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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.

Eukaryotes utilize distinct mitogen/messenger-activated protein kinase (MAPK) pathways to evoke appropriate responses when confronted with different stimuli. In yeast, hyperosmotic stress activates MAPK Hog1, whereas mating pheromones activate MAPK Fus3 (and MAPK Kss1). Because these pathways share several upstream components, including the small guanosine-5'-triphosphate phosphohydrolase (GTPase) cell-division-cycle-42 (Cdc42), mechanisms must exist to prevent inadvertent cross-pathway activation. Hog1 activity is required to prevent crosstalk to Fus3 and Kss1. To identify other factors required to maintain signaling fidelity during hypertonic stress, we devised an unbiased genetic selection for mutants unable to prevent such crosstalk even when active Hog1 is present. We repeatedly isolated truncated alleles of RGA1, a Cdc42-specific GTPase-activating protein (GAP), each lacking its C-terminal catalytic domain, that permit activation of the mating MAPKs under hyperosmotic conditions despite Hog1 being present. We show that Rga1 down-regulates Cdc42 within the high-osmolarity glycerol (HOG) pathway, but not the mating pathway. Because induction of mating pathway output via crosstalk from the HOG pathway takes significantly longer than induction of HOG pathway output, our findings suggest that, under normal conditions, Rga1 contributes to signal insulation by limiting availability of the GTP-bound Cdc42 pool generated by hypertonic stress. Thus, Rga1 action contributes to squelching crosstalk by imposing a type of "kinetic proofreading". Although Rga1 is a Hog1 substrate in vitro, we eliminated the possibility that its direct Hog1-mediated phosphorylation is necessary for its function in vivo. Instead, we found first that, like its paralog Rga2, Rga1 is subject to inhibitory phosphorylation by the S. cerevisiae cyclin-dependent protein kinase 1 (Cdk1) ortholog Cdc28 and that hyperosmotic shock stimulates its dephosphorylation and thus Rga1 activation. Second, we found that Hog1 promotes Rga1 activation by blocking its Cdk1-mediated phosphorylation, thereby allowing its phosphoprotein phosphatase 2A (PP2A)-mediated dephosphorylation. These findings shed light on why Hog1 activity is required to prevent crosstalk from the HOG pathway to the mating pheromone response pathway.

Members of the Puf family of RNA-binding proteins typically associate via their Pumilio homology domain with specific short motifs in the 3'-UTR of an mRNA and thereby influence the stability, localization and/or efficiency of translation of the bound transcript. In our prior unbiased proteome-wide screen for targets of the TORC2-stimulated protein kinase Ypk1, we identified the paralogs Puf1/Jsn1 and Puf2 as high-confidence substrates. Earlier work by others had demonstrated that Puf1 and Puf2 exhibit a marked preference for interaction with mRNAs encoding plasma membrane-associated proteins, consistent with our previous studies documenting that a primary physiological role of TORC2-Ypk1 signaling is maintenance of plasma membrane homeostasis. Here, we show, first, that both Puf1 and Puf2 are authentic Ypk1 substrates both in vitro and in vivo. Fluorescently tagged Puf1 localizes constitutively in cortical puncta closely apposed to the plasma membrane, whereas Puf2 does so in the absence of its Ypk1 phosphorylation, but is dispersed in the cytosol when phosphorylated. We further demonstrate that Ypk1-mediated phosphorylation of Puf1 and Puf2 upregulates production of the protein products of the transcripts to which they bind, with a concomitant increase in the level of the cognate mRNAs. Thus, Ypk1 phosphorylation relieves Puf1- and Puf2-mediated post-transcriptional repression mainly by counteracting their negative effect on transcript stability. Using a heterologous protein-RNA tethering and fluorescent protein reporter assay, the consequence of Ypk1 phosphorylation in vivo was recapitulated for full-length Puf1 and even for N-terminal fragments (residues 1-340 and 143-295) corresponding to the region upstream of its dimerization domain (an RNA-recognition motif fold) encompassing its two Ypk1 phosphorylation sites (both also conserved in Puf2). This latter result suggests that alleviation of Puf1-imposed transcript destabilization does not obligatorily require dissociation of Ypk1-phosphorylated Puf1 from a transcript. Our findings add new insight about how the TORC2-Ypk1 signaling axis regulates the content of plasma membrane-associated proteins to promote maintenance of the integrity of the cell envelope.

Membrane-tethered sterol-binding Lam/Ltc proteins localize at junctions between the endoplasmic reticulum (ER) membrane and other organelles. Two of the six family members-Lam2/Ltc4 (initially Ysp2) and paralog Lam4/Ltc3-localize to ER-plasma membrane (PM) contact sites (CSs) and mediate retrograde ergosterol transport from the PM to the ER. Our prior work demonstrated that Lam2 and Lam4 are substrates of TORC2-regulated protein kinase Ypk1, that Ypk1-mediated phosphorylation inhibits their function in retrograde sterol transport, and that PM sterol retention bolsters cell survival under stressful conditions. At ER-PM CSs, Lam2 and Lam4 associate with Laf1/Ymr102c and Dgr2/Ykl121w (paralogous WD40 repeat-containing proteins) that reportedly bind sterol. Using fluorescent tags, we found that Lam2 and Lam4 remain at ER-PM CSs when Laf1 and Dgr2 are absent, whereas neither Laf1 nor Dgr2 remain at ER-PM CSs when Lam2 and Lam4 are absent. Loss of Laf1 (but not Dgr2) impedes retrograde ergosterol transport, and a laf1∆ mutation does not exacerbate the transport defect of lam2∆ lam4∆ cells, indicating a shared function. Lam2 and Lam4 bind Laf1 and Dgr2 in vitro in a pull-down assay, and the PH domain in Lam2 hinders its interaction with Laf1. Lam2 phosphorylated by Ypk1, and Lam2 with phosphomimetic (Glu) replacements at its Ypk1 sites, exhibited a marked reduction in Laf1 binding. Thus, phosphorylation prevents Lam2 interaction with Laf1 at ER-PM CSs, providing a mechanism by which Ypk1 action inhibits retrograde sterol transport.

