Endoplasmic reticulum stress response in yeast and humans

Stress pathways monitor intracellular systems and deploy a range of regulatory mechanisms in response to stress. One of the best-characterized pathways, the UPR (unfolded protein response), is an intracellular signal transduction pathway that monitors ER (endoplasmic reticulum) homoeostasis. Its activation is required to alleviate the effects of ER stress and is highly conserved from yeast to human. Although metazoans have three UPR outputs, yeast cells rely exclusively on the Ire1 (inositol-requiring enzyme-1) pathway, which is conserved in all Eukaryotes. In general, the UPR program activates hundreds of genes to alleviate ER stress but it can lead to apoptosis if the system fails to restore homoeostasis. In this review, we summarize the major advances in understanding the response to ER stress in Sc (Saccharomyces cerevisiae), Sp (Schizosaccharomyces pombe) and humans. The contribution of solved protein structures to a better understanding of the UPR pathway is discussed. Finally, we cover the interplay of ER stress in the development of diseases.     

N-glycans are not required for the efficient degradation of the mutant Saccharomyces cerevisiae CPY* in Schizosaccharomyces pombe

In eukaryotic cells, aberrant proteins generated in the endoplasmic reticulum (ER) are degraded by the ER-associated degradation (ERAD) pathway. Here, we report on the ERAD pathway of the fission yeast Schizosaccharomyces pombe. We constructed and expressed Saccharomyces cerevisiae wild-type CPY (ScCPY) and CPY-G255R mutant (ScCPY*) in S. pombe. While ScCPY was glycosylated and efficiently transported to the vacuoles in S. pombe, ScCPY* was retained in the ER and was not processed to the matured form in these cells. Cycloheximide chase experiments revealed that ScCPY* was rapidly degraded in S. pombe, and its degradation depended on Hrd1p and Ubc7p homologs. We also found that Mnl1p and Yos9p, proteins that are essential for ERAD in S. cerevisiae, were not required for ScCPY* degradation in S. pombe. Moreover, the null-glycosylation mutant of ScCPY, CPY*0000, was rapidly degraded by the ERAD pathway. These results suggested that N-linked oligosaccharides are not important for the recognition of luminal proteins for ERAD in S. pombe cells.     

Calnexin is involved in apoptosis induced by endoplasmic reticulum stress in the fission yeast

Stress conditions affecting the functions of the endoplasmic reticulum (ER) cause the accumulation of unfolded proteins. ER stress is counteracted by the unfolded-protein response (UPR). However, under prolonged stress the UPR initiates a proapoptotic response. Mounting evidence indicate that the ER chaperone calnexin is involved in apoptosis caused by ER stress. Here, we report that overexpression of calnexin in Schizosaccharomyces pombe induces cell death with apoptosis markers. Cell death was partially dependent on the Ire1p ER-stress transducer. Apoptotic death caused by calnexin overexpression required its transmembrane domain (TM), and involved sequences on either side of the ER membrane. Apoptotic death caused by tunicamycin was dramatically reduced in a strain expressing endogenous levels of calnexin lacking its TM and cytosolic tail. This demonstrates the involvement of calnexin in apoptosis triggered by ER stress. A genetic screen identified the S. pombe homologue of the human antiapoptotic protein HMGB1 as a suppressor of apoptotic death due to calnexin overexpression. Remarkably, overexpression of human calnexin in S. pombe also provoked apoptotic death. Our results argue for the conservation of the role of calnexin in apoptosis triggered by ER stress, and validate S. pombe as a model to elucidate the mechanisms of calnexin-mediated cell death.     

P bodies promote stress granule assembly in Saccharomyces cerevisiae

Recent results indicate that nontranslating mRNAs in eukaryotic cells exist in distinct biochemical states that accumulate in P bodies and stress granules, although the nature of interactions between these particles is unknown. We demonstrate in Saccharomyces cerevisiae that RNA granules with similar protein composition and assembly mechanisms as mammalian stress granules form during glucose deprivation. Stress granule assembly is dependent on P-body formation, whereas P-body assembly is independent of stress granule formation. This suggests that stress granules primarily form from mRNPs in preexisting P bodies, which is also supported by the kinetics of P-body and stress granule formation both in yeast and mammalian cells. These observations argue that P bodies are important sites for decisions of mRNA fate and that stress granules, at least in yeast, primarily represent pools of mRNAs stalled in the process of reentry into translation from P bodies.     

