Mechanism In Yeast Research Articles

The unfolded protein response: no longer just a special teams player.

The endoplasmic reticulum stress pathway known as the unfolded protein response is currently the best understood model of interorganellar signal transduction. Bridging a physical separation, the pathway provides a direct line of communication between the endoplasmic reticulum lumen and the nucleus. With the unfolded protein response, the cell has the means to monitor and respond to the changing needs of the endoplasmic reticulum. Beginning with the discovery of its remarkable signaling mechanism in yeast, the unfolded protein response has not ceased to reveal more of its many secrets. By applying powerful biochemical, genetic, genomic, and cytological approaches, the recent efforts of many groups have buried the long-held notion that the unfolded protein response is simply a regulatory platform for endoplasmic reticulum chaperones. We now know that the unfolded protein response regulates many genes that affect diverse aspects of cellular physiology. In addition, studies in mammals have revealed novel unfolded protein response signaling factors that may contribute to the specialized needs of multicellular organisms. This article focuses on these and other recent developments in the field.

Identification of the yeast cytidine deaminase CDD1 as an orphan C-->U RNA editase.

Yeast co-expressing rat APOBEC-1 and a fragment of human apolipoprotein B (apoB) mRNA assembled functional editosomes and deaminated C6666 to U in a mooring sequence-dependent fashion. The occurrence of APOBEC-1-complementing proteins suggested a naturally occurring mRNA editing mechanism in yeast. Previously, a hidden Markov model identified seven yeast genes encoding proteins possessing putative zinc-dependent deaminase motifs. Here, only CDD1, a cytidine deaminase, is shown to have the capacity to carry out C-->U editing on a reporter mRNA. This is only the second report of a cytidine deaminase that can use mRNA as a substrate. CDD1-dependent editing was growth phase regulated and demonstrated mooring sequence-dependent editing activity. Candidate yeast mRNA substrates were identified based on their homology with the mooring sequence-containing tripartite motif at the editing site of apoB mRNA and their ability to be edited by ectopically expressed APOBEC-1. Naturally occurring yeast mRNAs edited to a significant extent by CDD1 were, however, not detected. We propose that CDD1 be designated an orphan C-->U editase until its native RNA substrate, if any, can be identified and that it be added to the CDAR (cytidine deaminase acting on RNA) family of editing enzymes.

Calcium Influx and Signaling in Yeast Stimulated by Intracellular Sphingosine 1-Phosphate Accumulation

In mammalian cells, intracellular sphingosine 1-phosphate (S1P) can stimulate calcium release from intracellular organelles, resulting in the activation of downstream signaling pathways. The budding yeast Saccharomyces cerevisiae expresses enzymes that can synthesize and degrade S1P and related molecules, but their possible role in calcium signaling has not yet been tested. Here we examine the effects of S1P accumulation on calcium signaling using a variety of yeast mutants. Treatment of yeast cells with exogenous sphingosine stimulated Ca(2+) accumulation through two distinct pathways. The first pathway required the Cch1p and Mid1p subunits of a Ca(2+) influx channel, depended upon the function of sphingosine kinases (Lcb4p and Lcb5p), and was inhibited by the functions of S1P lyase (Dpl1p) and the S1P phosphatase (Lcb3p). The biologically inactive stereoisomer of sphingosine did not activate this Ca(2+) influx pathway, suggesting that the active S1P isomer specifically stimulates a calcium-signaling mechanism in yeast. The second Ca(2+) influx pathway stimulated by the addition of sphingosine was not stereospecific, was not dependent on the sphingosine kinases, occurred only at higher doses of added sphingosine, and therefore was likely to be nonspecific. Mutants lacking both S1P lyase and phosphatase (dpl1 lcb3 double mutants) exhibited constitutively high Ca(2+) accumulation and signaling in the absence of added sphingosine, and these effects were dependent on the sphingosine kinases. These results show that endogenous S1P-related molecules can also trigger Ca(2+) accumulation and signaling. Several stimuli previously shown to evoke calcium signaling in wild-type cells were examined in lcb4 lcb5 double mutants. All of the stimuli produced calcium signals independent of sphingosine kinase activity, suggesting that phosphorylated sphingoid bases might serve as messengers of calcium signaling in yeast during an unknown cellular response.

