Regulation of TORC2 Function and Localization in Yeast.

Every eukaryotic cell contains two distinct multisubunit protein kinase complexes that each contain a TOR (target of rapamycin) protein as the catalytic subunit. These ensembles, designated TORC1 and TORC2, serve as nutrient and stress sensors, signal integrators, and regulators of cell growth and homeostasis, but they differ in their composition, localization, and function. TORC1, activated on the cytosolic surface of the vacuole (or, in mammalian cells, on the cytosolic surface of the lysosome), promotes biosynthesis and suppresses autophagy. TORC2, located primarily at the plasma membrane (PM), maintains the proper levels and bilayer distribution of all PM components (sphingolipids, glycerophospholipids, sterols, and integral membrane proteins), which are needed for the membrane expansion that accompanies cell growth and division and for combating insults to PM integrity. This review summarizes our current understanding of the assembly, structural features, subcellular distribution, and function and regulation of TORC2, obtained largely through studies conducted with Saccharomyces cerevisiae.

Phosphorylation by the stress-activated MAPK Slt2 down-regulates the yeast TOR complex 2.

Saccharomyces cerevisiae target of rapamycin (TOR) complex 2 (TORC2) is an essential regulator of plasma membrane lipid and protein homeostasis. How TORC2 activity is modulated in response to changes in the status of the cell envelope is unclear. Here we document that TORC2 subunit Avo2 is a direct target of Slt2, the mitogen-activated protein kinase (MAPK) of the cell wall integrity pathway. Activation of Slt2 by overexpression of a constitutively active allele of an upstream Slt2 activator (Pkc1) or by auxin-induced degradation of a negative Slt2 regulator (Sln1) caused hyperphosphorylation of Avo2 at its MAPK phosphoacceptor sites in a Slt2-dependent manner and diminished TORC2-mediated phosphorylation of its major downstream effector, protein kinase Ypk1. Deletion of Avo2 or expression of a phosphomimetic Avo2 allele rendered cells sensitive to two stresses (myriocin treatment and elevated exogenous acetic acid) that the cell requires Ypk1 activation by TORC2 to survive. Thus, Avo2 is necessary for optimal TORC2 activity, and Slt2-mediated phosphorylation of Avo2 down-regulates TORC2 signaling. Compared with wild-type Avo2, phosphomimetic Avo2 shows significant displacement from the plasma membrane, suggesting that Slt2 inhibits TORC2 by promoting Avo2 dissociation. Our findings are the first demonstration that TORC2 function is regulated by MAPK-mediated phosphorylation.

TOR complex 2 is a master regulator of plasma membrane homeostasis.

As first demonstrated in budding yeast (Saccharomyces cerevisiae), all eukaryotic cells contain two, distinct multi-component protein kinase complexes that each harbor the TOR (Target Of Rapamycin) polypeptide as the catalytic subunit. These ensembles, dubbed TORC1 and TORC2, function as universal, centrally important sensors, integrators, and controllers of eukaryotic cell growth and homeostasis. TORC1, activated on the cytosolic surface of the lysosome (or, in yeast, on the cytosolic surface of the vacuole), has emerged as a primary nutrient sensor that promotes cellular biosynthesis and suppresses autophagy. TORC2, located primarily at the plasma membrane, plays a major role in maintaining the proper levels and bilayer distribution of all plasma membrane components (sphingolipids, glycerophospholipids, sterols, and integral membrane proteins). This article surveys what we have learned about signaling via the TORC2 complex, largely through studies conducted in S. cerevisiae. In this yeast, conditions that challenge plasma membrane integrity can, depending on the nature of the stress, stimulate or inhibit TORC2, resulting in, respectively, up-regulation or down-regulation of the phosphorylation and thus the activity of its essential downstream effector the AGC family protein kinase Ypk1. Through the ensuing effect on the efficiency with which Ypk1 phosphorylates multiple substrates that control diverse processes, membrane homeostasis is maintained. Thus, the major focus here is on TORC2, Ypk1, and the multifarious targets of Ypk1 and how the functions of these substrates are regulated by their Ypk1-mediated phosphorylation, with emphasis on recent advances in our understanding of these processes.

Heterotrimeric G Protein-coupled Receptor Signaling in Yeast Mating Pheromone Response.

The DNAs encoding the receptors that respond to the peptide mating pheromones of the budding yeastSaccharomyces cerevisiaewere isolated in 1985, and were the very first genes for agonist-binding heterotrimeric G protein-coupled receptors (GPCRs) to be cloned in any organism. Now, over 30 years later, this yeast and its receptors continue to provide a pathfinding experimental paradigm for investigating GPCR-initiated signaling and its regulation, as described in this retrospective overview.