Functional conservation of Dhh1p,a cytoplasmic DExD/H-box protein present in large complexes

The DHH1 gene in the yeast Saccharomyces cerevisiae encodes a putative RNA helicase of remarkable sequence similarity to several other DExD/H-box proteins, including Xp54 in Xenopus laevis and Ste13p in Schizosaccharomyces pombe. We show here that over-expression of Xp54, an integral component of the stored messenger ribonucleoprotein (mRNP) particles, can rescue the loss of Dhh1p in yeast. Localization and sedimentation studies showed that Dhh1p exists predominantly in the cytoplasm and is present in large complexes whose sizes appear to vary according to the growth stage of the cell culture. In addition, deletion of dhh1, when placed in conjunction with the mutant dbp5 and ded1 alleles, resulted in a synergistically lethal effect, suggesting that Dhh1p may have a role in mRNA export and translation. Finally, similar to Ste13p, Dhh1p is required for sporulation in the budding yeast. Taken together, our data provide evidence that the functions of Dhh1p are conserved through evolution.     

Pdc1 Functions in the Assembly of P Bodies in Schizosaccharomyces pombe

P bodies are cytoplasmic RNA granules containing the Dcp1-Dcp2 decapping enzymes where mRNA decay can occur. Here, we describe the characterization of P bodies in the fission yeast Schizosaccharomyces pombe. Most information on the property and function of P bodies stems from studies in the distantly related budding yeast Saccharomyces cerevisiae, and Edc3 was identified as a scaffold protein required for P-body assembly. However, we found that, unlike in S. cerevisiae, fission yeast Edc3 was dispensable for P-body formation. Pdc1, a novel partner of the fission yeast decapping enzyme, with a limited similarity to plant Edc4/Varicose that is required for the assembly of P bodies, was identified (tandem affinity purification–matrix-assisted laser desorption ionization tandem mass spectrometry [TAP-MALDI MS/MS]). Pdc1 interacts with Dcp2 through its C terminus and contains a coiled-coil region for self-interaction to mediate P-body formation. In line with the model that Pdc1 cross-bridges different proteins, additional interactions can be demonstrated with components such as Edc3 and Ste13. Although Pdc1 is not required for the interaction between Dcp1 and Dcp2, our data suggest that Pdc1 acts as a functional homologue of Edc4, a third component of the decapping enzymes that is thought to be absent from fungi. Together, these results highlight the diverse P-body protein compositions between different species and might help to provide insight into their evolutionary paths.     

Calnexin Is Essential for Survival under Nitrogen Starvation and Stationary Phase in Schizosaccharomyces pombe

Cell fate is determined by the balance of conserved molecular mechanisms regulating death (apoptosis) and survival (autophagy). Autophagy is a process by which cells recycle their organelles and macromolecules through degradation within the vacuole in yeast and plants, and lysosome in metazoa. In the yeast Schizosaccharomyces pombe, autophagy is strongly induced under nitrogen starvation and in aging cells. Previously, we demonstrated that calnexin (Cnx1p), a highly conserved transmembrane chaperone of the endoplasmic reticulum (ER), regulates apoptosis under ER stress or inositol starvation. Moreover, we showed that in stationary phase, Cnx1p is cleaved into two moieties, L_Cnx1p and S_Cnx1p. Here, we show that the processing of Cnx1p is regulated by autophagy, induced by nitrogen starvation or cell aging. The cleavage of Cnx1p involves two vacuolar proteases: Isp6, which is essential for autophagy, and its paralogue Psp3. Blocking autophagy through the knockout of autophagy-related genes (atg) results in inhibition of both, the cleavage and the trafficking of Cnx1p from the ER to the vacuole. We demonstrate that Cnx1p is required for cell survival under nitrogen-starvation and in chronological aging cultures. The death of the mini_cnx1 mutant (overlapping S_cnx1p) cells is accompanied by accumulation of high levels of reactive-oxygen species (ROS), a slowdown in endocytosis and severe cell-wall defects. Moreover, mutant cells expressing only S_Cnx1p showed cell wall defects. Co-expressing mutant overlapping the L_Cnx1p and S_Cnx1p cleavage products reverses the death, ROS phenotype and cell wall defect to wild-type levels. As it is involved in both apoptosis and autophagy, Cnx1p could be a nexus for the crosstalk between these pro-death and pro-survival mechanisms. Ours, and observations in mammalian systems, suggest that the multiple roles of calnexin depend on its sub-cellular localization and on its cleavage. The use of S. pombe should assist in further shedding light on the multiple roles of calnexin.     