More Pairs of SOCS

Capacitative calcium entry (CCE) refers to Ca 2+ influx across the plasma membrane (PM) that occurs in response to calcium release from intracellular stores. One proposed regulatory mechanism in vertebrate cells involves the physical and functional coupling of IP 3 -activated calcium release channels (IP 3 Rs) present in store membranes with store-operated channels (SOCs). When activated, IP 3 Rs are thought to undergo a conformational change that couples to the opening of SOCs in the PM. Two papers suggest that there may be additional channels and mechanisms that can contribute to CCE: Kiselyov et al . have found gating of SOCs by ryanodine receptors (RyRs) and Locke et al . have identified a CCE mechanism in yeast that involves a channel with homology to voltage-gated Ca 2+ channels. Kiselyov et al . demonstrate that, like IP 3 Rs, RyRs appeared to gate the store-operated calcium channels hTrp3 and I crac in whole cells and in membrane patches when RyRs were stimulated with caffeine. hTrp3 channels coimmunoprecipitated with either IP 3 Rs or RyRs but not both, suggesting that although both receptors are present in store membranes, their interaction with SOCs is mutually exclusive. The authors also found that cell membrane patches contained SOCs coupled to either IP 3 Rs or RyRs, but not to both simultaneously, and suggest that the pairs are present in separate membrane microdomains. Yeasts are also capable of using calcium as a second messenger and have increased concentrations of calcium in the organelles of the secretory pathway. Using a genetic screen designed to detect mutants defective in CCE, Locke et al. identified two proteins required for CCE in yeast: Cch1p, a PM SOC that has structural homology to voltage-gated Ca 2+ channels and a PM glycoprotein, Mid1p. Disruption of the genes for CCH1 or MID1 abolished high-affinity Ca 2+ influx associated with conditions that stimulated CCE. The yeast model of CCE involves the Golgi Ca 2+ -ATPase, which sequesters calcium in the secretory organelles, and Cch1p and Mid1p, which are localized to the PM and stimulate Ca 2+ influx required for store replenishment. Kiselyov, K.I., Shin, D.M., Wang, Y., Pessah, I.N., Allen, P.D., and Muallem, S. (2000) Gating of store-operated channels by conformational coupling to ryanodine receptors. Mol. Cell 6 : 421-431. [Online Journal] Locke, E.G., Bonilla, M., Liang, L., Takita, Y., and Cunningham, K.W. (2000) A homolog of voltage-gated Ca 2+ channels stimulated by depletion of secretory Ca 2+ in yeast. Mol. Cell. Biol. 20 : 6686-6694. [Abstract] [Full Text]

