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.Support via the research grants G.0694.13, G.0A63.15 and SBO-S006617N. We also thank KU Leuven for support by granting the C14/17/063 projectinfo:eu-repo/semantics/publishedVersio

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

The evolutionary conserved TOR complex 1 controls growth in response to the quality and quantity of nutrients such as carbon and amino acids. The protein kinase Sch9 is the main TORC1 effector in yeast. However, only few of its direct targets are known. In this study, we performed a genome-wide screening looking for mutants which require Sch9 function for their survival and growth. In this way, we identified multiple components of the highly conserved vacuolar proton pump (V-ATPase) which mediates the luminal acidification of multiple biosynthetic and endocytic organelles. Besides a genetic interaction, we found Sch9 also physically interacts with the V- ATPase to regulate its assembly state in response to glucose availability and TORC1 activity. Moreover, the interaction with the V-ATPase has consequences for ageing as it allowed Sch9 to control vacuolar pH and thereby trigger either lifespan extension or lifespan shortening. Hence, our results provide insights into the signaling mechanism coupling glucose availability, TORC1 signaling, pH homeostasis and longevity. As both Sch9 and the V-ATPase are highly conserved and implicated in various pathologies, these results offer fertile ground for further research in higher eukaryotes

TORC1 determines Fab1 lipid kinase function at signaling endosomes and vacuoles

Acknowledgments: We thank Lars Langemeyer for feedback, all members from the Ungermann lab for discussions, and Kathrin Auffarth, Angela Perz, and Malika Jaquenoud for expert technical assistance. This work was supported by the DFG (UN111/10-1 to C.U.), the Canton of Fribourg (to J.D. and C.D.V.), and the Swiss National Science Foundation (310030_166474/184671 to C.D.V. and 310030_184781 and 316030_177088 to J.D.). Z.C. received support from a travel stipend of the Boehringer Ingelheim Fonds. P.C.M. received additional support from the graduate program of the Collaborative Research Center 944 (SFB 944) and Department of Biology/Chemistry Osnabrück. E.E. received a fellowship of FWO Vlaanderen, Belgium (SB-FWO 1S06419N). Author Contributions: Z.C. and P.C.M. conducted all experiments on Fab1 localization and function; R.H. conducted experiments on development and analysis of the Sch91–183 probe; R.N., Z.H., M.-P.P.-G., and J.D. did the phosphorylation assays and analyses; and E.E. and J.W. conceived and performed the initial Sch9 mapping. T.N. and C.J.S. did the lipid analysis of the mutant alleles. J.G. analyzed microcopy data with Z.C. C.D.V. and C.U. conceived the study and wrote the manuscript with support of J.W.Peer reviewedPublisher PD

Hexokinase 2; Tangled between sphingolipid and sugar metabolism

peerreview_statement: The publishing and review policy for this title is described in its Aims & Scope. aims_and_scope_url: http://www.tandfonline.com/action/journalInformation?show=aimsScope&journalCode=kccy20status: publishe

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.status: publishe

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.status: publishe

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

Funding: Research was funded by fellowships of FWO-Vlaanderen (Fonds Wetenschappelijk Onderzoek) to RG and EE, a grant of the Biotechnology and Biological Sciences Research Council (BB/V016334/1) to RH, grants of the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) through Project-ID 403222702, SFB 1381, TP B08 to SR and RO 1028/5-2 to SR, the Germany’s Excellence Strategy, (BIOSS) EXC 949 and CIBSS (EXC 2189) to SR, the DFG projects UN111/10-2 and SFB 1557, TP14 to CU, the Swiss National Science Foundation (310030_166474/184671) to CDV, FWO-Vlaanderen (G069413, G0C7222N) to JW and KU Leuven (C14/17/063, C14/21/095) to JW. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

Sch9 physically interacts with the V-ATPase.

