Target of rapamycin signaling mediates vacuolar fragmentation.

In eukaryotic cells, cellular homeostasis requires that different organelles respond to intracellular as well as environmental signals and modulate their behavior as conditions demand. Understanding the molecular mechanisms required for these changes remains an outstanding goal. One such organelle is the lysosome/vacuole, which undergoes alterations in size and number in response to environmental and physiological stimuli. Changes in the morphology of this organelle are mediated in part by the equilibrium between fusion and fission processes. While the fusion of the yeast vacuole has been studied intensively, the regulation of vacuolar fission remains poorly characterized by comparison. In recent years, a number of studies have incorporated genome-wide visual screens and high-throughput microscopy to identify factors required for vacuolar fission in response to diverse cellular insults, including hyperosmotic and endoplasmic reticulum stress. Available evidence now demonstrates that the rapamycin-sensitive TOR network, a master regulator of cell growth, is required for vacuolar fragmentation in response to stress. Importantly, many of the genes identified in these studies provide new insights into potential links between the vacuolar fission machinery and TOR signaling. Together these advances both extend our understanding of the regulation of vacuolar fragmentation in yeast as well as underscore the role of analogous events in mammalian cells.

TOR complex 2-Ypk1 signaling is an essential positive regulator of the general amino acid control response and autophagy.

The highly conserved Target of Rapamycin (TOR) kinase is a central regulator of cell growth and metabolism in response to nutrient availability. TOR functions in two structurally and functionally distinct complexes, TOR Complex 1 (TORC1) and TOR Complex 2 (TORC2). Through TORC1, TOR negatively regulates autophagy, a conserved process that functions in quality control and cellular homeostasis and, in this capacity, is part of an adaptive nutrient deprivation response. Here we demonstrate that during amino acid starvation TOR also operates independently as a positive regulator of autophagy through the conserved TORC2 and its downstream target protein kinase, Ypk1. Under these conditions, TORC2-Ypk1 signaling negatively regulates the Ca(2+)/calmodulin-dependent phosphatase, calcineurin, to enable the activation of the amino acid-sensing eIF2α kinase, Gcn2, and to promote autophagy. Our work reveals that the TORC2 pathway regulates autophagy in an opposing manner to TORC1 to provide a tunable response to cellular metabolic status.

Regulation of ceramide synthase by casein kinase 2-dependent phosphorylation in Saccharomyces cerevisiae.

Complex sphingolipids are important components of eukaryotic cell membranes and, together with their biosynthetic precursors, including sphingoid long chain bases and ceramides, have important signaling functions crucial for cell growth and survival. Ceramides are produced at the endoplasmic reticulum (ER) membrane by a multicomponent enzyme complex termed ceramide synthase (CerS). In budding yeast, this complex is composed of two catalytic subunits, Lac1 and Lag1, as well as an essential regulatory subunit, Lip1. Proper formation of ceramides by CerS has been shown previously to require the Cka2 subunit of casein kinase 2 (CK2), a ubiquitous enzyme with multiple cellular functions, but the precise mechanism involved has remained unidentified. Here we present evidence that Lac1 and Lag1 are direct targets for CK2 and that phosphorylation at conserved positions within the C-terminal cytoplasmic domain of each protein is required for optimal CerS activity. Our data suggest that phosphorylation of Lac1 and Lag1 is important for proper localization and distribution of CerS within the ER membrane and that phosphorylation of these sites is functionally linked to the COP I-dependent C-terminal dilysine ER retrieval pathway. Together, our data identify CK2 as an important regulator of sphingolipid metabolism, and additionally, because both ceramides and CK2 have been implicated in the regulation of cancer, our findings may lead to an enhanced understanding of their relationship in health and disease.

Cell growth control: mTOR takes on fat.

In a recent issue of Cell Metabolism, Porstmann et al. (2008) demonstrate that fatty acid biosynthesis, under the transcriptional control of SREBP1, is regulated by the rapamycin-sensitive mTOR signaling network, thus expanding the scope of biosynthetic processes integrated by mTOR.

Plasma membrane recruitment and activation of the AGC kinase Ypk1 is mediated by target of rapamycin complex 2 (TORC2) and its effector proteins Slm1 and Slm2.

