Pib2 as an Emerging Master Regulator of Yeast TORC1

Funding: The APC was funded by the Biotechnology and Biological Sciences Research Council (BB/V016334/1). Acknowledgments: I thank Tatsuya Maeda and Mirai Tanigawa (Hamamatsu University School of Medicine) and Andreas Milias-Argeitis (University of Groningen) for critical reading of the manuscript. I thank Katy Betchetti for English editing.Peer reviewedPublisher PD

Ypk1/Ypk2 kinases maintain Rho1 at the plasma membrane by flippase-dependent lipid remodelling after membrane stresses

The plasma membrane (PM) is frequently challenged by mechanical stresses. In budding yeast, TORC2-Ypk1/Ypk2 kinase cascade plays a critical role in PM stress responses by reorganizing the actin cytoskeleton via Rho1 GTPase. However, the molecular mechanism by which TORC2-Ypk1/Ypk2 regulates Rho1 is not well defined. Here, we found that Ypk1/Ypk2 maintain PM localization of Rho1 under PM stress via spatial reorganization of the lipids including phosphatidylserine (PS). Genetic evidence suggests that this process is mediated by the Lem3-containing lipid flippase. We propose that TORC2-Ypk1/Ypk2-Lem3 axis-mediated lipid remodelling is a backup mechanism for PM anchoring of Rho1 after PM stress-induced acute degradation of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), which is responsible for Rho1 localization in a normal condition. Since all the signaling molecules studied here are conserved in higher eukaryotes, our findings may represent a general mechanism to cope with PM stress

A spatially and functionally distinct pool of TORC1 defines signaling endosomes in yeast

The evolutionarily conserved target of rapamycin complex 1 (TORC1) regulates cell growth in a homeostatic manner by tuning anabolic and catabolic processes in response to nutritional and hormonal cues. Interestingly, rather than being localized at the plasma membrane as perhaps expected for an integrator of extracellular signals, TORC1 mainly localizes at vacuolar (in yeast) and lysosomal (in more complex eukaryotes) membranes where it seems optimally placed to sense both the nutrient status within the cytoplasm and the vacuolar/lysosomal compartment. How TORC1 controls downstream targets that are distant from the vacuole/lysosome, is currently poorly understood. In this context, we recently identified and characterized 2 spatially and functionally distinct pools of TORC1 in the budding yeast Saccharomyces cerevisiae: one at the vacuole that promotes protein synthesis, and another one at endosomes that inhibits protein degradation. Thus, our findings highlight the presence of spatially separated pools of TORC1 that are commissioned with functionally specific tasks within cells. In addition, they pinpoint the existence of signaling endosomes in yeast, which raises numerous new questions that are warranted to direct future research in this area

TORC1 specifically inhibits microautophagy through ESCRT-0

Nutrient starvation induces the degradation of specific plasma membrane proteins through the multivesicular body (MVB) sorting pathway and of vacuolar membrane proteins through microautophagy. Both of these processes require the gateway protein Vps27, which recognizes ubiquitinated cargo proteins at phosphatidylinositol 3-phosphate-rich membranes as part of a heterodimeric complex coined endosomal sorting complex required for transport 0. The target of rapamycin complex 1 (TORC1), a nutrient-activated central regulator of cell growth, directly phosphorylates Vps27 to antagonize its function in microautophagy, but whether this also serves to restrain MVB sorting at endosomes is still an open question. Here, we show that TORC1 inhibits both the MVB pathway-driven turnover of the plasma membrane-resident high-affinity methionine permease Mup1 and the inositol transporter Itr1 and the microautophagy-dependent degradation of the vacuolar membrane-associated v- ATPase subunit Vph1. Using a Vps277D variant that mimics the TORC1- phosphorylated state of Vps27, we further show that cargo sorting of Vph1 at the vacuolar membrane, but not of Mup1 and Itr1 at endosomes, is sensitive to the TORC1-controlled modifications of Vps27. Thus, TORC1 specifically modulates microautophagy through phosphorylation of Vps27, but controls MVB sorting through alternative mechanisms

