EGOC inhibits TOROID polymerization by structurally activating TORC1.

Target of rapamycin complex 1 (TORC1) is a protein kinase controlling cell homeostasis and growth in response to nutrients and stresses. In Saccharomyces cerevisiae, glucose depletion triggers a redistribution of TORC1 from a dispersed localization over the vacuole surface into a large, inactive condensate called TOROID (TORC1 organized in inhibited domains). However, the mechanisms governing this transition have been unclear. Here, we show that acute depletion and repletion of EGO complex (EGOC) activity is sufficient to control TOROID distribution, independently of other nutrient-signaling pathways. The 3.9-Å-resolution structure of TORC1 from TOROID cryo-EM data together with interrogation of key interactions in vivo provide structural insights into TORC1-TORC1' and TORC1-EGOC interaction interfaces. These data support a model in which glucose-dependent activation of EGOC triggers binding to TORC1 at an interface required for TOROID assembly, preventing TORC1 polymerization and promoting release of active TORC1.

Ultrastructure expansion microscopy reveals the cellular architecture of budding and fission yeast.

The budding and fission yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe have served as invaluable model organisms to study conserved fundamental cellular processes. Although super-resolution microscopy has in recent years paved the way to a better understanding of the spatial organization of molecules in cells, its wide use in yeasts has remained limited due to the specific know-how and instrumentation required, contrasted with the relative ease of endogenous tagging and live-cell fluorescence microscopy. To facilitate super-resolution microscopy in yeasts, we have extended the ultrastructure expansion microscopy (U-ExM) method to both S. cerevisiae and S. pombe, enabling a 4-fold isotropic expansion. We demonstrate that U-ExM allows imaging of the microtubule cytoskeleton and its associated spindle pole body, notably unveiling the Sfi1p-Cdc31p spatial organization on the appendage bridge structure. In S. pombe, we validate the method by monitoring the homeostatic regulation of nuclear pore complex number through the cell cycle. Combined with NHS-ester pan-labelling, which provides a global cellular context, U-ExM reveals the subcellular organization of these two yeast models and provides a powerful new method to augment the already extensive yeast toolbox. This article has an associated First Person interview with Kerstin Hinterndorfer and Felix Mikus, two of the joint first authors of the paper.

TOR Signaling Is Going through a Phase.

Recently in Cell, Kato et al. (2019) and Yang et al. (2019) report that reversible oxidation of multiple methionines in a region of Pbp1, the yeast paralog of ataxin-2 protein, couples metabolic redox status to phase separation of Pbp1 into liquid-like condensates. In turn, Pbp1 condensates inhibit target of rapamycin complex 1 (TORC1) signaling and thereby induce autophagy and restore metabolic homeostasis.

Regulation of Cellular Metabolism through Phase Separation of Enzymes.

Metabolism is the sum of the life-giving chemical processes that occur within a cell. Proper regulation of these processes is essential for all organisms to thrive and prosper. When external factors are too extreme, or if internal regulation is corrupted through genetic or epigenetic changes, metabolic homeostasis is no longer achievable and diseases such as metabolic syndrome or cancer, aging, and, ultimately, death ensue. Metabolic reactions are catalyzed by proteins, and the in vitro kinetic properties of these enzymes have been studied by biochemists for many decades. These efforts led to the appreciation that enzyme activities can be acutely regulated and that this regulation is critical to metabolic homeostasis. Regulation can be mediated through allosteric interactions with metabolites themselves or via post-translational modifications triggered by intracellular signal transduction pathways. More recently, enzyme regulation has attracted the attention of cell biologists who noticed that change in growth conditions often triggers the condensation of diffusely localized enzymes into one or more discrete foci, easily visible by light microscopy. This reorganization from a soluble to a condensed state is best described as a phase separation. As summarized in this review, stimulus-induced phase separation has now been observed for dozens of enzymes suggesting that this could represent a widespread mode of activity regulation, rather than, or in addition to, a storage form of temporarily superfluous enzymes. Building on our recent structure determination of TOROIDs (TORc1 Organized in Inhibited Domain), the condensate formed by the protein kinase Target Of Rapamycin Complex 1 (TORC1), we will highlight that the molecular organization of enzyme condensates can vary dramatically and that future work aimed at the structural characterization of enzyme condensates will be critical to understand how phase separation regulates enzyme activity and consequently metabolic homeostasis. This information may ultimately facilitate the design of strategies to target the assembly or disassembly of specific enzymes condensates as a therapeutic approach to restore metabolic homeostasis in certain diseases.