α-Arrestins participate in cargo selection for both clathrin-independent and clathrin-mediated endocytosis.

Clathrin-mediated endocytosis (CME) is a well-studied mechanism to internalize plasma membrane proteins; however, to endocytose such cargo, most eukaryotic cells also use alternative clathrin-independent endocytic (CIE) pathways, which are less well characterized. The budding yeast Saccharomyces cerevisiae, a widely used model for studying CME, was recently shown to have a CIE pathway that requires the GTPase Rho1, the formin Bni1, and their regulators. Nevertheless, in both yeast and mammalian cells, the mechanisms underlying cargo selection in CME and CIE are only beginning to be understood. For CME in yeast, particular α-arrestins contribute to recognition of specific cargos and promote their ubiquitylation by recruiting the E3 ubiquitin protein ligase Rsp5. Here, we show that the same α-arrestin-cargo pairs promote internalization through the CIE pathway by interacting with CIE components. Notably, neither expression of Rsp5 nor its binding to α-arrestins is required for CIE. Thus, α-arrestins are important for cargo selection in both the CME and CIE pathways, but function by distinct mechanisms in each pathway.

Sphingolipid biosynthesis upregulation by TOR complex 2-Ypk1 signaling during yeast adaptive response to acetic acid stress.

Acetic acid-induced inhibition of yeast growth and metabolism limits the productivity of industrial fermentation processes, especially when lignocellulosic hydrolysates are used as feedstock in industrial biotechnology. Tolerance to acetic acid of food spoilage yeasts is also a problem in the preservation of acidic foods and beverages. Thus understanding the molecular mechanisms underlying adaptation and tolerance to acetic acid stress is increasingly important in industrial biotechnology and the food industry. Prior genetic screens for Saccharomyces cerevisiae mutants with increased sensitivity to acetic acid identified loss-of-function mutations in the YPK1 gene, which encodes a protein kinase activated by the target of rapamycin (TOR) complex 2 (TORC2). We show in the present study by several independent criteria that TORC2-Ypk1 signaling is stimulated in response to acetic acid stress. Moreover, we demonstrate that TORC2-mediated Ypk1 phosphorylation and activation is necessary for acetic acid tolerance, and occurs independently of Hrk1, a protein kinase previously implicated in the cellular response to acetic acid. In addition, we show that TORC2-Ypk1-mediated activation of l-serine:palmitoyl-CoA acyltransferase, the enzyme complex that catalyzes the first committed step of sphingolipid biosynthesis, is required for acetic acid tolerance. Furthermore, analysis of the sphingolipid pathway using inhibitors and mutants indicates that it is production of certain complex sphingolipids that contributes to conferring acetic acid tolerance. Consistent with that conclusion, promoting sphingolipid synthesis by adding exogenous long-chain base precursor phytosphingosine to the growth medium enhanced acetic acid tolerance. Thus appropriate modulation of the TORC2-Ypk1-sphingolipid axis in industrial yeast strains may have utility in improving fermentations of acetic acid-containing feedstocks.

A Förster Resonance Energy Transfer (FRET)-based System Provides Insight into the Ordered Assembly of Yeast Septin Hetero-octamers.

Prior studies in both budding yeast (Saccharomyces cerevisiae) and in human cells have established that septin protomers assemble into linear hetero-octameric rods with 2-fold rotational symmetry. In mitotically growing yeast cells, five septin subunits are expressed (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1) and assemble into two types of rods that differ only in their terminal subunit: Cdc11-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Cdc11 and Shs1-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Shs1. EM analysis has shown that, under low salt conditions, the Cdc11-capped rods polymerize end to end to form long paired filaments, whereas Shs1-capped rods form arcs, spirals, and rings. To develop a facile method to study septin polymerization in vitro, we exploited our previous work in which we generated septin complexes in which all endogenous cysteine (Cys) residues were eliminated by site-directed mutagenesis, except an introduced E294C mutation in Cdc11 in these experiments. Mixing samples of a preparation of such single-Cys containing Cdc11-capped rods that have been separately derivatized with organic dyes that serve as donor and acceptor, respectively, for FRET provided a spectroscopic method to monitor filament assembly mediated by Cdc11-Cdc11 interaction and to measure its affinity under specified conditions. Modifications of this same FRET scheme also allow us to assess whether Shs1-capped rods are capable of end to end association either with themselves or with Cdc11-capped rods. This FRET approach also was used to follow the binding to septin filaments of a septin-interacting protein, the type II myosin-binding protein Bni5.