Septin family proteins are quite similar to each other both within and between eukaryotic species. Typically, multiple discrete septins co-assemble into linear heterooligomers (usually hexameric or octameric rods) with a variety of cellular functions. We know little about how incorporation of different septins confers different properties to such complexes. This issue is especially acute in human cells where 13 separate septin gene products (often produced in multiple forms arising from alternative start codons and differential splicing) are expressed in a tissue-specific manner. Based on sequence alignments and phylogenetic criteria, human septins fall into four distinct groups predictive of their interactions, that is, members of the same group appear to occupy the same position within oligomeric septin protomers, which are palindromic (have twofold rotational symmetry about a central homodimeric pair). Many such protomers are capable of end-to-end polymerization, generating filaments. Over a decade ago, a study using X-ray crystallography and single-particle electron microscopy deduced the arrangement within recombinant heterohexamers comprising representatives of three human septin groups-SEPT2, SEPT6, and SEPT7. This model greatly influenced subsequent studies of human and other septin complexes, including how incorporating a septin from a fourth group forms heterooctamers, as first observed in budding yeast. Two recent studies, including one in this issue of Cytoskeleton, provide clear evidence that, in fact, the organization of subunits within human septin heterohexamers and heterooctamers is inverted relative to the original model. These findings are discussed here in a broader context, including possible causes for the initial confusion.

Eukaryotic cell survival requires maintenance of plasma membrane (PM) homeostasis in response to environmental insults and changes in lipid metabolism. In yeast, a key regulator of PM homeostasis is target of rapamycin (TOR) complex 2 (TORC2), a multiprotein complex containing the evolutionarily conserved TOR protein kinase isoform Tor2. PM localization is essential for TORC2 function. One core TORC2 subunit (Avo1) and two TORC2--associated regulators (Slm1 and Slm2) contain pleckstrin homology (PH) domains that exhibit specificity for binding phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P2). To investigate the roles of PtdIns4,5P2 and constituent subunits of TORC2, we used auxin-inducible degradation to systematically eliminate these factors and then examined localization, association, and function of the remaining TORC2 components. We found that PtdIns4,5P2 depletion significantly reduced TORC2 activity, yet did not prevent PM localization or disassembly of TORC2. Moreover, truncated Avo1 (lacking its C-terminal PH domain) was still recruited to the PM and supported growth. Even when all three PH-containing proteins were absent, the remaining TORC2 subunits were PM-bound. Revealingly, Avo3 localized to the PM independent of both Avo1 and Tor2, whereas both Tor2 and Avo1 required Avo3 for their PM anchoring. Our findings provide new mechanistic information about TORC2 and pinpoint Avo3 as pivotal for TORC2 PM localization and assembly in vivo.

Eukaryotic cell survival requires maintenance of plasma membrane (PM) homeostasis in response to environmental insults and changes in lipid metabolism. In yeast, a key regulator of PM homeostasis is target of rapamycin (TOR) complex 2 (TORC2), a multiprotein complex containing the evolutionarily conserved TOR protein kinase isoform Tor2. PM localization is essential for TORC2 function. One core TORC2 subunit (Avo1) and two TORC2--associated regulators (Slm1 and Slm2) contain pleckstrin homology (PH) domains that exhibit specificity for binding phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P2). To investigate the roles of PtdIns4,5P2 and constituent subunits of TORC2, we used auxin-inducible degradation to systematically eliminate these factors and then examined localization, association, and function of the remaining TORC2 components. We found that PtdIns4,5P2 depletion significantly reduced TORC2 activity, yet did not prevent PM localization or disassembly of TORC2. Moreover, truncated Avo1 (lacking its C-terminal PH domain) was still recruited to the PM and supported growth. Even when all three PH-containing proteins were absent, the remaining TORC2 subunits were PM-bound. Revealingly, Avo3 localized to the PM independent of both Avo1 and Tor2, whereas both Tor2 and Avo1 required Avo3 for their PM anchoring. Our findings provide new mechanistic information about TORC2 and pinpoint Avo3 as pivotal for TORC2 PM localization and assembly in vivo.

The evolutionarily conserved Target of Rapamycin (TOR) complex-2 (TORC2) is an essential regulator of plasma membrane homeostasis in budding yeast (Saccharomyces cerevisiae). In this yeast, TORC2 phosphorylates and activates the effector protein kinase Ypk1 and its paralog Ypk2. These protein kinases, in turn, carry out all the crucial functions of TORC2 by phosphorylating and thereby controlling the activity of at least a dozen downstream substrates. A previously uncharacterized interplay between the Rab5 GTPases and TORC2 signaling was uncovered through analysis of a newly suspected Ypk1 target. Muk1, one of two guanine nucleotide exchange factors for the Rab5 GTPases, was found to be a physiologically relevant Ypk1 substrate; and, genetic analysis indicates that Ypk1-mediated phosphorylation activates the guanine nucleotide exchange activity of Muk1. Second, it was demonstrated both in vivo and in vitro that the GTP-bound state of the Rab5 GTPase Vps21/Ypt51 physically associates with TORC2 and acts as a direct positive effector required for full TORC2 activity. These interrelationships provide a self-reinforcing control circuit for sustained up-regulation of TORC2-Ypk1 signaling. In this overview, we summarize the experimental basis of these findings, their implications, and speculate as to the molecular basis for Rab5-mediated TORC2 activation.