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

Multilayered control of protein turnover by torc1 and atg1

The target of rapamycin complex 1 (TORC1) is a master regulator of cell homeostasis, which promotes anabolic reactions and synchronously inhibits catabolic processes such as autophagy-mediated protein degradation. Its prime autophagy target is Atg13, a subunit of the Atg1 kinase complex that acts as the gatekeeper of canonical autophagy. To study whether the activities of TORC1 and Atg1 are coupled through additional, more intricate control mechanisms than simply this linear pathway, we analyzed the epistatic relationship between TORC1 and Atg1 by using quantitative phosphoproteomics. Our in vivo data, combined with targeted in vitro TORC1 and Atg1 kinase assays, not only uncover numerous TORC1 and Atg1 effectors, but also suggest distinct bi-directional regulatory feedback loops and characterize Atg29 as a commonly regulated downstream target of both TORC1 and Atg1. Thus, an exquisitely multilayered regulatory network appears to coordinate TORC1 and Atg1 activities to robustly tune autophagy in response to nutritional cues

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

Spatially distinct pools of TORC1 balance protein homeostasis

The eukaryotic TORC1 kinase is a homeostatic controller of growth that integrates nutritional cues and mediates signals primarily from the surface of lysosomes or vacuoles. Amino acids activate TORC1 via the Rag GTPases that combine into structurally conserved multi-protein complexes such as the EGO complex (EGOC) in yeast. Here we show that Ego1, which mediates membrane-anchoring of EGOC via lipid modifications that it acquires while traveling through the trans-Golgi network, is separately sorted to vacuoles and perivacuolar endosomes. At both surfaces, it assembles EGOCs, which regulate spatially distinct pools of TORC1 that impinge on functionally divergent effectors: vacuolar TORC1 predominantly targets Sch9 to promote protein synthesis, whereas endosomal TORC1 phosphorylates Atg13 and Vps27 to inhibit macroautophagy and ESCRT-driven microautophagy, respectively. Thus, the coordination of three key regulatory nodes in protein synthesis and degradation critically relies on a division of labor between spatially sequestered populations of TORC

Zds1/Zds2-PP2ACdc55 complex specifies signaling output from Rho1 GTPase

Acknowledgments We thank David Pellman, John Pringle, Daniel Lew, Masaki Mizunuma, Kenji Irie, and the Yeast Genome Resource Center for yeast strains and plasmids and members of Yoshida Laboratory and Keiko Kono for their support. Multicopy suppressor screening for gef∆ was initiated in the Pellman Laboratory with the help of Didem Ilter. This research was supported by Sprout grant from Brandeis University (E.M. Jonasson and S. Yoshida), an American-Italian Cancer Foundation Postdoctoral fellowship (V. Rossio), and a Massachusetts Life Sciences Center grant (S. Yoshida).Peer reviewedPublisher PD

Pib2 as an Emerging Master Regulator of Yeast TORC1

Cell growth is dynamically regulated in response to external cues such as nutrient availability, growth factor signals, and stresses. Central to this adaptation process is the Target of Rapamycin Complex 1 (TORC1), an evolutionarily conserved kinase complex that fine-tunes an enormous number of cellular events. How upstream signals are sensed and transmitted to TORC1 has been intensively studied in major model organisms including the budding yeast Saccharomyces cerevisiae. This field recently saw a breakthrough: the identification of yeast phosphatidylInositol(3)-phosphate binding protein 2 (Pib2) protein as a critical regulator of TORC1. Although the study of Pib2 is still in its early days, multiple groups have provided important mechanistic insights on how Pib2 relays nutrient signals to TORC1. There remain, on the other hand, significant gaps in our knowledge and mysteries that warrant further investigations. This is the first dedicated review on Pib2 that summarizes major findings and outstanding questions around this emerging key player in cell growth regulation