Internalization of Heterologous Sugar Transporters by Endogenous α-Arrestins in the Yeast Saccharomyces cerevisiae.

When expressed in Saccharomyces cerevisiae using either of two constitutive yeast promoters (PGK1prom and CCW12prom), the transporters CDT-1 and CDT-2 from the filamentous fungus Neurospora crassa are able to catalyze, respectively, active transport and facilitated diffusion of cellobiose (and, for CDT-2, also xylan and its derivatives). In S. cerevisiae, endogenous permeases are removed from the plasma membrane by clathrin-mediated endocytosis and are marked for internalization through ubiquitinylation catalyzed by Rsp5, a HECT class ubiquitin:protein ligase (E3). Recruitment of Rsp5 to specific targets is mediated by a 14-member family of endocytic adaptor proteins, termed α-arrestins. Here we demonstrate that CDT-1 and CDT-2 are subject to α-arrestin-mediated endocytosis, that four α-arrestins (Rod1, Rog3, Aly1, and Aly2) are primarily responsible for this internalization, that the presence of the transport substrate promotes transporter endocytosis, and that, at least for CDT-2, residues located in its C-terminal cytosolic domain are necessary for its efficient endocytosis. Both α-arrestin-deficient cells expressing CDT-2 and otherwise wild-type cells expressing CDT-2 mutants unresponsive to α-arrestin-driven internalization exhibit an increased level of plasma membrane-localized transporter compared to that of wild-type cells, and they grow, utilize the transport substrate, and generate ethanol anaerobically better than control cells. IMPORTANCE: Ethanolic fermentation of the breakdown products of plant biomass by budding yeast Saccharomyces cerevisiae remains an attractive biofuel source. To achieve this end, genes for heterologous sugar transporters and the requisite enzyme(s) for subsequent metabolism have been successfully expressed in this yeast. For one of the heterologous transporters examined in this study, we found that the amount of this protein residing in the plasma membrane was the rate-limiting factor for utilization of the cognate carbon source (cellobiose) and its conversion to ethanol.

Membrane-protein binding measured with solution-phase plasmonic nanocube sensors.

We describe a solution-phase sensor of lipid-protein binding based on localized surface plasmon resonance (LSPR) of silver nanocubes. When silica-coated nanocubes are mixed in a suspension of lipid vesicles, supported membranes spontaneously assemble on their surfaces. Using a standard laboratory spectrophotometer, we calibrated the LSPR peak shift due to protein binding to the membrane surface and then characterized the lipid-binding specificity of a pleckstrin homology domain protein.

A calcineurin-dependent switch controls the trafficking function of α-arrestin Aly1/Art6.

Proper regulation of plasma membrane protein endocytosis by external stimuli is required for cell growth and survival. In yeast, excess levels of certain nutrients induce endocytosis of the cognate permeases to prevent toxic accumulation of metabolites. The α-arrestins, a family of trafficking adaptors, stimulate ubiquitin-dependent and clathrin-mediated endocytosis by interacting with both a client permease and the ubiquitin ligase Rsp5. However, the molecular mechanisms that control α-arrestin function are not well understood. Here, we show that α-arrestin Aly1/Art6 is a phosphoprotein that specifically interacts with and is dephosphorylated by the Ca(2+)- and calmodulin-dependent phosphoprotein phosphatase calcineurin/PP2B. Dephosphorylation of Aly1 by calcineurin at a subset of phospho-sites is required for Aly1-mediated trafficking of the aspartic acid and glutamic acid transporter Dip5 to the vacuole, but it does not alter Rsp5 binding, ubiquitinylation, or stability of Aly1. In addition, dephosphorylation of Aly1 by calcineurin does not regulate the ability of Aly1 to promote the intracellular sorting of the general amino acid permease Gap1. These results suggest that phosphorylation of Aly1 inhibits its vacuolar trafficking function and, conversely, that dephosphorylation of Aly1 by calcineurin serves as a regulatory switch to promote Aly1-mediated trafficking to the vacuole.

Protein kinase Ypk1 phosphorylates regulatory proteins Orm1 and Orm2 to control sphingolipid homeostasis in Saccharomyces cerevisiae.