Target of rapamycin complex-2 (TORC2), a conserved protein kinase complex, is an indispensable regulator of plasma membrane homeostasis. In budding yeast (Saccharomyces cerevisiae), the essential downstream effector of TORC2 is protein kinase Ypk1 and its paralog Ypk2. Muk1, a Rab5-specific guanine nucleotide exchange factor (GEF), was identified in our prior global screen for candidate Ypk1 targets. We confirm here that Muk1 is a substrate of Ypk1 and demonstrate that Ypk1-mediated phosphorylation stimulates Muk1 function in vivo. Strikingly, yeast lacking its two Rab5 GEFs (Muk1 and Vps9) or its three Rab5 paralogs (Vps21/Ypt51, Ypt52, and Ypt53) or overexpressing Msb3, a Rab5-directed GTPase-activating protein, all exhibit pronounced reduction in TORC2-mediated phosphorylation and activation of Ypk1. Vps21 coimmunoprecipitates with TORC2, and immuno-enriched TORC2 is less active in vitro in the absence of Rab5 GTPases. Thus, TORC2-dependent and Ypk1-mediated activation of Muk1 provides a control circuit for positive (self-reinforcing) up-regulation to sustain TORC2-Ypk1 signaling.

In budding yeast, a collar of septin filaments at the neck between a mother cell and its bud marks the incipient site for cell division and serves as a scaffold that recruits proteins required for proper spatial and temporal execution of cytokinesis. A set of interacting proteins that localize at or near the bud neck, including Aim44/Gps1, Nba1 and Nis1, also has been implicated in preventing Cdc42-dependent bud site re-establishment at the division site. We found that, at their endogenous level, Aim44 and Nis1 robustly localize sequentially at the septin collar. Strikingly, however, when overproduced, both proteins shift their subcellular distribution predominantly to the nucleus. Aim44 localizes with the inner nuclear envelope, as well as at the plasma membrane, whereas Nis1 accumulates within the nucleus, indicating that these proteins normally undergo nucleocytoplasmic shuttling. Of the 14 yeast karyopherins, Kap123/Yrb4 is the primary importin for Aim44, whereas several importins mediate Nis1 nuclear entry. Conversely, Kap124/Xpo1/Crm1 is the primary exportin for Nis1, whereas both Xpo1 and Cse1/Kap109 likely contribute to Aim44 nuclear export. Even when endogenously expressed, Nis1 accumulates in the nucleus when Nba1 is absent. When either Aim44 or Nis1 are overexpressed, Nba1 is displaced from the bud neck, further consistent with the mutual interactions of these proteins. Collectively, our results indicate that a previously unappreciated level at which localization of septin-associated proteins is controlled is via regulation of their nucleocytoplasmic shuttling, which places constraints on their availability for complex formation with other partners at the bud neck.

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.

To observe internalization of the yeast pheromone receptor Ste2 by fluorescence microscopy in live cells in real time, we visualized only those molecules present at the cell surface at the time of agonist engagement (rather than the total cellular pool) by tagging this receptor at its N-terminus with an exocellular fluorogen-activating protein (FAP). A FAP is a single-chain antibody engineered to bind tightly a nonfluorescent, cell-impermeable dye (fluorogen), thereby generating a fluorescent complex. The utility of FAP tagging to study trafficking of integral membrane proteins in yeast, which possesses a cell wall, had not been examined previously. A diverse set of signal peptides and propeptide sequences were explored to maximize expression. Maintenance of the optimal FAP-Ste2 chimera intact required deletion of two, paralogous, glycosylphosphatidylinositol (GPI)-anchored extracellular aspartyl proteases (Yps1 and Mkc7). FAP-Ste2 exhibited a much brighter and distinct plasma membrane signal than Ste2-GFP or Ste2-mCherry yet behaved quite similarly. Using FAP-Ste2, new information was obtained about the mechanism of its internalization, including novel insights about the roles of the cargo-selective endocytic adaptors Ldb19/Art1, Rod1/Art4, and Rog3/Art7.

In our proteome-wide screen, Ysp2 (also known as Lam2/Ltc4) was identified as a likely physiologically relevant target of the TOR complex 2 (TORC2)-dependent protein kinase Ypk1 in the yeast Saccharomyces cerevisiae. Ysp2 was subsequently shown to be one of a new family of sterol-binding proteins located at plasma membrane (PM)-endoplasmic reticulum (ER) contact sites. Here we document that Ysp2 and its paralogue Lam4/Ltc3 are authentic Ypk1 substrates in vivo and show using genetic and biochemical criteria that Ypk1-mediated phosphorylation inhibits the ability of these proteins to promote retrograde transport of sterols from the PM to the ER. Furthermore, we provide evidence that a change in PM sterol homeostasis promotes cell survival under membrane-perturbing conditions known to activate TORC2-Ypk1 signaling. These observations define the underlying molecular basis of a new regulatory mechanism for cellular response to plasma membrane stress.