The yeast AGC kinase orthologs Ypk1 and Ypk2 control several important cellular processes, including actin polarization, endocytosis, and sphingolipid metabolism. Activation of Ypk1/2 requires phosphorylation by kinases localized at the plasma membrane (PM), including the 3-phosphoinositide-dependent kinase 1 orthologs Pkh1/Pkh2 and the target of rapamycin complex 2 (TORC2). Unlike their mammalian counterparts SGK and Akt, Ypk1 and Ypk2 lack an identifiable lipid-targeting motif; therefore, how these proteins are recruited to the PM has remained elusive. To explore Ypk1/2 function, we constructed ATP analog-sensitive alleles of both kinases and monitored global changes in gene expression following their inhibition, where we observed increased expression of stress-responsive target genes controlled by Ca(2+)-dependent phosphatase calcineurin. TORC2 has been shown previously to negatively regulate calcineurin in part by phosphorylating two related proteins, Slm1 and Slm2, which associate with the PM via plextrin homology domains. We therefore investigated the relationship between Slm1 and Ypk1 and discovered that these proteins interact physically and that Slm1 recruits Ypk1 to the PM for phosphorylation by TORC2. We observed further that these steps facilitate subsequent phosphorylation of Ypk1 by Pkh1/2. Remarkably, a requirement for Slm1, can be bypassed by fusing the plextrin homology domain of Slm1 alone onto Ypk1, demonstrating that the essential function of Slm1 is largely attributable to its role in Ypk1 activation. These findings both extend the scope of cellular processes regulated by Ypk1/2 to include negative regulation of calcineurin and broaden the repertoire of mechanisms for membrane recruitment and activation of a protein kinase.

Regulation of ceramide biosynthesis by TOR complex 2.

Ceramides and sphingoid long-chain bases (LCBs) are precursors to more complex sphingolipids and play distinct signaling roles crucial for cell growth and survival. Conserved reactions within the sphingolipid biosynthetic pathway are responsible for the formation of these intermediates. Components of target of rapamycin complex 2 (TORC2) have been implicated in the biosynthesis of sphingolipids in S. cerevisiae; however, the precise step regulated by this complex remains unknown. Here we demonstrate that yeast cells deficient in TORC2 activity are impaired for de novo ceramide biosynthesis both in vivo and in vitro. We find that TORC2 regulates this step in part by activating the AGC kinase Ypk2 and that this step is antagonized by the Ca2+/calmodulin-dependent phosphatase calcineurin. Because Ypk2 is activated independently by LCBs, the direct precursors to ceramides, our data suggest a model wherein TORC2 signaling is coupled with LCB levels to control Ypk2 activity and, ultimately, regulate ceramide formation.

TOR signaling and S6 kinase 1: Yeast catches up.

Conservation of the rapamycin-sensitive TOR signaling network among eukaryotes has been instrumental to the rapid progress made in this field in recent years. A recent report in Molecular Cell (Urban et al., 2007) now extends this conservation to include Sch9, an AGC protein kinase family member from S. cerevisiae, which appears to be the long sought after yeast ortholog of mammalian S6 kinase 1 (S6K1) and a direct target for the rapamycin-sensitive TOR complex I.

The origin story of rapamycin: systemic bias in biomedical research and cold war politics.

METEI (Medical Expedition to Easter Island) was a Canadian-led expedition to Easter Island in 1964 that led to the discovery of rapamycin, launching a billion-dollar drug industry and major field of biomedical research. Stanley's Dream, by medical historian Jacalyn Duffin, provides remarkable details about METEI and raises important and timely questions about systemic bias in biomedical studies, the relationship between science and geopolitics, as well as obligations of pharmaceutical companies to indigenous communities. As such, this book is a must-read for those interested in the intersection of science and society as well as anyone who has used rapamycin, or one of many derivatives, in their laboratory or clinic.

Identification of defined structural elements within TOR2 kinase required for TOR complex 2 assembly and function in Saccharomyces cerevisiae.