The Orm family proteins are conserved integral membrane proteins of the endoplasmic reticulum that are key homeostatic regulators of sphingolipid biosynthesis. Orm proteins bind to and inhibit serine:palmitoyl-coenzyme A transferase, the first enzyme in sphingolipid biosynthesis. In Saccharomyces cerevisiae, Orm1 and Orm2 are inactivated by phosphorylation in response to compromised sphingolipid synthesis (e.g., upon addition of inhibitor myriocin), thereby restoring sphingolipid production. We show here that protein kinase Ypk1, one of an essential pair of protein kinases, is responsible for this regulatory modification. Myriocin-induced hyperphosphorylation of Orm1 and Orm2 does not occur in ypk1 cells, and immunopurified Ypk1 phosphorylates Orm1 and Orm2 robustly in vitro exclusively on three residues that are known myriocin-induced sites. Furthermore, the temperature-sensitive growth of ypk1(ts) ypk2 cells is substantially ameliorated by deletion of ORM genes, confirming that a primary physiological role of Ypk1-mediated phosphorylation is to negatively regulate Orm function. Ypk1 immunoprecipitated from myriocin-treated cells displays a higher specific activity for Orm phosphorylation than Ypk1 from untreated cells. To identify the mechanism underlying Ypk1 activation, we systematically tested several candidate factors and found that the target of rapamycin complex 2 (TORC2) kinase plays a key role. In agreement with prior evidence that a TORC2-dependent site in Ypk1(T662) is necessary for cells to exhibit a wild-type level of myriocin resistance, a Ypk1(T662A) mutant displays only weak Orm phosphorylation in vivo and only weak activation in vitro in response to sphingolipid depletion. Additionally, sphingolipid depletion increases phosphorylation of Ypk1 at T662. Thus, Ypk1 is both a sensor and effector of sphingolipid level, and reduction in sphingolipids stimulates Ypk1, at least in part, via TORC2-dependent phosphorylation.

Native cysteine residues are dispensable for the structure and function of all five yeast mitotic septins.

Budding yeast septins assemble into hetero-octamers and filaments required for cytokinesis. Solvent-exposed cysteine (Cys) residues provide sites for attaching substituents useful in assessing assembly kinetics and protein interactions. To introduce Cys at defined locations, site-directed mutagenesis was used, first, to replace the native Cys residues in Cdc3 (C124 C253 C279), Cdc10 (C266), Cdc11 (C43 C137 C138), Cdc12 (C40 C278), and Shs1 (C29 C148) with Ala, Ser, Val, or Phe. When plasmid-expressed, each Cys-less septin mutant rescued the cytokinesis defects caused by absence of the corresponding chromosomal gene. When integrated and expressed from its endogenous promoter, the same mutants were fully functional, except Cys-less Cdc12 mutants (which were viable, but exhibited slow growth and aberrant morphology) and Cdc3(C124V C253V C279V) (which was inviable). No adverse phenotypes were observed when certain pairs of Cys-less septins were co-expressed as the sole source of these proteins. Cells grew less well when three Cys-less septins were co-expressed, suggesting some reduction in fitness. Nonetheless, cells chromosomally expressing Cys-less Cdc10, Cdc11, and Cdc12, and expressing Cys-less Cdc3 from a plasmid, grew well at 30°C. Moreover, recombinant Cys-less septins--or where one of the Cys-less septins contained a single Cys introduced at a new site--displayed assembly properties in vitro indistinguishable from wild-type.

Activation of the DExD/H-box protein Dbp5 by the nuclear-pore protein Gle1 and its coactivator InsP6 is required for mRNA export.

The DExD/H-box ATPase Dbp5 is essential for nuclear mRNA export, although its precise role in this process remains poorly understood. Here, we identify the nuclear pore protein Gle1 as a cellular activator of Dbp5. Dbp5 alone is unable to stably bind RNA or effectively hydrolyse ATP under physiological conditions, but addition of Gle1 dramatically stimulates these activities. A gle1 point mutant deficient for Dbp5 stimulation in vitro displays an mRNA export defect in vivo, indicating that activation of Dbp5 is an essential function of Gle1. Interestingly, Gle1 binds directly to inositol hexakisphosphate (InsP6) and InsP6 potentiates the Gle1-mediated stimulation of Dbp5. Dominant mutations in DBP5 and GLE1 that rescue mRNA export phenotypes associated with the lack of InsP6 mimic the InsP6 effects in vitro. Our results define specific functions for Gle1 and InsP6 in mRNA export and suggest that local activation of Dbp5 at the nuclear pore is critical for mRNA export.

A protein kinase network regulates the function of aminophospholipid flippases.

