The nutrient-responsive CDK Pho85 primes the Sch9 kinase for its activation by TORC1.

Yeast cells maintain an intricate network of nutrient signaling pathways enabling them to integrate information on the availability of different nutrients and adjust their metabolism and growth accordingly. Cells that are no longer capable of integrating this information, or that are unable to make the necessary adaptations, will cease growth and eventually die. Here, we studied the molecular basis underlying the synthetic lethality caused by loss of the protein kinase Sch9, a key player in amino acid signaling and proximal effector of the conserved growth-regulatory TORC1 complex, when combined with either loss of the cyclin-dependent kinase (CDK) Pho85 or loss of its inhibitor Pho81, which both have pivotal roles in phosphate sensing and cell cycle regulation. We demonstrate that it is specifically the CDK-cyclin pair Pho85-Pho80 or the partially redundant CDK-cyclin pairs Pho85-Pcl6/Pcl7 that become essential for growth when Sch9 is absent. Interestingly, the respective three CDK-cyclin pairs regulate the activity and distribution of the phosphatidylinositol-3 phosphate 5-kinase Fab1 on endosomes and vacuoles, where it generates phosphatidylinositol-3,5 bisphosphate that serves to recruit both TORC1 and its substrate Sch9. In addition, Pho85-Pho80 directly phosphorylates Sch9 at Ser726, and to a lesser extent at Thr723, thereby priming Sch9 for its subsequent phosphorylation and activation by TORC1. The TORC1-Sch9 signaling branch therefore integrates Pho85-mediated information at different levels. In this context, we also discovered that loss of the transcription factor Pho4 rescued the synthetic lethality caused by loss of Pho85 and Sch9, indicating that both signaling pathways also converge on Pho4, which appears to be wired to a feedback loop involving the high-affinity phosphate transporter Pho84 that fine-tunes Sch9-mediated responses.

Deficiency of the Thyroid Hormone Transporter Monocarboxylate Transporter 8 in Neural Progenitors Impairs Cellular Processes Crucial for Early Corticogenesis.

Thyroid hormones (THs) are essential for establishing layered brain structures, a process called corticogenesis, by acting on transcriptional activity of numerous genes. In humans, deficiency of the monocarboxylate transporter 8 (MCT8), involved in cellular uptake of THs before their action, results in severe neurological abnormalities, known as the Allan-Herndon-Dudley syndrome. While the brain lesions predominantly originate prenatally, it remains unclear how and when exactly MCT8 dysfunction affects cellular processes crucial for corticogenesis. We investigated this by inducing in vivo RNAi vector-based knockdown of MCT8 in neural progenitors of the chicken optic tectum, a layered structure that shares many developmental features with the mammalian cerebral cortex. MCT8 knockdown resulted in cellular hypoplasia and a thinner optic tectum. This could be traced back to disrupted cell-cycle kinetics and a premature shift to asymmetric cell divisions impairing progenitor cell pool expansion. Birth-dating experiments confirmed diminished neurogenesis in the MCT8-deficient cell population as well as aberrant migration of both early-born and late-born neuroblasts, which could be linked to reduced reelin signaling and disorganized radial glial cell fibers. Impaired neurogenesis resulted in a reduced number of glutamatergic and GABAergic neurons, but the latter additionally showed decreased differentiation. Moreover, an accompanying reduction in untransfected GABAergic neurons suggests hampered intercellular communication. These results indicate that MCT8-dependent TH uptake in the neural progenitors is essential for early events in corticogenesis, and help to understand the origin of the problems in cortical development and function in Allan-Herndon-Dudley syndrome patients.SIGNIFICANCE STATEMENT Thyroid hormones (THs) are essential to establish the stereotypical layered structure of the human forebrain during embryonic development. Before their action on gene expression, THs require cellular uptake, a process facilitated by the TH transporter monocarboxylate transporter 8 (MCT8). We investigated how and when dysfunctional MCT8 can induce brain lesions associated with the Allan-Herndon-Dudley syndrome, characterized by psychomotor retardation. We used the layered chicken optic tectum to model cortical development, and induced MCT8 deficiency in neural progenitors. Impaired cell proliferation, migration, and differentiation resulted in an underdeveloped optic tectum and a severe reduction in nerve cells. Our data underline the need for MCT8-dependent TH uptake in neural progenitors and stress the importance of local TH action in early development.

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.

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.

Snf1/AMPK fine-tunes TORC1 signaling in response to glucose starvation.