The mammalian target of rapamycin (mTOR) is a large protein kinase that assembles into two multisubunit protein complexes, mTORC1 and mTORC2, to regulate cell growth in eukaryotic cells. While significant progress has been made in our understanding of the composition and structure of these complexes, important questions remain regarding the role of specific sequences within mTOR important for complex formation and activity. To address these issues, we have used a molecular genetic approach to explore TOR complex assembly in budding yeast, where two closely related TOR paralogues, TOR1 and TOR2, partition preferentially into TORC1 versus TORC2, respectively. We previously identified an ∼500-amino-acid segment within the N-terminal half of each protein, termed the major assembly specificity (MAS) domain, which can govern specificity in formation of each complex. In this study, we have extended the use of chimeric TOR1-TOR2 genes as a "sensitized" genetic system to identify specific subdomains rendered essential for TORC2 function, using synthetic lethal interaction analyses. Our findings reveal important design principles underlying the dimeric assembly of TORC2 as well as identifying specific segments within the MAS domain critical for TORC2 function, to a level approaching single-amino-acid resolution. Together these findings highlight the complex and cooperative nature of TOR complex assembly and function.

Regulation of ribosome biogenesis: where is TOR?

Li et al., (2006) have shown that TOR complex 1 in yeast binds directly to the rDNA promoter and thereby activates Pol I-dependent synthesis of 35S RNA. This is an important advance in the understanding of how ribosome biogenesis is regulated in response to environmental conditions.

Probing the membrane environment of the TOR kinases reveals functional interactions between TORC1, actin, and membrane trafficking in Saccharomyces cerevisiae.

The TOR kinases are regulators of growth in eukaryotic cells that assemble into two distinct protein complexes, TORC1 and TORC2, where TORC1 is inhibited by the antibiotic rapamycin. Present models favor a view wherein TORC1 regulates cell mass accumulation, and TORC2 regulates spatial aspects of growth, including organization of the actin cytoskeleton. Here, we demonstrate that in yeast both TORC1 and TORC2 fractionate with a novel form of detergent-resistant membranes that are distinct from detergent-resistant plasma membrane "rafts." Proteomic analysis of these TOR-associated membranes revealed the presence of regulators of endocytosis and the actin cytoskeleton. Genetic analyses revealed a significant number of interactions between these components and TORC1, demonstrating a functional link between TORC1 and actin/endocytosis-related genes. Moreover, we found that inhibition of TORC1 by rapamycin 1) disrupted actin polarization, 2) delayed actin repolarization after glucose starvation, and 3) delayed accumulation of lucifer yellow within the vacuole. By combining our genetic results with database mining, we constructed a map of interactions that led to the identification of additional genetic interactions between TORC1 and components involved in membrane trafficking. Together, these results reveal the broad scope of cellular processes influenced by TORC1, and they underscore the functional overlap between TORC1 and TORC2.

Redesigning TOR Kinase to Explore the Structural Basis for TORC1 and TORC2 Assembly.

TOR is a serine/threonine protein kinase that assembles into distinct TOR Complexes 1 and 2 (TORC1 or TORC2) to regulate cell growth. In mammalian cells, a single mTOR incorporates stably into mTORC1 and mTORC2. By contrast, in Saccharomyces cerevisiae, two highly similar Tor1 and Tor2 proteins exist, where Tor1 assembles exclusively into TORC1 and Tor2 assembles preferentially into TORC2. To gain insight into TOR complex assembly, we used this bifurcation in yeast to identify structural elements within Tor1 and Tor2 that govern their complex specificity. We have identified a concise region of ~500 amino acids within the N-terminus of Tor2, which we term the Major Assembly Specificity (MAS) domain, that is sufficient to confer significant TORC2 activity when placed into an otherwise Tor1 protein. Consistently, introduction of the corresponding MAS domain from Tor1 into an otherwise Tor2 is sufficient to confer stable association with TORC1-specific components. Remarkably, much like mTOR, this latter chimera also retains stable interactions with TORC2 components, indicating that determinants throughout Tor1/Tor2 contribute to complex specificity. Our findings are in excellent agreement with recent ultrastructural studies of TORC1 and TORC2, where the MAS domain is involved in quaternary interactions important for complex formation and/or stability.

Comment on "Sterilizing immunity in the lung relies on targeting fungal apoptosis-like programmed cell death".