Target of rapamycin complex 2-dependent phosphorylation of the coat protein Pan1 by Akl1 controls endocytosis dynamics in Saccharomyces cerevisiae.

Target of rapamycin complex 2 (TORC2) is a widely conserved serine/threonine protein kinase. In the yeast Saccharomyces cerevisiae, TORC2 is essential, playing a key role in plasma membrane homeostasis. In this role, TORC2 regulates diverse processes, including sphingolipid synthesis, glycerol production and efflux, polarization of the actin cytoskeleton, and endocytosis. The major direct substrate of TORC2 is the AGC-family kinase Ypk1. Ypk1 connects TORC2 signaling to actin polarization and to endocytosis via the flippase kinases Fpk1 and Fpk2. Here, we report that Fpk1 mediates TORC2 signaling to control actin polarization, but not endocytosis, via aminophospholipid flippases. To search for specific targets of these flippase kinases, we exploited the fact that Fpk1 prefers to phosphorylate Ser residues within the sequence RXS(L/Y)(D/E), which is present ∼90 times in the yeast proteome. We observed that 25 of these sequences are phosphorylated by Fpk1 in vitro We focused on one sequence hit, the Ark/Prk-family kinase Akl1, as this kinase previously has been implicated in endocytosis. Using a potent ATP-competitive small molecule, CMB4563, to preferentially inhibit TORC2, we found that Fpk1-mediated Akl1 phosphorylation inhibits Akl1 activity, which, in turn, reduces phosphorylation of Pan1 and of other endocytic coat proteins and ultimately contributes to a slowing of endocytosis kinetics. These results indicate that the regulation of actin polarization and endocytosis downstream of TORC2 is signaled through separate pathways that bifurcate at the level of the flippase kinases.

Cryo-EM structure of Saccharomyces cerevisiae target of rapamycin complex 2.

The target of rapamycin (TOR) kinase assembles into two distinct multiprotein complexes, conserved across eukaryote evolution. In contrast to TOR complex 1 (TORC1), TORC2 kinase activity is not inhibited by the macrolide rapamycin. Here, we present the structure of Saccharomyces cerevisiae TORC2 determined by electron cryo-microscopy. TORC2 contains six subunits assembling into a 1.4 MDa rhombohedron. Tor2 and Lst8 form the common core of both TOR complexes. Avo3/Rictor is unique to TORC2, but interacts with the same HEAT repeats of Tor2 that are engaged by Kog1/Raptor in mammalian TORC1, explaining the mutual exclusivity of these two proteins. Density, which we conclude is Avo3, occludes the FKBP12-rapamycin-binding site of Tor2's FRB domain rendering TORC2 rapamycin insensitive and recessing the kinase active site. Although mobile, Avo1/hSin1 further restricts access to the active site as its conserved-region-in-the-middle (CRIM) domain is positioned along an edge of the TORC2 active-site-cleft, consistent with a role for CRIM in substrate recruitment.

TORC1 organized in inhibited domains (TOROIDs) regulate TORC1 activity.