<p>(A, B) Physical interaction of Sch9 with Vma1 depends on glucose availability. Cells expressing HA₆-Sch9 were grown as in <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006835#pgen.1006835.g006" target="_blank">Fig 6A</a></b>, followed by re-addition of 2% glucose (A) or 0.2% glutamine (B). Total lysates (input) and anti-Vma1 immunoprecipitates (IP) were analyzed by immunoblotting. (C, D) Sch9 regulates V-ATPase assembly downstream of TORC1. (C) WT and <i>sch9Δ</i> cells were grown to mid-log phase in YPD, pH 5. Half of the culture was treated with 200 nM rapamycin for 30 min and subsequently starved for glucose in the presence of rapamycin. The untreated half was further grown for 30 min and subsequently starved for glucose. (D) V-ATPase assembly was assessed in the <i>sch9Δ</i> strain expressing the empty vector (pRS416), the wild-type <i>SCH9</i> gene (Sch9^WT), or one of the <i>SCH9</i> mutant genes in which its TORC1 phosphorylation sites are mutated (Sch9^5A and Sch9^2D3E). The WT strain expressing the empty vector was taken as an additional control. Precultures were grown overnight in minimal medium lacking uracil buffered at pH 5 and inoculated in YPD medium (50 mM MES, pH 5). Once cells reached exponential phase, half of the culture was treated with 200 nM rapamycin (rapa) for 30 min. To quantify V-ATPase assembly, complexes were IPed with antibodies against Vma1 and Vph1. Results are depicted as mean values ± SEM from at least three independent experiments. One- or two-way ANOVA analyses were performed to determine statistical significances. Unless indicated otherwise, asterisks indicate a statistical significance compared to the WT strain grown in YPD without rapamycin. See also <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006835#pgen.1006835.t002" target="_blank">Table 2</a></b>.</p

Function of Sch9 in regulating ageing is dependent on V-ATPase activity.

<p>With the exception of panel E, all chronological ageing data represent measurements performed on cells at day 8 in stationary phase. (A) Cell survival and (B) ROS determination of strains aged in non-buffered fully supplemented medium as determined by flow cytometry. (C) Cell survival and (D) ROS levels of strains aged in fully supplemented medium buffered at pH 5.5 as determined by flow cytometry. (E) Cell survival of strains grown in buffered medium at day 23 in stationary phase as determined by CFU counting. (F) pH of the culture medium of ageing cells grown in buffered medium. (G) Cell survival and (H) ROS determination of strains grown in medium containing the indicated concentration of methionine as determined by flow cytometry. Results depicted are mean values ± SD. (I) Sch9 affects pHv. Vacuolar pH was measured during exponential growth and during glucose starvation using the ratiometric fluorescent pH indicator BCECF-AM. Results depicted are mean values ± SEM of four independent experiments. All differences between strains and conditions are statistically significant unless stated as ns (not significant). A detailed statistical analysis is presented in <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006835#pgen.1006835.s013" target="_blank">S3 Table</a></b>. See also <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006835#pgen.1006835.s007" target="_blank">S7</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006835#pgen.1006835.s008" target="_blank">S8</a> Figs</b>.</p

Effects on colony size and growth by deletion of <i>VPH1</i>, <i>STV1</i> and/or <i>SCH9</i>.

<p>(A, B) A synthetic sick phenotype arises when deletion of <i>SCH9</i> is combined with a fully dysfunctional V-ATPase. (A) Tetrad dissection of the diploid strain JW 04 952 (<i>sch9Δ/SCH9 vph1Δ/VPH1 stv1Δ/STV1</i>). (B) Colony sizes were calculated, normalized relative to WT and are shown as mean values ± SD. Letters indicate groups of strains with a significant difference in colony size (p < 0.001, one-way ANOVA). (C, D) Strains combining deletion of <i>SCH9</i> with a fully dysfunctional V-ATPase show a deteriorated growth phenotype. OD_600nm was followed over time in fully supplemented medium without buffer (C) or buffered at pH 5 (D). A representative experiment with at least 4 independent colonies for each strain is shown. Error bars represent SD from the mean. See also <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006835#pgen.1006835.s004" target="_blank">S4</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006835#pgen.1006835.s005" target="_blank">S5</a> Figs</b>.</p