Shlezinger et al (Reports, 8 September 2017, p. 1037) report that the common fungus Aspergillus fumigatus, a cause of aspergillosis, undergoes caspase-dependent apoptosis-like cell death triggered by lung neutrophils. However, the technologies they used do not provide reliable evidence that fungal cells die via a protease signaling cascade thwarted by a fungal caspase inhibitor homologous to human survivin.

Mks1 in concert with TOR signaling negatively regulates RTG target gene expression in S. cerevisiae.

The target of rapamycin (TOR) signaling pathway allows eukaryotic cells to regulate their growth in response to nutritional cues. In S. cerevisiae, TOR controls the expression of genes involved in several nutrient-responsive biosynthetic pathways. In particular, we have demonstrated that TOR negatively regulates a concise cluster of genes (termed RTG target genes) that encode mitochondrial and peroxisomal enzymes required for de novo amino acid biosynthesis. TOR acts in part by regulating the subcellular localization of the Rtg1/Rtg3 transcription factor complex. Nuclear entry of this complex requires the cytoplasmic protein Rtg2, whose precise function has remained ill defined. Here we establish that the likely role of Rtg2 is to antagonize the activity of another protein, Mks1, which we demonstrate is itself a negative regulator of RTG target gene activation. Results of epistasis analyses suggest that Rtg2 and Mks1 act downstream of TOR and upstream of Rtg1 and Rtg3. Moreover, we find that Mks1 phosphorylation responds to TOR as well as to each of the Rtg1-Rtg3 proteins, indicative of complex regulation within this branch of TOR signaling. In addition to RTG target genes, microarray analysis reveals robust expression of lysine biosynthetic genes in mks1Delta cells, which depends on a functional RTG pathway. This latter result provides a molecular explanation for the previous identification of MKS1 as LYS80, a negative regulator of lysine biosynthesis [8].

Tor kinases are in distinct membrane-associated protein complexes in Saccharomyces cerevisiae.

Tor1p and Tor2p kinases, targets of the immune-suppressive antibiotic rapamycin, are components of a highly conserved signaling network that couples nutrient availability and cell growth. To gain insight into the molecular basis underlying Tor-dependent signaling, we used cell fractionation and immunoaffinity chromatography to examine the physical environment of Tor2p. We found that the majority of Tor2p associates with a membrane-bound compartment along with at least four other proteins, Avo1p-Avo3p and Lst8p. Using immunogold electron microscopy, we observed that Tor2p, as well as Tor1p, localizes in punctate clusters to regions adjacent to the plasma membrane and within the cell interior, often in association with characteristic membranous tracks. Cell fractionation, coimmunoprecipitation, and immunogold electron microscopy experiments confirmed that Lst8 associates with both Tor2p as well as Tor1p at these membranous sites. In contrast, we find that Kog1, the yeast homologue of the mammalian Tor regulatory protein Raptor, interacts preferentially with Tor1p. These findings provide evidence for the existence of Tor signaling complexes that contain distinct as well as overlapping components. That these complexes colocalize to a membrane-bound compartment suggests an intimate relationship between membrane-mediated signaling and Tor activity.

Stress-response transcription factors Msn2 and Msn4 couple TORC2-Ypk1 signaling and mitochondrial respiration to ATG8 gene expression and autophagy.

Macroautophagy/autophagy is a starvation and stress-induced catabolic process critical for cellular homeostasis and adaptation. Several Atg proteins are involved in the formation of the autophagosome and subsequent degradation of cytoplasmic components, a process termed autophagy flux. Additionally, the expression of several Atg proteins, in particular Atg8, is modulated transcriptionally, yet the regulatory mechanisms involved remain poorly understood. Here we demonstrate that the AGC kinase Ypk1, target of the rapamycin-insensitive TORC2 signaling pathway, controls ATG8 expression by repressing the heterodimeric Zinc-finger transcription factors Msn2 and Msn4. We find that Msn2 and Msn4 promote ATG8 expression downstream of the histone deacetylase complex (HDAC) subunit Ume6, a previously identified negative regulator of ATG8 expression. Moreover, we demonstrate that TORC2-Ypk1 signaling is functionally linked to distinct mitochondrial respiratory complexes. Surprisingly, we find that autophagy flux during amino acid starvation is also dependent upon Msn2-Msn4 activity, revealing a broad role for these transcription factors in the autophagy response.

