The architecture of the Rag GTPase signaling network

The evolutionarily conserved target of rapamycin complex 1 (TORC1) couples an array of intra- and extracellular stimuli to cell growth, proliferation and metabolism, and its deregulation is associated with various human pathologies such as immunodeficiency, epilepsy, and cancer. Among the diverse stimuli impinging on TORC1, amino acids represent essential input signals, but how they control TORC1 has long remained a mystery. The recent discovery of the Rag GTPases, which assemble as heterodimeric complexes on vacuolar/lysosomal membranes, as central elements of an amino acid signaling network upstream of TORC1 in yeast, flies, and mammalian cells represented a breakthrough in this field. Here, we review the architecture of the Rag GTPase signaling network with a special focus on structural aspects of the Rag GTPases and their regulators in yeast and highlight both the evolutionary conservation and divergence of the mechanisms that control Rag GTPases

Conventional and emerging roles of the energy sensor Snf1/AMPK in Saccharomyces cerevisiae

All proliferating cells need to match metabolism, growth and cell cycle progression with nutrient availability to guarantee cell viability in spite of a changing environment. In yeast, a signaling pathway centered on the effector kinase Snf1 is required to adapt to nutrient limitation and to utilize alternative carbon sources, such as sucrose and ethanol. Snf1 shares evolutionary conserved functions with the AMP-activated Kinase (AMPK) in higher eukaryotes which, activated by energy depletion, stimulates catabolic processes and, at the same time, inhibits anabolism. Although the yeast Snf1 is best known for its role in responding to a number of stress factors, in addition to glucose limitation, new unconventional roles of Snf1 have recently emerged, even in glucose repressing and unstressed conditions. Here, we review and integrate available data on conventional and non-conventional functions of Snf1 to better understand the complexity of cellular physiology which controls energy homeostasis

TORC1 coordinates the conversion of Sic1 from a target to an inhibitor of cyclin-CDK-Cks1

Eukaryotic cell cycle progression through G1–S is driven by hormonal and growth- related signals that are transmitted by the target of rapamycin complex 1 (TORC1) pathway. In yeast, inactivation of TORC1 restricts G1–S transition due to the rapid clearance of G1 cyclins (Cln) and the stabilization of the B-type cyclin (Clb) cyclin- dependent kinase (CDK) inhibitor Sic1. The latter mechanism remains mysterious but requires the phosphorylation of Sic1-Thr173 by Mpk1 and inactivation of the Sic1- pThr173-targeting phosphatase (PP2ACdc55) through greatwall kinase-activated endosulfines. Here we show that the Sic1-pThr173 residue serves as a specific docking site for the CDK phospho-acceptor subunit Cks1 that sequesters, together with a C-terminal Clb5-binding motif in Sic1, Clb5-CDK-Cks1 complexes, thereby preventing them from flagging Sic1 for ubiquitin-dependent proteolysis. Interestingly, this functional switch of Sic1 from a target to an inhibitor of cyclin-CDK-Cks1 also operates in proliferating cells and is coordinated by the greatwall kinase, which responds to both Cln-CDK-dependent cell-cycle and TORC1-mediated nutritional cues

The Yeast Protein Kinase Sch9 Functions as a Central Nutrient-Responsive Hub That Calibrates Metabolic and Stress-Related Responses

Funding Information: This research was funded by the Canton of Fribourg and the Swiss National Science Foundation (310030_166474/184671) to C.D.V., the Fonds Wetenschappelijk Onderzoek (FWO)-Vlaanderen (G069413, G0C7222N) and KU Leuven (C14/17/063, C14/21/095) to J.W., Biotechnology and Biological Sciences Research Council (BB/V016334/1) to RH, and Portuguese National funds, through the Foundation for Science and Technology (FCT)—project UIDB/50026/2020 and UIDP/50026/2020 for P.L. and B.S.-M. B.S.-M. was funded by FCT, grant number DL 57/2016.Peer reviewedPublisher PD

Expanded metabolite coverage of Saccharomyces cerevisiae extract through improved chloroform/methanol extraction and tert-butyldimethylsilyl derivatization

AbstractWe present an improved extraction and derivatization protocol for GC–MS analysis of amino/non-amino acids in Saccharomyces cerevisiae. Yeast cells were extracted with chloroform: aqueous-methanol (1:1, v/v) and the resulting non-polar and polar extracts combined and dried for derivatization. Polar and non-polar metabolites were derivatized using tert-butyldimethylsilyl (t-BDMS) dissolved in acetonitrile. Using microwave treatment of the samples, the derivatization process could be completed within 2 h (from >20 h of the conventional method), providing fully derivatized metabolites that contain multiple derivatizable organic functional groups. This results in a single derivative from one metabolite, leading to increased accuracy and precision for identification and quantification of the method. Analysis of combined fractions allowed the method to expand the coverage of detected metabolites from polar metabolites i.e. amino acids, organic acids and non-polar metabolites i.e. fatty alcohols and long-chain fatty acids which are normally non detectable. The recoveries of the extraction method was found at 88 ± 4%, RSD, N = 3 using anthranilic acid as an internal standard. The method promises to be a very useful tool in various aspects of biotechnological applications i.e. development of cell factories, metabolomics profiling, metabolite identification, 13C-labeled flux analysis or semi-quantitative analysis of metabolites in yeast samples

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

The GTPase activating protein Gyp7 regulates Rab7/Ypt7 activity on late endosomes

Organelles of the endomembrane system contain Rab GTPases as identity markers. Their localization is determined by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). It remains largely unclear how these regulators are specifically targeted to organelles and how their activity is regulated. Here, we focus on the GAP Gyp7, which acts on the Rab7-like Ypt7 protein in yeast, and surprisingly observe the protein exclusively in puncta proximal to the vacuole. Mistargeting of Gyp7 to the vacuole strongly affects vacuole morphology, suggesting that endosomal localization is needed for function. In agreement, efficient endolysosomal transport requires Gyp7. In vitro assays reveal that Gyp7 requires a distinct lipid environment for membrane binding and activity. Overexpression of Gyp7 concentrates Ypt7 in late endosomes and results in resistance to rapamycin, an inhibitor of the target of rapamycin complex 1 (TORC1), suggesting that these late endosomes are signaling endosomes. We postulate that Gyp7 is part of regulatory machinery involved in late endosome function

Multicentre Italian study of SARS-CoV-2 infection in children and adolescents, preliminary data as at 10 April 2020

Data on features of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in children and adolescents are scarce. We report preliminary results of an Italian multicentre study comprising 168 laboratory-confirmed paediatric cases (median: 2.3 years, range: 1 day-17.7 years, 55.9% males), of which 67.9% were hospitalised and 19.6% had comorbidities. Fever was the most common symptom, gastrointestinal manifestations were frequent; two children required intensive care, five had seizures, 49 received experimental treatments and all recovered