Limited exposure of aminophospholipids on the outer leaflet of the plasma membrane is a fundamental feature of eukaryotic cells and is maintained by the action of inward-directed P-type ATPases ("flippases"). Yeast S. cerevisiae has five flippases (Dnf1, Dnf2, Dnf3, Drs2, and Neo1), but their regulation is poorly understood. Two paralogous plasma membrane-associated protein kinases, Pkh1 and Pkh2 (orthologs of mammalian PDK1), are required for viability of S. cerevisiae cells because they activate several essential downstream protein kinases by phosphorylating a critical Thr in their activation loops. Two such targets are related protein kinases Ypk1 and Ypk2 (orthologs of mammalian SGK1), which have been implicated in multiple processes, including endocytosis and coupling of membrane expansion to cell wall remodeling, but the downstream effector(s) of these kinases have been elusive. Here we show that a physiologically relevant substrate of Ypk1 is another protein kinase, Fpk1, a known flippase activator. We show that Ypk1 phosphorylates and thereby down-regulates Fpk1, and further that a complex sphingolipid counteracts the down-regulation of Fpk1 by Ypk1. Our findings delineate a unique regulatory mechanism for imposing a balance between sphingolipid content and aminophospholipid asymmetry in eukaryotic plasma membranes.

Pheromone-induced anisotropy in yeast plasma membrane phosphatidylinositol-4,5-bisphosphate distribution is required for MAPK signaling.

During response of budding yeast to peptide mating pheromone, the cell becomes markedly polarized and MAPK scaffold protein Ste5 localizes to the resulting projection (shmoo tip). We demonstrated before that this recruitment is essential for sustained MAPK signaling and requires interaction of a pleckstrin homology (PH) domain in Ste5 with phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] in the plasma membrane. Using fluorescently tagged high-affinity probes specific for PtdIns(4,5)P(2), we have now found that this phosphoinositide is highly concentrated at the shmoo tip in cells responding to pheromone. Maintenance of this strikingly anisotropic distribution of PtdIns(4,5)P(2), stable tethering of Ste5 at the shmoo tip, downstream MAPK activation, and expression of a mating pathway-specific reporter gene all require continuous function of the plasma membrane-associated PtdIns 4-kinase Stt4 and the plasma membrane-associated PtdIns4P 5-kinase Mss4 (but not the Golgi-associated PtdIns 4-kinase Pik1). Our observations demonstrate that PtdIns(4,5)P(2) is the primary determinant for restricting localization of Ste5 within the plasma membrane and provide direct evidence that an extracellular stimulus-evoked self-reinforcing mechanism generates a spatially enriched pool of PtdIns(4,5)P(2) necessary for the membrane anchoring and function of a signaling complex.

Mitogen-activated protein kinase (MAPK) cascades-A yeast perspective.

Discovery of the class of protein kinase now dubbed a mitogen (or messenger)-activated protein kinase (MAPK) is an illustrative example of how disparate lines of investigation can converge and reveal an enzyme family universally conserved among eukaryotes, from single-celled microbes to humans. Moreover, elucidation of the circuitry controlling MAPK function defined a now overarching principle in enzyme regulation-the concept of an activation cascade mediated by sequential phosphorylation events. Particularly ground-breaking for this field of exploration were the contributions of genetic approaches conducted using several model organisms, but especially the budding yeast Saccharomyces cerevisiae. Notably, examination of how haploid yeast cells respond to their secreted peptide mating pheromones was crucial in pinpointing genes encoding MAPKs and their upstream activators. Fully contemporaneous biochemical analysis of the activities elicited upon stimulation of mammalian cells by insulin and other growth- and differentiation-inducing factors lead eventually to the demonstration that components homologous to those in yeast were involved. Continued studies of these pathways in yeast were integral to other foundational discoveries in MAPK signaling, including the roles of tethering, scaffolding and docking interactions.

Structure of a Ca2+-myristoyl switch protein that controls activation of a phosphatidylinositol 4-kinase in fission yeast.