More Pairs of SOCS

Capacitative calcium entry (CCE) refers to Ca2+ influx across the plasma membrane (PM) that occurs in response to calcium release from intracellular stores. One proposed regulatory mechanism in vertebrate cells involves the physical and functional coupling of IP3-activated calcium release channels (IP3Rs) present in store membranes with store-operated channels (SOCs). When activated, IP3Rs are thought to undergo a conformational change that couples to the opening of SOCs in the PM. Two papers suggest that there may be additional channels and mechanisms that can contribute to CCE: Kiselyov et al. have found gating of SOCs by ryanodine receptors (RyRs) and Locke et al. have identified a CCE mechanism in yeast that involves a channel with homology to voltage-gated Ca2+ channels. Kiselyov et al. demonstrate that, like IP3Rs, RyRs appeared to gate the store-operated calcium channels hTrp3 and Icrac in whole cells and in membrane patches when RyRs were stimulated with caffeine. hTrp3 channels coimmunoprecipitated with either IP3Rs or RyRs but not both, suggesting that although both receptors are present in store membranes, their interaction with SOCs is mutually exclusive. The authors also found that cell membrane patches contained SOCs coupled to either IP3Rs or RyRs, but not to both simultaneously, and suggest that the pairs are present in separate membrane microdomains. Yeasts are also capable of using calcium as a second messenger and have increased concentrations of calcium in the organelles of the secretory pathway. Using a genetic screen designed to detect mutants defective in CCE, Locke et al. identified two proteins required for CCE in yeast: Cch1p, a PM SOC that has structural homology to voltage-gated Ca2+ channels and a PM glycoprotein, Mid1p. Disruption of the genes for CCH1 or MID1 abolished high-affinity Ca2+ influx associated with conditions that stimulated CCE. The yeast model of CCE involves the Golgi Ca2+ -ATPase, which sequesters calcium in the secretory organelles, and Cch1p and Mid1p, which are localized to the PM and stimulate Ca2+ influx required for store replenishment.Kiselyov, K.I., Shin, D.M., Wang, Y., Pessah, I.N., Allen, P.D., and Muallem, S. (2000) Gating of store-operated channels by conformational coupling to ryanodine receptors. Mol. Cell 6: 421-431. [Online Journal]Locke, E.G., Bonilla, M., Liang, L., Takita, Y., and Cunningham, K.W. (2000) A homolog of voltage-gated Ca2+ channels stimulated by depletion of secretory Ca2+ in yeast. Mol. Cell. Biol. 20: 6686-6694. [Abstract] [Full Text]

Dissecting transcriptional circuitry and mechanisms in yeast

Dissecting transcriptional circuitry and mechanisms in yeast

Oligomerized Ced-4 Kills Budding Yeast through a Caspase-Independent Mechanism

In Caenorhabdtis elegans, Ced-3, Ced-4, and Ced-9 are components of a cell suicide program. Ced-4 facilitates the proteolytic activation of the caspase, Ced-3, while Ced-9 opposes Ced-3/Ced-4 killing. To examine the interactions among these proteins they were expressed in Saccharomyces cerevisiae. Ced-3 and Ced-4 were lethal when expressed alone, revealing an intrinsic Ced-4 killing activity. Coexpression of Ced-9 blocked Ced-3- and Ced-4-induced killing, showing Ced-9 can independently antagonize the action of both proteins. Ced-3- but not Ced-4-toxicity was attenuated by coexpression of the caspase inhibitors, CrmA and p35. Thus, besides its Ced-3- and Ced-9-dependent action in C. elegans, Ced-4 has an additional Ced-9-dependent, Ced-3-independent killing mechanism in yeast. Two-hybrid analysis confirmed that Ced-4 formed heteromers with Ced-9. In addition, Ced-4 formed homomers and mutation of its nucleoside triphosphate binding motif eliminated both homomerization and cell killing. We suggest the caspase-independent lethality of Ced-4 in yeast is mediated by a Ced-4 homomer.

A Yeast Homologue of the Human Phosphotyrosyl Phosphatase Activator PTPA Is Implicated in Protection against Oxidative DNA Damage Induced by the Model Carcinogen 4-Nitroquinoline 1-Oxide