To grow, eukaryotic cells must expand by inserting glycerolipids, sphingolipids, sterols, and proteins into their plasma membrane, and maintain the proper levels and bilayer distribution. A fungal cell must coordinate growth with enlargement of its cell wall. In Saccharomyces cerevisiae, a plasma membrane-localized protein kinase complex, Target of Rapamicin (TOR) complex-2 (TORC2) (mammalian ortholog is mTORC2), serves as a sensor and masterregulator of these plasma membrane- and cell wall-associated events by directly phosphorylating and thereby stimulating the activity of two types of effector protein kinases: Ypk1 (mammalian ortholog is SGK1), along with a paralog (Ypk2); and, Pkc1 (mammalian ortholog is PKN2/PRK2). Ypk1 is a central regulator of pathways and processes required for plasma membrane lipid and protein homeostasis, and requires phosphorylation on its T-loop by eisosome-associated protein kinase Pkh1 (mammalian ortholog is PDK1) and a paralog (Pkh2). For cell survival under various stresses, Ypk1 function requires TORC2-mediated phosphorylation at multiple sites near its C terminus. Pkc1 controls diverse processes, especially cell wall synthesis and integrity. Pkc1 is also regulated by Pkh1- and TORC2-dependent phosphorylation, but, in addition, by interaction with Rho1-GTP and lipids phosphatidylserine (PtdSer) and diacylglycerol (DAG). We also describe here what is currently known about the downstream substrates modulated by Ypk1-mediated and Pkc1-mediated phosphorylation.

Yeast (Saccharomyces cerevisiae) target of rapamycin (TOR) complex 2 (TORC2) is a multi-subunit plasma membrane-associated protein kinase and vital growth regulator. Its essential functions are exerted via phosphorylation and stimulation of downstream protein kinase Ypk1 (and its paralog Ypk2). Ypk1 phosphorylates multiple substrates to regulate plasma membrane lipid and protein composition. Ypk1 function requires phosphorylation of Thr504 in its activation loop by eisosome-associated Pkh1 (and its paralog Pkh2). For cell survival under certain stresses, however, Ypk1 activity requires further stimulation by TORC2-mediated phosphorylation at C-terminal sites, dubbed the "turn" (Ser644) and "hydrophobic" (Thr662) motifs. Here we show that four additional C-terminal sites are phosphorylated in a TORC2-dependent manner, collectively defining a minimal consensus. We found that the newly identified sites are as important for Ypk1 activity, stability, and biological function as Ser644 and Thr662. Ala substitutions at the four new sites abrogated the ability of Ypk1 to rescue the phenotypes of Ypk1 deficiency, whereas Glu substitutions had no ill effect. Combining the Ala substitutions with an N-terminal mutation (D242A), which has been demonstrated to bypass the need for TORC2-mediated phosphorylation, restored the ability to complement a Ypk1-deficient cell. These findings provide new insights about the molecular basis for TORC2-dependent activation of Ypk1.

Depending on the stress, plasma membrane alterations activate or inhibit yeast target of rapamycin (TOR) complex 2, which, in turn, upregulates or downregulates the activity of its essential downstream effector, protein kinase Ypk1. Through phosphorylation of multiple substrates, Ypk1 controls many processes that restore homeostasis. One such substrate is protein kinase Fpk1, which is negatively regulated by Ypk1. Fpk1 phosphorylates and stimulates flippases that translocate aminoglycerophospholipids from the outer to the inner leaflet of the plasma membrane. Fpk1 has additional roles, but other substrates were uncharacterized. We show that Fpk1 phosphorylates and inhibits protein kinase Akl1, related to protein kinases Ark1 and Prk1, which modulate the dynamics of actin patch-mediated endocytosis. Akl1 has two Fpk1 phosphorylation sites (Ark1 and Prk1 have none) and is hypophosphorylated when Fpk1 is absent. Conversely, under conditions that inactivate TORC2-Ypk1 signaling, which alleviates Fpk1 inhibition, Akl1 is hyperphosphorylated. Monitoring phosphorylation of known Akl1 substrates (Sla1 and Ent2) confirmed that Akl1 is hyperactive when not phosphorylated by Fpk1. Fpk1-mediated negative regulation of Akl1 enhances endocytosis, because an Akl1 mutant immune to Fpk1 phosphorylation causes faster dissociation of Sla1 from actin patches, confers elevated resistance to doxorubicin (a toxic compound whose entry requires endocytosis), and impedes Lucifer yellow uptake (a marker of fluid phase endocytosis). Thus, TORC2-Ypk1, by regulating Fpk1-mediated phosphorylation of Akl1, adjusts the rate of endocytosis.

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.ImportanceEthanolic 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.

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.

Septins are a protein family found in all eukaryotes (except higher plants) that have roles in membrane remodeling and formation of diffusion barriers and as a scaffold to recruit other proteins. In budding yeast, proper execution of cytokinesis and cell division requires the formation of a collar of circumferential filaments at the bud neck. These filaments are assembled from apolar septin hetero-octamers. Currently, little is known about the mechanisms that control the arrangement and dynamics of septin structures. In this study, we utilized both Förster resonance energy transfer and electron microscopy to analyze the biophysical properties of the septin-binding protein Bni5 and how its association with septin filaments affects their organization. We found that the interaction of Bni5 with the terminal subunit (Cdc11) at the junctions between adjacent hetero-octamers in paired filaments is highly cooperative. Both the C-terminal end of Bni5 and the C-terminal extension of Cdc11 make important contributions to their interaction. Moreover, this binding may stabilize the dimerization of Bni5, which, in turn, forms cross-filament braces that significantly narrow, and impose much more uniform spacing on, the gap between paired filaments.

