticulum (ER) to form ceramide and continues in the Golgi compartment with synthesis of complex sphingolipids. Most reactions are reversible, allowing for a rapid interconversion of metabolic intermediates. Ceramide can also be catabolized by ceramidases to regenerate sphingosine, while complex sphingolipids are hydrolyzed by glycohydrolases and sphingomyelinases to recycle ceramide. Both ceramide and sphingosine can be phosphorylated to produce ceramide-1-phosphate and sphingosine-1phosphate. Besides biosynthesis and breakdown, sphingolipids are also subject to uptake and secretion. In general, a relative increase in the level of ceramide or sphingosine is associated to anti-proliferation, senescence and apoptosis, while an augmentation of ceramide-1-phosphate or sphingosine-1-phosphate levels usually promotes cell growth and survival. Sphingolipid biology is complex and the current picture is far from clear. In fact, sphingolipid metabolism and signaling coordinate a network that is strongly dependent on other metabolic inputs. The latter is evidenced by the targets and effector pathways that act in conjunction with sphingolipid signaling. These include key players known to operate at the intersect of different signaling routes, like 14-3-3 proteins, the protein phosphatase PP2A, the protein kinases AKT-PKB/Sch9 and AMPK/Snf1 (mammalian/yeast), or the multiprotein complexes, TORC1 and TORC2. 2 Recent work by the group of Vitor Costa extended the coordinative role of sphingolipid signaling toward sugar metabolism and glucose signaling. By performing a comparative phosphoproteomic analysis of wild-type yeast cells or cells lacking the ceramide-activated type 2A-like protein phosphatase Sit4, they identified several proteins with significantly altered expression levels, most of which are involved in carbohydrate metabolism and energy production. They also identified some proteins with altered phosphorylation and this included the hexokinase Hxk2, which displays increased serine-15 (S15) phosphorylation in glucose grown sit4D cells. Hxk2 is a dual-function kinase that shuttles between the cytosol and nucleus. Besides its role in sugar uptake and the initial step of glycolysis, it has a regulatory role in glucose signaling. During fermentative growth, when glucose levels are high, Hxk2 interacts with the transcription factor Mig1 and the AMPK-ortholog Snf1 to form a nuclear complex that represses genes involved in the utilization of alternative carbon sources, like SUC2, gluconeogenesis and respiratory growth. Upon glucose limitation, Hxk2-S15 phosphorylation and the subsequent phosphorylation of Mig1 by Snf1 trigger disintegration of the repressor complex and exit of both Hxk2 and Mig1 from the nucleus. The enhanced phosphorylation of Hxk2 in sit4D cells is consistent with previous reports showing that already during growth on glucose respiration is derepressed in sit4D cells and that mitochondrial respiration is indeed essential because sit4D cells fail to grow anaerobically. Moreover, Costa’s group also linked the Hxk2 phosphorylation status to other phenotypes, such as the enhanced oxidative stress resistance and extended lifespan of the sit4D mutant and, conversely, the premature aging of mutant cells lacking the sphingomyelinase Isc1. Notably, Isc1 translocates in a Sch9-dependent manner from the ER to the mitochondria during the diauxic shift, thereby gaining activity to allow for the adaptation from fermentation to respiration. How Sit4 controls Hxk2 phosphorylation still needs to be clarified. The Costa group found no evidence for the possibilities that Hxk2 would be a direct target of Sit4 or that Sit4 would influence Hxk2 phosphorylation indirectly through inhibition of Snf1. Whether Sit4 acts via the protein phosphatase complex Reg1-Glc7, known to dephosphorylate Hxk2, or via the protein kinase Tda1, known to be essential for Hxk2-S15 phosphorylation, still needs to be confirmed (Fig. 1). Also Sch9 could be involved since this kinase appears to modulate Hxk2 phosphorylation in response to glucose availability. Moreover, Sch9 is an established effector of sphingolipid signaling that is