Mitochondrial respiration links TOR complex 2 signaling to calcium regulation and autophagy.

The target of rapamycin (TOR) kinase is a conserved regulator of cell growth and functions within 2 different protein complexes, TORC1 and TORC2, where TORC2 positively controls macroautophagy/autophagy during amino acid starvation. Under these conditions, TORC2 signaling inhibits the activity of the calcium-regulated phosphatase calcineurin and promotes the general amino acid control (GAAC) response and autophagy. Here we demonstrate that TORC2 regulates calcineurin by controlling the respiratory activity of mitochondria. In particular, we find that mitochondrial oxidative stress affects the calcium channel regulatory protein Mid1, which we show is an essential upstream activator of calcineurin. Thus, these findings describe a novel regulation for autophagy that involves TORC2 signaling, mitochondrial respiration, and calcium homeostasis.

Sterol transporters at membrane contact sites regulate TORC1 and TORC2 signaling.

Membrane contact sites (MCSs) function to facilitate the formation of membrane domains composed of specialized lipids, proteins, and nucleic acids. In cells, membrane domains regulate membrane dynamics and biochemical and signaling pathways. We and others identified a highly conserved family of sterol transport proteins (Ltc/Lam) localized at diverse MCSs. In this study, we describe data indicating that the yeast family members Ltc1 and Ltc3/4 function at the vacuole and plasma membrane, respectively, to create membrane domains that partition upstream regulators of the TORC1 and TORC2 signaling pathways to coordinate cellular stress responses with sterol homeostasis.

Calcium channel regulator Mid1 links TORC2-mediated changes in mitochondrial respiration to autophagy.

Autophagy is a catabolic process that recycles cytoplasmic contents and is crucial for cell survival during stress. The target of rapamycin (TOR) kinase regulates autophagy as part of two distinct protein complexes, TORC1 and TORC2. TORC1 negatively regulates autophagy according to nitrogen availability. In contrast, TORC2 functions as a positive regulator of autophagy during amino acid starvation, via its target kinase Ypk1, by repressing the activity of the calcium-dependent phosphatase calcineurin and promoting the general amino acid control (GAAC) response. Precisely how TORC2-Ypk1 signaling regulates calcineurin within this pathway remains unknown. Here we demonstrate that activation of calcineurin requires Mid1, an endoplasmic reticulum-localized calcium channel regulatory protein implicated in the oxidative stress response. We find that normal mitochondrial respiration is perturbed in TORC2-Ypk1-deficient cells, which results in the accumulation of mitochondrial-derived reactive oxygen species that signal to Mid1 to activate calcineurin, thereby inhibiting the GAAC response and autophagy. These findings describe a novel pathway involving TORC2, mitochondrial oxidative stress, and calcium homeostasis for autophagy regulation.

Target of rapamycin signaling mediates vacuolar fission caused by endoplasmic reticulum stress in Saccharomyces cerevisiae.

The yeast vacuole is equivalent to the mammalian lysosome and, in response to diverse physiological and environmental stimuli, undergoes alterations both in size and number. Here we demonstrate that vacuoles fragment in response to stress within the endoplasmic reticulum (ER) caused by chemical or genetic perturbations. We establish that this response does not involve known signaling pathways linked previously to ER stress but instead requires the rapamycin-sensitive TOR Complex 1 (TORC1), a master regulator of cell growth, together with its downstream effectors, Tap42/Sit4 and Sch9. To identify additional factors required for ER stress-induced vacuolar fragmentation, we conducted a high-throughput, genome-wide visual screen for yeast mutants that are refractory to ER stress-induced changes in vacuolar morphology. We identified several genes shown previously to be required for vacuolar fusion and/or fission, validating the utility of this approach. We also identified a number of new components important for fragmentation, including a set of proteins involved in assembly of the V-ATPase. Remarkably, we find that one of these, Vph2, undergoes a change in intracellular localization in response to ER stress and, moreover, in a manner that requires TORC1 activity. Together these results reveal a new role for TORC1 in the regulation of vacuolar behavior.