The target of rapamycin (TOR) is a eukaryotic serine/threonine protein kinase that functions in two distinct complexes, TORC1 and TORC2, to regulate growth and metabolism. GTPases, responding to signals generated by abiotic stressors, nutrients, and, in metazoans, growth factors, play an important but poorly understood role in TORC1 regulation. Here we report that, in budding yeast, glucose withdrawal (which leads to an acute loss of TORC1 kinase activity) triggers a similarly rapid Rag GTPase-dependent redistribution of TORC1 from being semi-uniform around the vacuolar membrane to a single, vacuole-associated cylindrical structure visible by super-resolution optical microscopy. Three-dimensional reconstructions of cryo-electron micrograph images of these purified cylinders demonstrate that TORC1 oligomerizes into a higher-level hollow helical assembly, which we name a TOROID (TORC1 organized in inhibited domain). Fitting of the recently described mammalian TORC1 structure into our helical map reveals that oligomerization leads to steric occlusion of the active site. Guided by the implications from our reconstruction, we present a TOR1 allele that prevents both TOROID formation and TORC1 inactivation in response to glucose withdrawal, demonstrating that oligomerization is necessary for TORC1 inactivation. Our results reveal a novel mechanism by which Rag GTPases regulate TORC1 activity and suggest that the reversible assembly and/or disassembly of higher-level structures may be an underappreciated mechanism for the regulation of protein kinases.

Compartmentalization of the endoplasmic reticulum in the early C. elegans embryos.

The one-cell Caenorhabditis elegans embryo is polarized to partition fate determinants between the cell lineages generated during its first division. Using fluorescence loss in photobleaching, we find that the endoplasmic reticulum (ER) of the C. elegans embryo is physically continuous throughout the cell, but its membrane is compartmentalized shortly before nuclear envelope breakdown into an anterior and a posterior domain, indicating that a diffusion barrier forms in the ER membrane between these two domains. Using mutants with disorganized ER, we show that ER compartmentalization is independent of the morphological transition that the ER undergoes in mitosis. In contrast, compartmentalization takes place at the position of the future cleavage plane in a par-3-dependent manner. Together, our data indicate that the ER membrane is compartmentalized in cells as diverse as budding yeast, mouse neural stem cells, and the early C. elegans embryo.

TORC2 Structure and Function.

The target of rapamycin (TOR) kinase functions in two multiprotein complexes, TORC1 and TORC2. Although both complexes are evolutionarily conserved, only TORC1 is acutely inhibited by rapamycin. Consequently, only TORC1 signaling is relatively well understood; and, at present, only mammalian TORC1 is a validated drug target, pursued in immunosuppression and oncology. However, the knowledge void surrounding TORC2 is dissipating. Acute inhibition of TORC2 with small molecules is now possible and structural studies of both TORC1 and TORC2 have recently been reported. Here we review these recent advances as well as observations made from tissue-specific mTORC2 knockout mice. Together these studies help define TORC2 structure-function relationships and suggest that mammalian TORC2 may one day also become a bona fide clinical target.

Molecular Basis of the Rapamycin Insensitivity of Target Of Rapamycin Complex 2.

Target of Rapamycin (TOR) plays central roles in the regulation of eukaryote growth as the hub of two essential multiprotein complexes: TORC1, which is rapamycin-sensitive, and the lesser characterized TORC2, which is not. TORC2 is a key regulator of lipid biosynthesis and Akt-mediated survival signaling. In spite of its importance, its structure and the molecular basis of its rapamycin insensitivity are unknown. Using crosslinking-mass spectrometry and electron microscopy, we determined the architecture of TORC2. TORC2 displays a rhomboid shape with pseudo-2-fold symmetry and a prominent central cavity. Our data indicate that the C-terminal part of Avo3, a subunit unique to TORC2, is close to the FKBP12-rapamycin-binding domain of Tor2. Removal of this sequence generated a FKBP12-rapamycin-sensitive TORC2 variant, which provides a powerful tool for deciphering TORC2 function in vivo. Using this variant, we demonstrate a role for TORC2 in G2/M cell-cycle progression.

The TAO kinase KIN-18 regulates contractility and establishment of polarity in the C. elegans embryo.

Cell polarity is crucial for many aspects of cell and developmental biology. Cytoskeleton remodeling plays an essential role in the establishment of cell polarity. In the Caenorhabditis elegans one-cell embryo, while the actomyosin cytoskeleton is required for asymmetric localization of the PAR proteins, anterior PAR proteins exert a feedback regulation on contractility. Here we identify the TAO kinase KIN-18 as a regulator of cortical contractility in the early embryo. KIN-18 negatively regulates cortical contractions in a RHO-1 dependent manner and regulates RHO-1 cortical localization. KIN-18 contributes to polarity establishment by regulating the position of the boundary between anterior and posterior PAR proteins. Although KIN-18 is involved in polarity establishment, depletion of KIN-18 restores contractions in a par-3 mutant indicating that kin-18 is epistatic to par-3. We suggest a model in which KIN-18 provides a link between the cytoskeleton remodeling and polarity machineries, uncovering a role for TAO kinases in the regulation of cell polarity.