The AMP-activated protein kinase (AMPK) and the target of rapamycin complex 1 (TORC1) are central kinase modules of two opposing signaling pathways that control eukaryotic cell growth and metabolism in response to the availability of energy and nutrients. Accordingly, energy depletion activates AMPK to inhibit growth, while nutrients and high energy levels activate TORC1 to promote growth. Both in mammals and lower eukaryotes such as yeast, the AMPK and TORC1 pathways are wired to each other at different levels, which ensures homeostatic control of growth and metabolism. In this context, a previous study (Hughes Hallett et al., 2015) reported that AMPK in yeast, that is Snf1, prevents the transient TORC1 reactivation during the early phase following acute glucose starvation, but the underlying mechanism has remained elusive. Using a combination of unbiased mass spectrometry (MS)-based phosphoproteomics, genetic, biochemical, and physiological experiments, we show here that Snf1 temporally maintains TORC1 inactive in glucose-starved cells primarily through the TORC1-regulatory protein Pib2. Our data, therefore, extend the function of Pib2 to a hub that integrates both glucose and, as reported earlier, glutamine signals to control TORC1. We further demonstrate that Snf1 phosphorylates the TORC1 effector kinase Sch9 within its N-terminal region and thereby antagonizes the phosphorylation of a C-terminal TORC1-target residue within Sch9 itself that is critical for its activity. The consequences of Snf1-mediated phosphorylation of Pib2 and Sch9 are physiologically additive and sufficient to explain the role of Snf1 in short-term inhibition of TORC1 in acutely glucose-starved cells.

Coordinated glucose-induced Ca2+ and pH responses in yeast Saccharomyces cerevisiae.

Ca2+ and pH homeostasis are closely intertwined and this interrelationship is crucial in the cells' ability to adapt to varying environmental conditions. To further understand this Ca2+-pH link, cytosolic Ca2+ was monitored using the aequorin-based bioluminescent assay in parallel with fluorescence reporter-based assays to monitor plasma membrane potentials and intracellular (cytosolic and vacuolar) pH in yeast Saccharomyces cerevisiae. At external pH 5, starved yeast cells displayed depolarized membrane potentials and responded to glucose re-addition with small Ca2+ transients accompanied by cytosolic alkalinization and profound vacuolar acidification. In contrast, starved cells at external pH 7 were hyperpolarized and glucose re-addition induced large Ca2+ transients and vacuolar alkalinization. In external Ca2+-free medium, glucose-induced pH responses were not affected but Ca2+ transients were abolished, indicating that the intracellular [Ca2+] increase was not prerequisite for activation of the two primary proton pumps, being Pma1 at the plasma membrane and the vacuolar and Golgi localized V-ATPases. A reduction in Pma1 expression resulted in membrane depolarization and reduced Ca2+ transients, indicating that the membrane hyperpolarization generated by Pma1 activation governed the Ca2+ influx that is associated with glucose-induced Ca2+ transients. Loss of V-ATPase activity through concanamycin A inhibition did not alter glucose-induced cytosolic pH responses but affected vacuolar pH changes and Ca2+ transients, indicating that the V-ATPase established vacuolar proton gradient is substantial for organelle H+/Ca2+ exchange. Finally, a systematic analysis of yeast deletion strains allowed us to reveal an essential role for both the vacuolar H+/Ca2+ exchanger Vcx1 and the Golgi exchanger Gdt1 in the dissipation of intracellular Ca2+.

The Role of Sch9 and the V-ATPase in the Adaptation Response to Acetic Acid and the Consequences for Growth and Chronological Lifespan.

Studies with Saccharomyces cerevisiae indicated that non-physiologically high levels of acetic acid promote cellular acidification, chronological aging, and programmed cell death. In the current study, we compared the cellular lipid composition, acetic acid uptake, intracellular pH, growth, and chronological lifespan of wild-type cells and mutants lacking the protein kinase Sch9 and/or a functional V-ATPase when grown in medium supplemented with different acetic acid concentrations. Our data show that strains lacking the V-ATPase are especially more susceptible to growth arrest in the presence of high acetic acid concentrations, which is due to a slower adaptation to the acid stress. These V-ATPase mutants also displayed changes in lipid homeostasis, including alterations in their membrane lipid composition that influences the acetic acid diffusion rate and changes in sphingolipid metabolism and the sphingolipid rheostat, which is known to regulate stress tolerance and longevity of yeast cells. However, we provide evidence that the supplementation of 20 mM acetic acid has a cytoprotective and presumable hormesis effect that extends the longevity of all strains tested, including the V-ATPase compromised mutants. We also demonstrate that the long-lived sch9Δ strain itself secretes significant amounts of acetic acid during stationary phase, which in addition to its enhanced accumulation of storage lipids may underlie its increased lifespan.

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.