The model carcinogen 4-nitroquinoline 1-oxide (4-NQO) has historically been characterized as "UV-mimetic" with respect to its genotoxic properties. However, recent evidence indicates that 4-NQO, unlike 254-nm UV light, may exert significant cytotoxic and/or mutagenic potential via the generation of reactive oxygen species. To elucidate the response of eukaryotic cells to 4-NQO-induced oxidative stress, we isolated Saccharomyces cerevisiae mutants exhibiting hypersensitivity to the cytotoxic effects of this mutagen. One such mutant, EBY1, was cross-sensitive to the oxidative agents UVA and diamide while retaining parental sensitivities to 254-nm UV light, methyl methanesulfonate, and ionizing radiation. A complementing gene (designated yPTPA1), restoring full UVA and 4-NQO resistance to EBY1 and encoding a protein that shares 40% identity with the human phosphotyrosyl phosphatase activator hPTPA, has been isolated. Targeted deletion of yPTPA1 in wild type yeast engendered the identical pattern of mutagen hypersensitivity as that manifested by EBY1, in addition to a spontaneous mutator phenotype that was markedly enhanced upon exposure to either UVA or 4-NQO but not to 254-nm UV or methyl methanesulfonate. Moreover, the yptpa1 deletion mutant exhibited a marked deficiency in the recovery of high molecular weight DNA following 4-NQO exposure, revealing a defect at the level of DNA repair. These data (i) strongly support a role for active oxygen intermediates in determining the genotoxic outcome of 4-NQO exposure and (ii) suggest a novel mechanism in yeast involving yPtpa1p-mediated activation of a phosphatase that participates in the repair of oxidative DNA damage, implying that hPTPA may exert a similar function in humans.

Human transferrin receptor is active and plasma membrane-targeted in yeast.

The human transferrin receptor, a type II plasma membrane protein which mediates iron transport in human cells, was expressed in the yeast Saccharomyces cerevisiae. The transferrin receptor synthesized by yeast cells was posttranslationally modified comparable to the native receptor with respect to glycosylation and dimer formation. The location of the expressed receptor in the yeast plasma membrane indicates that the targeting of this type II membrane protein shares similar mechanisms in yeast and mammalian cells. The yeast-expressed transferrin receptor showed binding activity towards its natural ligand, transferrin in an ELISA binding assay.

Human transferrin receptor is active and plasma membrane-targeted in yeast

The human transferrin receptor, a type II plasma membrane protein which mediates iron transport in human cells, was expressed in the yeast Saccharomyces cerevisiae. The transferrin receptor synthesized by yeast cells was posttranslationally modified comparable to the native receptor with respect to glycosylation and dimer formation. The location of the expressed receptor in the yeast plasma membrane indicates that the targeting of this type II membrane protein shares similar mechanisms in yeast and mammalian cells. The yeast-expressed transferrin receptor showed binding activity towards its natural ligand, transferrin in an ELISA binding assay.

Activation and repression mechanisms in yeast.

Activation and repression mechanisms in yeast.

Altered extent of cross-linking of beta1,6-glucosylated mannoproteins to chitin in Saccharomyces cerevisiae mutants with reduced cell wall beta1,3-glucan content.

The yeast cell wall contains beta1,3-glucanase-extractable and beta1,3-glucanase-resistant mannoproteins. The beta1,3-glucanase-extractable proteins are retained in the cell wall by attachment to a beta1,6-glucan moiety, which in its turn is linked to beta1,3-glucan (J. C. Kapteyn, R. C. Montijn, E. Vink, J. De La Cruz, A. Llobell, J. E. Douwes, H. Shimoi, P. N. Lipke, and F. M. Klis, Glycobiology 6:337-345, 1996). The beta1,3-glucanase-resistant protein fraction could be largely released by exochitinase treatment and contained the same set of beta1,6-glucosylated proteins, including Cwp1p, as the B1,3-glucanase-extractable fraction. Chitin was linked to the proteins in the beta1,3-glucanase-resistant fraction through a beta1,6-glucan moiety. In wild-type cell walls, the beta1,3-glucanase-resistant protein fraction represented only 1 to 2% of the covalently linked cell wall proteins, whereas in cell walls of fks1 and gas1 deletion strains, which contain much less beta1,3-glucan but more chitin, beta1,3-glucanase-resistant proteins represented about 40% of the total. We propose that the increased cross-linking of cell wall proteins via beta1,6-glucan to chitin represents a cell wall repair mechanism in yeast, which is activated in response to cell wall weakening.

Regulation of the protein kinase PKR by the vaccinia virus pseudosubstrate inhibitor K3L is dependent on residues conserved between the K3L protein and the PKR substrate eIF2alpha.