Neuronal calcium sensor (NCS) proteins transduce Ca2+ signals and are highly conserved from yeast to humans. We determined NMR structures of the NCS-1 homolog from fission yeast (Ncs1), which activates a phosphatidylinositol 4-kinase. Ncs1 contains an α-NH2-linked myristoyl group on a long N-terminal arm and four EF-hand motifs, three of which bind Ca2+, assembled into a compact structure. In Ca2+-free Ncs1, the N-terminal arm positions the fatty acyl chain inside a cavity near the C terminus. The C14 end of the myristate is surrounded by residues in the protein core, whereas its amide-linked (C1) end is flanked by residues at the protein surface. In Ca2+-bound Ncs1, the myristoyl group is extruded (Ca2+-myristoyl switch), exposing a prominent patch of hydrophobic residues that specifically contact phosphatidylinositol 4-kinase. The location of the buried myristate and structure of Ca2+-free Ncs1 are quite different from those in other NCS proteins. Thus, a unique remodeling of each NCS protein by its myristoyl group, and Ca2+-dependent unmasking of different residues, may explain how each family member recognizes distinct target proteins.

Septin stability and recycling during dynamic structural transitions in cell division and development.

Septins are conserved proteins found in hetero-oligomeric complexes that are incorporated into distinct structures during cell division and differentiation; yeast septins Cdc3, Cdc10, Cdc11, and Cdc12 form hetero-octamers and polymerize into filaments, which form a "collar" at the mother-bud neck [1]. Posttranslational modifications, nucleotide binding, and protein-protein and protein-lipid interactions influence assembly and disassembly of septin structures [2], but whether individual septins are used repeatedly to build higher-order assemblies was not known. We used fluorescence-based pulse-chase methods to visualize the fate of pre-existing (old) and newly synthesized (new) molecules of two septins, Cdc10 and Cdc12. They were recycled through multiple mitotic divisions, and old and new molecules were incorporated indistinguishably into the collar. Likewise, old and new subunits intermixed within hetero-octamers, indicating that exchange occurs at this organizational level. Remarkably, in meiosis, Cdc10 made during vegetative growth was reutilized to build sporulation-specific structures and reused again during spore germination for budding and during subsequent mitotic divisions. Although Cdc12 also persisted during sporulation, it was excluded from septin structures and replaced by another subunit, Spr3; only new Cdc12 populated the collar of germinating spores. Thus, mechanisms governing septin incorporation are specific to each subunit and to the developmental state of the cell.

Genetic interactions with mutations affecting septin assembly reveal ESCRT functions in budding yeast cytokinesis.

Membrane trafficking via targeted exocytosis to the Saccharomyces cerevisiae bud neck provides new membrane and membrane-associated factors that are critical for cytokinesis. It remains unknown whether yeast plasma membrane abscission, the final step of cytokinesis, occurs spontaneously following extensive vesicle fusion, as in plant cells, or requires dedicated membrane fission machinery, as in cultured mammalian cells. Components of the endosomal sorting complexes required for transport (ESCRT) pathway, or close relatives thereof, appear to participate in cytokinetic abscission in various cell types, but roles in cell division had not been documented in budding yeast, where ESCRTs were first characterized. By contrast, the septin family of filament-forming cytoskeletal proteins were first identified by their requirement for yeast cell division. We show here that mutations in ESCRT-encoding genes exacerbate the cytokinesis defects of cla4Δ or elm1Δ mutants, in which septin assembly is perturbed at an early stage in cell division, and alleviate phenotypes of cells carrying temperature-sensitive alleles of a septin-encoding gene, CDC10. Elevated chitin synthase II (Chs2) levels coupled with aberrant morphogenesis and chitin deposition in elm1Δ cells carrying ESCRT mutations suggest that ESCRTs normally enhance the efficiency of cell division by promoting timely endocytic turnover of key cytokinetic enzymes.

Stress resistance and signal fidelity independent of nuclear MAPK function.

Elevated external solute stimulates a conserved MAPK cascade that elicits responses that maintain osmotic balance. The yeast high-osmolarity glycerol (HOG) pathway activates Hog1 MAPK (mammalian ortholog p38alpha/SAPKalpha), which enters the nucleus and induces expression of >50 genes, implying that transcriptional up-regulation is necessary to cope with hyperosmotic stress. Contrary to this expectation, we show here that cells lacking the karyopherin required for Hog1 nuclear import or in which Hog1 is anchored at the plasma membrane (or both) can withstand long-term hyperosmotic challenge by ionic and nonionic solutes without exhibiting the normal change in transcriptional program (comparable with hog1Delta cells), as judged by mRNA hybridization and microarray analysis. For such cells to survive hyperosmotic stress, systematic genetic analysis ruled out the need for any Hog1-dependent transcription factor, the Hog1-activated MAPKAP kinases, or ion, glycerol, and water channels. By contrast, enzymes needed for glycerol production were essential for viability. Thus, control of intracellular glycerol formation by Hog1 is critical for maintenance of osmotic balance but not transcriptional induction of any gene.