Various methods can provide a readout of the physical interaction between two biomolecules. A recently described tripartite split-GFP system has the potential to report by direct visualization via a fluorescence signal the intimate association of minimally tagged proteins expressed at their endogenous level in their native cellular milieu and can capture transient or weak interactions. Here we document the utility of this tripartite split-GFP system to assess in living cells protein-protein interactions in a dynamic cytoskeletal structure-the septin collar at the yeast bud neck. We show, first, that for septin-septin interactions, this method yields a robust signal whose strength reflects the known spacing between the subunits in septin filaments and thus serves as a "molecular ruler." Second, the method yields little or no spurious signal even with highly abundant cytosolic proteins readily accessible to the bud neck (including molecular chaperone Hsp82 and glycolytic enzyme Pgk1). Third, using two proteins (Bni5 and Hsl1) that have been shown by other means to bind directly to septins at the bud neck in vivo, we validate that the tripartite split-GFP method yields the same conclusions and further insights about specificity. Finally, we demonstrate the capacity of this approach to uncover additional new information by examining whether three other proteins reported to localize to the bud neck (Nis1, Bud4, and Hof1) are able to interact physically with any of the subunits in the septin collar and, if so, with which ones.

Passage through the eukaryotic cell cycle requires processes that are tightly regulated both spatially and temporally. Surveillance mechanisms (checkpoints) exert quality control and impose order on the timing and organization of downstream events by impeding cell cycle progression until the necessary components are available and undamaged and have acted in the proper sequence. In budding yeast, a checkpoint exists that does not allow timely execution of the G2/M transition unless and until a collar of septin filaments has properly assembled at the bud neck, which is the site where subsequent cytokinesis will occur. An essential component of this checkpoint is the large (1518-residue) protein kinase Hsl1, which localizes to the bud neck only if the septin collar has been correctly formed. Hsl1 reportedly interacts with particular septins; however, the precise molecular determinants in Hsl1 responsible for its recruitment to this cellular location during G2 have not been elucidated. We performed a comprehensive mutational dissection and accompanying image analysis to identify the sequence elements within Hsl1 responsible for its localization to the septins at the bud neck. Unexpectedly, we found that this targeting is multipartite. A segment of the central region of Hsl1 (residues 611-950), composed of two tandem, semiredundant but distinct septin-associating elements, is necessary and sufficient for binding to septin filaments both in vitro and in vivo. However, in addition to 611-950, efficient localization of Hsl1 to the septin collar in the cell obligatorily requires generalized targeting to the cytosolic face of the plasma membrane, a function normally provided by the C-terminal phosphatidylserine-binding KA1 domain (residues 1379-1518) in Hsl1 but that can be replaced by other, heterologous phosphatidylserine-binding sequences.

Genome editing exploiting CRISPR/Cas9 has been adopted widely in academia and in the biotechnology industry to manipulate DNA sequences in diverse organisms. Molecular engineering of Cas9 itself and its guide RNA, and the strategies for using them, have increased efficiency, optimized specificity, reduced inappropriate off-target effects, and introduced modifications for performing other functions (transcriptional regulation, high-resolution imaging, protein recruitment, and high-throughput screening). Moreover, Cas9 has the ability to multiplex, i.e., to act at different genomic targets within the same nucleus. Currently, however, introducing concurrent changes at multiple loci involves: (i) identification of appropriate genomic sites, especially the availability of suitable PAM sequences; (ii) the design, construction, and expression of multiple sgRNA directed against those sites; (iii) potential difficulties in altering essential genes; and (iv) lingering concerns about "off-target" effects. We have devised a new approach that circumvents these drawbacks, as we demonstrate here using the yeast Saccharomyces cerevisiae First, any gene(s) of interest are flanked upstream and downstream with a single unique target sequence that does not normally exist in the genome. Thereafter, expression of one sgRNA and cotransformation with appropriate PCR fragments permits concomitant Cas9-mediated alteration of multiple genes (both essential and nonessential). The system we developed also allows for maintenance of the integrated, inducible Cas9-expression cassette or its simultaneous scarless excision. Our scheme-dubbed mCAL for " M: ultiplexing of C: as9 at A: rtificial L: oci"-can be applied to any organism in which the CRISPR/Cas9 methodology is currently being utilized. In principle, it can be applied to install synthetic sequences into the genome, to generate genomic libraries, and to program strains or cell lines so that they can be conveniently (and repeatedly) manipulated at multiple loci with extremely high efficiency.