The mammalian double-stranded RNA-activated protein kinase PKR is a component of the cellular antiviral defense mechanism and phosphorylates Ser-51 on the alpha subunit of the translation factor eIF2 to inhibit protein synthesis. To identify the molecular determinants that specify substrate recognition by PKR, we performed a mutational analysis on the vaccinia virus K3L protein, a pseudosubstrate inhibitor of PKR. High-level expression of PKR is lethal in the yeast Saccharomyces cerevisiae because PKR phosphorylates eIF2alpha and inhibits protein synthesis. We show that coexpression of vaccinia virus K3L can suppress the growth-inhibitory effects of PKR in yeast, and using this system, we identified both loss-of-function and hyperactivating mutations in K3L. Truncation of, or point mutations within, the C-terminal portion of the K3L protein, homologous to residues 79 to 83 in eIF2alpha, abolished PKR inhibitory activity, whereas the hyperactivating mutation, K3L-H47R, increased the homology between the K3L protein and eIF2alpha adjacent to the phosphorylation site at Ser-51. Biochemical and yeast two-hybrid analyses revealed that the suppressor phenotype of the K3L mutations correlated with the affinity of the K3L protein for PKR and was inversely related to the level of eIF2alpha phosphorylation in the cell. These results support the idea that residues conserved between the pseudosubstrate K3L protein and the authentic substrate eIF2alpha play an important role in substrate recognition, and they suggest that PKR utilizes sequences both near and over 30 residues from the site of phosphorylation for substrate recognition. Finally, by reconstituting part of the mammalian antiviral defense mechanism in yeast, we have established a genetically useful system to study viral regulators of PKR.

Modulation of Cell Death in Yeast by the Bcl-2 Family of Proteins

Bcl-2 family members are regulators of cell death. The precise biochemical properties of these proteins are unclear although intrafamily protein-protein association is thought to be involved. To elucidate structure-activity relationships among Bcl-2 proteins and identify the pathways in which they act, an inducible death suppressor assay was developed in yeast. Only Bax and Bak killed yeast via a process that did not require interleukin-1beta-converting enzyme-like proteases. Bax/Bak lethality was suppressed by coexpression of Bcl-2 family members that are anti-apoptotic in vertebrates, namely Bcl-xL, Bcl-2, Mcl-1, and A1. Furthermore, Bcl-xL and Bcl-2 suppressed Bax toxicity by distinct mechanisms in yeast. Bad, Bcl-xS, and Ced-9 lacked suppressor activity. These inactive proteins bound to anti-apoptotic members of the Bcl-2 family but not to Bax or Bak. In contrast, most Bcl-2 family proteins that attenuated death bound to Bax and Bak. However, two mutants of Bcl-xL suppressed Bax-induced cell death while having no Bax binding activity. Therefore, Bcl-xL functions independently of Bax binding, perhaps by interacting with a common target or promoting a pathway that antagonizes Bax. Thus, the pathways downstream of Bax and Bcl-xL may be conserved between vertebrates and yeast. This suppressor assay could be used to isolate components of these pathways.

Biochemical and Genetic Characterization of a 2-Deoxy-D-Glucose Resistant Mutant of Saccharomyces Cerevisiae

Biochemical and genetic characteristics of a 2-deoxy-d-glucose resistant mutant of Saccharomyces cerevisiae were examined. Fermentation studies indicated that the mutant possessed greater maltose uptake ability in the presence of glucose than did the parental strain. In addition, growth of cells on glucose and/or maltose resulted in differences in sugar transport and growth rates between the mutant and the parental strains. Gene expression monitored by 35S-methionine in vivo labeling of nascent solubilized cellular proteins under the various growth conditions revealed alterations in protein profile. Among the radiolabeled polypeptides, maltose-inducible and glucose-repressible products were observed. The identified proteins may play a role in the repression/derepression mechanism in yeast.

Nuclear transport and nuclear pores in yeast.