The highly conserved eukaryotic DRG factors are required for efficient translation in a manner redundant with the putative RNA helicase Slh1.

Eukaryotic and archaeal DRG factors are highly conserved proteins with characteristic GTPase motifs. This suggests their implication in a central biological process, which has so far escaped detection. We show here that the two Saccharomyces cerevisiae DRGs form distinct complexes, RBG1 and RBG2, and that the former co-fractionate with translating ribosomes. A genetic screen for triple synthetic interaction demonstrates that yeast DRGs have redundant function with Slh1, a putative RNA helicase also associating with translating ribosomes. Translation and cell growth are severely impaired in a triple mutant lacking both yeast DRGs and Slh1, but not in double mutants. This new genetic assay allowed us to characterize the roles of conserved motifs present in these proteins for efficient translation and/or association with ribosomes. Altogether, our results demonstrate for the first time a direct role of the highly conserved DRG factors in translation and indicate that this function is redundantly shared by three factors. Furthermore, our data suggest that important cellular processes are highly buffered against external perturbation and, consequently, that redundantly acting factors may escape detection in current high-throughput binary genetic interaction screens.

Regulation of ARE transcript 3' end processing by the yeast Cth2 mRNA decay factor.

Regulation of mRNA decay is an important step modulating gene expression. The stability of numerous eukaryotic mRNAs is controlled by adenosine/uridine-rich elements (AREs) located in their 3'UTR. In Saccharomyces cerevisiae, the Cth2 protein stimulates the decay of target ARE mRNAs on iron starvation. Cth2, and its mammalian homologue tristetraprolin, contains a characteristic tandem CCCH zinc-finger essential for ARE binding and mRNA decay. We have performed a structure-function analysis of Cth2 to understand the mechanism(s) by which it destabilizes mRNAs. This indicated that a conserved N-terminal region of Cth2 is essential for its decay function but dispensable for RNA binding. Unexpectedly, Cth2 mutants lacking this domain blocked the normal 3' end processing of ARE mRNAs leading to the formation of extended transcripts. These can also be detected in mutant of the polyadenylation machinery. Consistently, Cth2 localization in the nucleus suggests that it may interfere with poly(A) site selection. Our analysis reveal that ARE-binding protein may affect mRNA 3' end processing and that this contributes to mRNA destabilization.

[Epigenetic control, development and natural genetic variation in plants]. / Contrôles épigénétiques, développement et variation génétique naturelle chez les plantes.

Plant life strategies differ radically from those of most animals. Plants are not motile, and can only face stress by developing appropriate physiological responses. In addition, many developmental decisions take place during post-embryonic life in plants, whereas vertebrate and invertebrate development is nearly complete by the time of birth. For instance, while the germ line is typically set aside early during embryogenesis in animals, plants produce gametes from stem cell populations that were previously used for the vegetative growth of shoots. Nevertheless, plants and animals have similar nuclear organization, chromatin constitution and gene content, which raises the question as to whether or not fundamental differences in the use of genetic information underlie their distinct life strategies. More specifically, we would like to know if chromatin and the epigenetically defined, heritable cell fates that it can confer play comparable roles in plants and animals. Here we review our current knowledge on chromatin-mediated epigenetic processes in plants. Based on available evidence, we argue that epigenetic regulation of gene expression plays a relatively minor role in plants compared to mammals. Conversely, plants appear to be more prone than other multicellular organisms to the induction of chromatin-based, epigenetically modified gene activity states that can be transmitted over many generations. These so-called "epimutations" may therefore represent a significant proportion of the natural genetic variation seen in plants. In humans, epimutations are frequently observed in cancers, and given their metastable nature, they could also play an important role in familial disorders that do not demonstrate clear Mendelian inheritance.