G-protein-coupled receptors (GPCRs) are integral membrane proteins that initiate stimulus-dependent activation of cognate heterotrimeric G-proteins, triggering ensuing downstream cellular responses. Tight regulation of GPCR-evoked pathways is required because prolonged stimulation can be detrimental to an organism. Ste2, a GPCR in Saccharomyces cerevisiae that mediates response of MATa haploids to the peptide mating pheromone α-factor, is down-regulated by both constitutive and agonist-induced endocytosis. Efficient agonist-stimulated internalization of Ste2 requires its association with an adaptor protein, the α-arrestin Rod1/Art4, which recruits the HECT-domain ubiquitin ligase Rsp5, allowing for ubiquitinylation of the C-terminal tail of the receptor and its engagement by the clathrin-dependent endocytic machinery. We previously showed that dephosphorylation of Rod1 by calcineurin (phosphoprotein phosphatase 2B) is required for optimal Rod1 function in Ste2 down-regulation. We show here that negative regulation of Rod1 by phosphorylation is mediated by two distinct stress-activated protein kinases, Snf1/AMPK and Ypk1/SGK1, and demonstrate both in vitro and in vivo that this phospho-regulation impedes the ability of Rod1 to promote mating pathway desensitization. These studies also revealed that, in the absence of its phosphorylation, Rod1 can promote adaptation independently of Rsp5-mediated receptor ubiquitinylation, consistent with recent evidence that α-arrestins can contribute to cargo recognition by both clathrin-dependent and clathrin-independent mechanisms. However, in cells lacking a component (formin Bni1) required for clathrin-independent entry, Rod1 derivatives that are largely unphosphorylated and unable to associate with Rsp5 still promote efficient adaptation, indicating a third mechanism by which this α-arrestin promotes desensitization of the pheromone-response pathway.

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.

Septin complexes display remarkable plasticity in subunit composition, yet how a new subunit assembled into higher-order structures confers different functions is not fully understood. Here, this question is addressed in budding yeast, where during meiosis Spr3 and Spr28 replace the mitotic septin subunits Cdc12 and Cdc11 (and Shs1), respectively. In vitro, the sole stable complex that contains both meiosis-specific septins is a linear Spr28-Spr3-Cdc3-Cdc10-Cdc10-Cdc3-Spr3-Spr28 hetero-octamer. Only coexpressed Spr3 and Spr28 colocalize with Cdc3 and Cdc10 in mitotic cells, indicating that incorporation requires a Spr28-Spr3 protomer. Unlike their mitotic counterparts, Spr28-Spr3-capped rods are unable to form higher-order structures in solution but assemble to form long paired filaments on lipid monolayers containing phosphatidylinositol-4,5-bisphosphate, mimicking presence of this phosphoinositide in the prospore membrane. Spr28 and Spr3 fail to rescue the lethality of a cdc11Δ cdc12Δ mutant, and Cdc11 and Cdc12 fail to restore sporulation proficiency to spr3Δ/spr3Δ spr28Δ/spr28Δ diploids. Thus, specific meiotic and mitotic subunits endow septin complexes with functionally distinct properties.

Septins are a family of eukaryotic GTP-binding proteins that associate into linear rods, which, in turn, polymerize end-on-end into filaments, and further assemble into other, more elaborate super-structures at discrete subcellular locations. Hence, septin-based ensembles are considered elements of the cytoskeleton. One function of these structures that has been well-documented in studies conducted in budding yeast Saccharomyces cerevisiae is to serve as a scaffold that recruits regulatory proteins, which dictate the spatial and temporal control of certain aspects of the cell division cycle. In particular, septin-associated protein kinases couple cell cycle progression with cellular morphogenesis. Thus, septin-containing structures serve as signaling platforms that integrate a multitude of signals and coordinate key downstream networks required for cell cycle passage. This review summarizes what we currently understand about how the action of septin-associated protein kinases and their substrates control information flow to drive the cell cycle into and out of mitosis, to regulate bud growth, and especially to direct timely and efficient execution of cytokinesis and cell abscission. Thus, septin structures represent a regulatory node at the intersection of many signaling pathways. In addition, and importantly, the activities of certain septin-associated protein kinases also regulate the state of organization of the septins themselves, creating a complex feedback loop.

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.

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.

In eukaryotes, exposure to hypertonic conditions activates a MAPK (Hog1 in Saccharomyces cerevisiae and ortholog p38 in human cells). In yeast, intracellular glycerol accumulates to counterbalance the high external osmolarity. To prevent glycerol efflux, Hog1 action impedes the function of the aquaglyceroporin Fps1, in part, by displacing channel co-activators (Rgc1/2). However, Fps1 closes upon hyperosmotic shock even in hog1∆ cells, indicating another mechanism to prevent Fps1-mediated glycerol efflux. In our prior proteome-wide screen, Fps1 was identified as a target of TORC2-dependent protein kinase Ypk1 (Muir et al., 2014). We show here that Fps1 is an authentic Ypk1 substrate and that the open channel state of Fps1 requires phosphorylation by Ypk1. Moreover, hyperosmotic conditions block TORC2-dependent Ypk1-mediated Fps1 phosphorylation, causing channel closure, glycerol accumulation, and enhanced survival under hyperosmotic stress. These events are all Hog1-independent. Our findings define the underlying molecular basis of a new mechanism for responding to hypertonic conditions.

UnlabelledSeptins are a conserved family of GTP-binding proteins that form heterooctameric complexes that assemble into higher-order structures. In yeast, septin superstructure at the bud neck serves as a barrier to separate a daughter cell from its mother and as a scaffold to recruit the proteins that execute cytokinesis. However, how septins recruit specific factors has not been well characterized. In the accompanying article in this issue, (Finnigan et al. 2015), we demonstrated that the C-terminal extensions (CTEs) of the alternative terminal subunits of septin heterooctamers, Cdc11 and Shs1, share a role required for optimal septin function in vivo. Here we describe our use of unbiased genetic approaches (both selection of dosage suppressors and analysis of synthetic interactions) that pinpointed Bni5 as a protein that interacts with the CTEs of Cdc11 and Shs1. Furthermore, we used three independent methods-construction of chimeric proteins, noncovalent tethering mediated by a GFP-targeted nanobody, and imaging by fluorescence microscopy-to confirm that a physiologically important function of the CTEs of Cdc11 and Shs1 is optimizing recruitment of Bni5 and thereby ensuring efficient localization at the bud neck of Myo1, the type II myosin of the actomyosin contractile ring.Related article inGeneticsFinnigan, G. C. et al., 2015 Comprehensive Genetic Analysis of Paralogous Terminal Septin Subunits Shs1 and Cdc11 in Saccharomyces cerevisiae. Genetics 200: 841-861.