The central features of nuclear import have been conserved during evolution. In yeast the nuclear accumulation of proteins follows the same selective and active transport mechanisms known from higher eukaryotes. Yeast nuclear proteins contain nuclear localization sequences (NLS) which are presumably recognized by receptors in the cytoplasm and the nuclear envelope. Subsequent to this recognition step, nuclear proteins are translocated into the nucleus via the nuclear pore complexes. The structure of the yeast nuclear pore complex resembles that of higher eukaryotes. Recently, the first putative components of the yeast nuclear import machinery have been cloned and sequenced. The genetically amenable yeast system allows for an efficient structural and functional analysis of these components. Due to the evolutionary conservation potential insights into the nuclear import mechanisms in yeast can be transferred to higher eukaryotes. Thus, yeast can be considered as a eukaryotic model system to study nuclear transport.

Role of Signal Transduction Systems in Cell Proliferation in Yeast

Publisher Summary This chapter discusses the major progress made in the genetic analysis of the mutants altered in the signal-transduction system and in the studies of the function of second messenger in yeast cells. The responses of yeast cells to external signals are commonly mediated by intracellular second messengers, such as cAMP, and the breakdown products of phosphoinositides. An important approach is to use mutants defective in the signal-transduction systems as the critical controls for the biochemical analyses of the cascade reactions related to the second messenger. The cAMP-requiring mutants are isolated from strains utilizing cAMP with the genetic background of P-28-24C. Strains indicating low cAMP levels have three distinct phenotypes: (1) they fail to grow efficiently on nonfermentable carbon sources, (2) they hyperaccumulate the storage carbohydrate glycogen and trehalose, and (3) diploid cells sporulate in rich medium. The mutant analysis on the cAMP-cascade system reveals that the cAMP-dependent protein kinase is involved in the phosphorylation events of specific proteins that need to regulate growth and differentiation of yeast. cAMP-dependent phosphorylation and dephosphorylation mechanisms in yeast regulate activities of various enzymes, such as trehalase, NAD-dependent glutamatedehydrogenase, glycogen phosphorylase, glycogen synthetase, and RNA polymerase.

Control of yeast GAL genes by MIG1 repressor: a transcriptional cascade in the glucose response.

Glucose repression is a global regulatory mechanism in yeast. We have investigated how glucose regulates the GAL genes, which are required for galactose fermentation. We found that the GAL genes are controlled by a transcriptional cascade. Thus, GAL4, which encodes an activator of the GAL genes, is repressed by MIG1, a zinc finger protein that binds to the GAL4 promoter. MIG1 has a dual role in control of the GAL genes, since MIG1 also binds to the promoter of GAL1, a gene regulated by GAL4. A disruption of MIG1 interacts synergistically with a disruption of GAL80, a gene involved in galactose induction. This suggests that the MIG1-dependent response to glucose is amplified by down-regulation of the induction pathway.

2-Deoxy-D-glucose resistant yeast with altered sugar transport activity

The transport of glucose and maltose in Saccharomyces cerevisiae was observed to occur by both high and low affinity transport systems. A spontaneously isolated 2-deoxy-D-glucose resistant mutant was observed to transport glucose and maltose only by the high affinity transport systems. Associated with this was an increase in the V max values, indicating derepression of the high affinity transport systems. The low affinity transport systems could not be detected. This mutant will be important in examining the repression regulatory and sugar transport mechanisms in yeast.

Mammalian glucocorticoid receptor derivatives enhance transcription in yeast.

In mammalian cells, the glucocorticoid receptor binds specifically to glucocorticoid response element (GRE) DNA sequences and enhances transcription from linked promoters. It is shown here that derivatives of the glucocorticoid receptor also enhance transcription when expressed in yeast. Receptor-mediated enhancement in yeast was observed in fusions of GRE sequences to the yeast cytochrome c1 (CYC1) promoter; the CYC1 upstream activator sequences were not essential, since enhancement was observed in fusions of GREs to mutant CYC1 promoters retaining only the TATA region and transcription startpoints. It is concluded that the receptor operates by a common, highly conserved mechanism in yeast and mammalian cells.