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

The peptidyl prolyl cis/trans isomerase Pin1/Ess1 inhibits phosphorylation and toxicity of tau in a yeast model for Alzheimer’s disease

Since hyperphosphorylation of protein tau is a crucial event in Alzheimer’s disease, additional mechanisms besides the interplay of kinase and phosphatase activities are investigated, such as the effect of the peptidyl prolyl cis/trans isomerase Pin1. This isomerase was shown to bind and isomerize phosphorylated protein tau, thereby restoring the microtubule associated protein function of tau as well as promoting the dephosphorylation of the protein by the trans-dependent phosphatase PP2A. In this study we used models based on Saccharomyces cerevisiae to further elucidate the influence of Pin1 and its yeast ortholog Ess1 on tau phosphorylation and self-assembly. We could demonstrate that in yeast, a lack of Pin1 isomerase activity leads to an increase in phosphorylation of tau at Thr231, comparable to AD brain and consistent with earlier findings in other model organisms. However, we could also distinguish an effect by Pin1 on other residues of tau, i.e. Ser235 and Ser198/199/202. Furthermore, depletion of Pin1 isomerase activity results in reduced growth of the yeast cells, which is enhanced upon expression of tau. This suggests that the accumulation of hyperphosphorylated and aggregation-prone tau causes cytotoxicity in yeast. This study introduces yeast as a valuable model organism to characterize in detail the effect of Pin1 on the biochemical characteristics of protein tau, more specifically its phosphorylation and aggregation

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

A Novel Tau Antibody Detecting the First Amino-Terminal Insert Reveals Conformational Differences Among Tau Isoforms

As human Tau undergoes pathologically relevant post-translational modifications when expressed in yeast, the use of humanized yeast models for the generation of novel Tau monoclonal antibodies has previously been proven to be successful. In this study, human Tau2N4R-ΔK280 purified from yeast was used for the immunization of mice and subsequent selection of high affinity Tau-specific monoclonal antibodies. The characterization of four novel antibodies in different Tau model systems yielded a phosphorylation-dependent antibody (15A10), an antibody directed to the first microtubule-binding repeat domain (16B12), a carboxy-terminal antibody (20G10) and an antibody targeting an epitope on the hinge of the first and second amino-terminal insert (18F12). The latter was found to be conformation-dependent, suggesting structural differences between the Tau splicing isoforms and allowing insight in the roles played by the amino-terminal inserts. As this monoclonal antibody also has the capacity to detect tangle-like structures in different transgenic Tau mice and neurofibrillary tangles in brain sections of patients diagnosed with Alzheimer's disease, we also tested the diagnostic potential of 18F12 in a pilot study and found this monoclonal antibody to have the ability to discriminate Alzheimer's disease patients from control individuals based on increased Tau levels in the cerebrospinal fluid.status: Published onlin

A Novel Tau Antibody Detecting the First Amino-Terminal Insert Reveals Conformational Differences Among Tau Isoforms

International audienceAs human Tau undergoes pathologically relevant post-translational modifications when expressed in yeast, the use of humanized yeast models for the generation of novel Tau monoclonal antibodies has previously been proven to be successful. In this study, human Tau2N4R-ΔK280 purified from yeast was used for the immunization of mice and subsequent selection of high affinity Tau-specific monoclonal antibodies. The characterization of four novel antibodies in different Tau model systems yielded a phosphorylation-dependent antibody (15A10), an antibody directed to the first microtubule-binding repeat domain (16B12), a carboxy-terminal antibody (20G10) and an antibody targeting an epitope on the hinge of the first and second amino-terminal insert (18F12). The latter was found to be conformation-dependent, suggesting structural differences between the Tau splicing isoforms and allowing insight in the roles played by the amino-terminal inserts. As this monoclonal antibody also has the capacity to detect tangle-like structures in different transgenic Tau mice and neurofibrillary tangles in brain sections of patients diagnosed with Alzheimer's disease, we also tested the diagnostic potential of 18F12 in a pilot study and found this monoclonal antibody to have the ability to discriminate Alzheimer's disease patients from control individuals based on increased Tau levels in the cerebrospinal fluid

Tau Monoclonal Antibody Generation Based on Humanized Yeast Models: IMPACT ON TAU OLIGOMERIZATION AND DIAGNOSTICS

A link between Tau phosphorylation and aggregation has been shown in different models for Alzheimer disease, including yeast. We used human Tau purified from yeast models to generate new monoclonal antibodies, of which three were further characterized. The first antibody, ADx201, binds the Tau proline-rich region independently of the phosphorylation status, whereas the second, ADx215, detects an epitope formed by the Tau N terminus when Tau is not phosphorylated at Tyr18. For the third antibody, ADx210, the binding site could not be determined because its epitope is probably conformational. All three antibodies stained tangle-like structures in different brain sections of THY-Tau22 transgenic mice and Alzheimer patients, and ADx201 and ADx210 also detected neuritic plaques in the cortex of the patient brains. In hippocampal homogenates from THY-Tau22 mice and cortex homogenates obtained from Alzheimer patients, ADx215 consistently stained specific low order Tau oligomers in diseased brain, which in size correspond to Tau dimers. ADx201 and ADx210 additionally reacted to higher order Tau oligomers and presumed prefibrillar structures in the patient samples. Our data further suggest that formation of the low order Tau oligomers marks an early disease stage that is initiated by Tau phosphorylation at N-terminal sites. Formation of higher order oligomers appears to require additional phosphorylation in the C terminus of Tau. When used to assess Tau levels in human cerebrospinal fluid, the antibodies permitted us to discriminate patients with Alzheimer disease or other dementia like vascular dementia, indicative that these antibodies hold promising diagnostic potential

Sch9 does not impact on vesicular trafficking.

<p>(A-E) Sorting and processing of vacuolar proteases is not impaired in exponentially growing <i>sch9Δ</i> cells. Processing of CPY (A), ALP (C) and Ape1 (E) was examined by Western blot. * represents cross-reacting band. Intracellular localization of CPY-GFP (B) and GFP-Pho8 (D) was examined by fluorescence microscopy. (F) Sch9 affects basal and non-specific autophagy. Exponentially growing cells expressing Pho8<i>Δ</i>60 were shifted to nitrogen starvation medium. Samples were taken at the indicated time points, proteins extracted, and specific activities determined. Results are normalized to the activity of a WT strain starved for 24h. Mean values ± SD are shown (unpaired t-test). See also <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006835#pgen.1006835.s001" target="_blank">S1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006835#pgen.1006835.s002" target="_blank">S2</a> Figs</b>.</p

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

Systematic analysis of genetic interaction partners of <i>SCH9</i>.

<p>(A) <i>SCH9</i> genetic interaction network. Osprey software was used to graphically display the relationships between genes identified in the SGA screening. Synthetic lethal interactions are connected by green lines; protein-protein interactions are shown in gray. Nodes are colored by process. Circles indicate well-defined protein complexes or group of genes that are functionally related. For clarity reasons, not all identified genes, nor all known interactions are shown. (B) Hypergeometric enrichment organized according to GO function, process and component. See also <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006835#pgen.1006835.s011" target="_blank">S1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006835#pgen.1006835.s012" target="_blank">S2</a> Tables</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