Septins are a family of GTP-binding proteins considered to be cytoskeletal elements because they self-assemble into filaments and other higher-order structures in vivo. In budding yeast, septins establish a diffusion barrier at the bud neck between a mother and daughter cell, promote membrane curvature there, and serve as a scaffold to recruit other proteins to the site of cytokinesis. However, the mechanism by which any septin engages a partner protein has been unclear. The two most related and recently evolved subunits appear to be Cdc11 and Shs1, and the basic building blocks for assembling septin structures are hetero-octameric rods (Cdc11-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Cdc11 and Shs1-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Shs1). Loss of Cdc11 is not normally tolerated, whereas cells lacking Shs1 do not appear grossly abnormal. We established several different sensitized genetic backgrounds wherein Shs1 is indispensable, which allowed us to carry out the first comprehensive and detailed genetic analysis of Shs1 in vivo. Our analysis revealed several novel insights, including: (i) the sole portion of Shs1 essential for its function is a predicted coiled-coil-forming segment in its C-terminal extension (CTE); (ii) the CTE of Cdc11 shares this function; (iii) this role for the CTEs of Cdc11 and Shs1 is quite distinct from that of the CTEs of Cdc3 and Cdc12; and (iv) heterotypic Cdc11 and Shs1 junctions likely occur in vivo.

Septin hetero-oligomers polymerize into cytoskeletal filaments with essential functions in many eukaryotic cell types. Mutations within the oligomerization interface that encompasses the GTP-binding pocket of a septin (its "G interface") cause thermoinstability of yeast septin hetero-oligomer assembly, and human disease. When coexpressed with its wild-type counterpart, a G interface mutant is excluded from septin filaments, even at moderate temperatures. We show that this quality control mechanism is specific to G interface mutants, operates during de novo septin hetero-oligomer assembly, and requires specific cytosolic chaperones. Chaperone overexpression lowers the temperature permissive for proliferation of cells expressing a G interface mutant as the sole source of a given septin. Mutations that perturb the septin G interface retard release from these chaperones, imposing a kinetic delay on the availability of nascent septin molecules for higher-order assembly. Un-expectedly, the disaggregase Hsp104 contributes to this delay in a manner that does not require its "unfoldase" activity, indicating a latent "holdase" activity toward mutant septins. These findings provide new roles for chaperone-mediated kinetic partitioning of non-native proteins and may help explain the etiology of septin-linked human diseases.

The glucose analog 2-deoxyglucose (2DG) inhibits the growth of Saccharomyces cerevisiae and human tumor cells, but its modes of action have not been fully elucidated. Yeast cells lacking Snf1 (AMP-activated protein kinase) are hypersensitive to 2DG. Overexpression of either of two low-affinity, high-capacity glucose transporters, Hxt1 and Hxt3, suppresses the 2DG hypersensitivity of snf1Δ cells. The addition of 2DG or the loss of Snf1 reduces HXT1 and HXT3 expression levels and stimulates transporter endocytosis and degradation in the vacuole. 2DG-stimulated trafficking of Hxt1 and Hxt3 requires Rod1/Art4 and Rog3/Art7, two members of the α-arrestin trafficking adaptor family. Mutations in ROD1 and ROG3 that block binding to the ubiquitin ligase Rsp5 eliminate Rod1- and Rog3-mediated trafficking of Hxt1 and Hxt3. Genetic analysis suggests that Snf1 negatively regulates both Rod1 and Rog3, but via different mechanisms. Snf1 activated by 2DG phosphorylates Rod1 but fails to phosphorylate other known targets, such as the transcriptional repressor Mig1. We propose a novel mechanism for 2DG-induced toxicity whereby 2DG stimulates the modification of α-arrestins, which promote glucose transporter internalization and degradation, causing glucose starvation even when cells are in a glucose-rich environment.

Plasma membrane function requires distinct leaflet lipid compositions. Two of the P-type ATPases (flippases) in yeast, Dnf1 and Dnf2, translocate aminoglycerophospholipids from the outer to the inner leaflet, stimulated via phosphorylation by cortically localized protein kinase Fpk1. By monitoring Fpk1 activity in vivo, we found that Fpk1 was hyperactive in cells lacking Gin4, a protein kinase previously implicated in septin collar assembly. Gin4 colocalized with Fpk1 at the cortical site of future bud emergence and phosphorylated Fpk1 at multiple sites, which we mapped. As judged by biochemical and phenotypic criteria, a mutant (Fpk1(11A)), in which 11 sites were mutated to Ala, was hyperactive, causing increased inward transport of phosphatidylethanolamine. Thus, Gin4 is a negative regulator of Fpk1 and therefore an indirect negative regulator of flippase function. Moreover, we found that decreasing flippase function rescued the growth deficiency of four different cytokinesis mutants, which suggests that the primary function of Gin4 is highly localized control of membrane lipid asymmetry and is necessary for optimal cytokinesis.

The protocols presented here allow for the facile generation of a wide variety of complex multipart DNA constructs (tagged gene products, gene fusions, chimeric proteins, and other variants) using homologous recombination and in vivo ligation in budding yeast (Saccharomyces cerevisiae). This method is straightforward, efficient and cost-effective, and can be used both for vector creation and for subsequent one-step, high frequency integration into a chromosomal locus in yeast. The procedure utilizes PCR with extended oligonucleotide "tails" of homology between multiple fragments to allow for reassembly in yeast in a single transformation followed by a method for highly efficient plasmid extraction from yeast (for transformation into bacteria). The latter is an improvement on existing methods of yeast plasmid extraction, which, historically, has been a limiting step in recovery of desired constructs. We describe the utility and convenience of our techniques, and provide several examples.

The class 4 P-type ATPases ("flippases") maintain membrane asymmetry by translocating phosphatidylethanolamine and phosphatidylserine from the outer leaflet to the cytosolic leaflet of the plasma membrane. In Saccharomyces cerevisiae, five related gene products (Dnf1, Dnf2, Dnf3, Drs2, and Neo1) are implicated in flipping of phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine. In MAT A: cells responding to α-factor, we found that Dnf1, Dnf2, and Dnf3, as well as the flippase-activating protein kinase Fpk1, localize at the projection ("shmoo") tip where polarized growth is occurring and where Ste5 (the central scaffold protein of the pheromone-initiated MAPK cascade) is recruited. Although viable, a MAT A: dnf1∆ dnf2∆ dnf3∆ triple mutant exhibited a marked decrease in its ability to respond to α-factor, which we could attribute to pronounced reduction in Ste5 stability resulting from an elevated rate of its Cln2⋅Cdc28-initiated degradation. Similarly, a MAT A: dnf1∆ dnf3∆ drs2∆ triple mutant also displayed marked reduction in its ability to respond to α-factor, which we could attribute to inefficient recruitment of Ste5 to the plasma membrane due to severe mislocalization of the cellular phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate pools. Thus proper remodeling of plasma membrane aminoglycerolipids and phosphoinositides is necessary for efficient recruitment, stability, and function of the pheromone signaling apparatus.

We have come a long way in the 55 years since Edmond Fischer and the late Edwin Krebs discovered that the activity of glycogen phosphorylase is regulated by reversible protein phosphorylation. Many of the fundamental molecular mechanisms that operate in biological signaling have since been characterized and the vast web of interconnected pathways that make up the cellular signaling network has been mapped in considerable detail. Nonetheless, it is important to consider how fast this field is still moving and the issues at the current boundaries of our understanding. One must also appreciate what experimental strategies have allowed us to attain our present level of knowledge. We summarize here some key issues (both conceptual and methodological), raise unresolved questions, discuss potential pitfalls, and highlight areas in which our understanding is still rudimentary. We hope these wide-ranging ruminations will be useful to investigators who carry studies of signal transduction forward during the rest of the 21st century.

Plasma membrane lipid composition must be maintained during growth and under environmental insult. In yeast, signaling mediated by TOR Complex 2 (TORC2)-dependent protein kinase Ypk1 controls lipid abundance and distribution in response to membrane stress. Ypk1, among other actions, alleviates negative regulation of L-serine:palmitoyl-CoA acyltransferase, upregulating production of long-chain base precursors to sphingolipids. To explore other roles for TORC2-Ypk1 signaling in membrane homeostasis, we devised a three-tiered genome-wide screen to identify additional Ypk1 substrates, which pinpointed both catalytic subunits of the ceramide synthase complex. Ypk1-dependent phosphorylation of both proteins increased upon either sphingolipid depletion or heat shock and was important for cell survival. Sphingolipidomics, other biochemical measurements and genetic analysis demonstrated that these modifications of ceramide synthase increased its specific activity and stimulated channeling of long-chain base precursors into sphingolipid end-products. Control at this branch point also prevents accumulation of intermediates that could compromise cell growth by stimulating autophagy.

G-protein-coupled receptors (GPCRs) are integral membrane proteins that initiate responses to extracellular stimuli by mediating ligand-dependent activation of cognate heterotrimeric G proteins. In yeast, occupancy of GPCR Ste2 by peptide pheromone α-factor initiates signaling by releasing a stimulatory Gβγ complex (Ste4-Ste18) from its inhibitory Gα subunit (Gpa1). Prolonged pathway stimulation is detrimental, and feedback mechanisms have evolved that act at the receptor level to limit the duration of signaling and stimulate recovery from pheromone-induced G1 arrest, including upregulation of the expression of an α-factor-degrading protease (Bar1), a regulator of G-protein signaling protein (Sst2) that stimulates Gpa1-GTP hydrolysis, and Gpa1 itself. Ste2 is also downregulated by endocytosis, both constitutive and ligand induced. Ste2 internalization requires its phosphorylation and subsequent ubiquitinylation by membrane-localized protein kinases (Yck1 and Yck2) and a ubiquitin ligase (Rsp5). Here, we demonstrate that three different members of the α-arrestin family (Ldb19/Art1, Rod1/Art4, and Rog3/Art7) contribute to Ste2 desensitization and internalization, and they do so by discrete mechanisms. We provide genetic and biochemical evidence that Ldb19 and Rod1 recruit Rsp5 to Ste2 via PPXY motifs in their C-terminal regions; in contrast, the arrestin fold domain at the N terminus of Rog3 is sufficient to promote adaptation. Finally, we show that Rod1 function requires calcineurin-dependent dephosphorylation.

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.

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.