Schizosaccharomyces pombe cardiolipin synthase is part of a mitochondrial fusion protein regulated by intron retention

Cardiolipin (CL) is a unique lipid component of mitochondria in all eukaryotes. It is important for the architecture of mitochondrial membranes and for mitochondrial dynamics. CL also creates a highly specific microenvironment of mitochondrial protein machineries. CL biosynthetic pathway is, however, only partially characterized in the fission yeast Schizosaccharomyces pombe. Here we show that CL synthase is an essential protein in S. pombe. It is encoded by the ORF SPAC22A12.08c as a C terminal part of a tandem fusion protein together with a mitochondrial hydrolase of unknown function. Expression of S. pombe CL synthase is able to complement deletion of the CRD1 gene of Saccharomyces cerevisiae and, vice versa, S. cerevisiae CRD1 gene complements deletion of S. pombe SPAC22A12.08c. The proper expression of CL synthase and its partner in the tandem protein, the mitochondrial hydrolase, is regulated at the level of alternate intron splicing. The first part of the SPAC22A12.08c fusion protein could be translated from both major SPAC22A12.08c derived mRNAs, with and without intron IV. Functional CL synthase, however, is produced only from the minor SPAC22A12.08c derived mRNA that has intron IV retained. Thus, intron retention is a novel mechanism for the differential expression of two proteins that evolved as a fusion protein and are under the control of the same promoter.     

Inhibition of Protein Translocation in Permeabilized Cells of Schizosaccharomyces pombe by Puromycin

To investigate protein translocation in eukaryotes, we reconstituted a protein translocation system using the permeabilized spheroplasts (P-cells) of the fission yeast Schizosaccharomyces pombe. The precursor of a sex pheromone of Saccharomyces cerevisiae, prepro-α-factor, was translocated across the endoplasmic reticulum (ER) of S. pombe posttranslationally, and glycosylated to the same extent as in the ER of S. cerevisiae. This suggested that the size of N-linked core-oligosaccharide in the ER of S. pombe is similar to that in S. cerevisiae. This translocation into the ER of S. pombe was inhibited by puromycin, but the translocation in the P-cells of S. cerevisiae was not inhibited. This difference in sensitivity to puromycin was due to the membrane but not the cytosolic fraction. Our results suggested that the translocation machinery of S. pombe was sensitive to puromycin and different from that of S. cerevisiae.     

Selected Schizosaccharomyces pombe Strains Have Characteristics That Are Beneficial for Winemaking

At present, wine is generally produced using Saccharomyces yeast followed by Oenococus bacteria to complete malolactic fermentation. This method has some unsolved problems, such as the management of highly acidic musts and the production of potentially toxic products including biogenic amines and ethyl carbamate. Here we explore the potential of the fission yeast Schizosaccharomyces pombe to solve these problems. We characterise an extensive worldwide collection of S. pombe strains according to classic biochemical parameters of oenological interest. We identify three genetically different S. pombe strains that appear suitable for winemaking. These strains compare favourably to standard Saccharomyces cerevisiae winemaking strains, in that they perform effective malic acid deacidification and significantly reduce levels of biogenic amines and ethyl carbamate precursors without the need for any secondary bacterial malolactic fermentation. These findings indicate that the use of certain S. pombe strains could be advantageous for winemaking in regions where malic acid is problematic, and these strains also show superior performance with respect to food safety.     

Dcp2 phosphorylation by Ste20 modulates stress granule assembly and mRNA decay in Saccharomyces cerevisiae

Translation and messenger RNA (mRNA) degradation are important sites of gene regulation, particularly during stress where translation and mRNA degradation are reprogrammed to stabilize bulk mRNAs and to preferentially translate mRNAs required for the stress response. During stress, untranslating mRNAs accumulate both in processing bodies (P-bodies), which contain some translation repressors and the mRNA degradation machinery, and in stress granules, which contain mRNAs stalled in translation initiation. How signal transduction pathways impinge on proteins modulating P-body and stress granule formation and function is unknown. We show that during stress in Saccharomyces cerevisiae, Dcp2 is phosphorylated on serine 137 by the Ste20 kinase. Phosphorylation of Dcp2 affects the decay of some mRNAs and is required for Dcp2 accumulation in P-bodies and specific protein interactions of Dcp2 and for efficient formation of stress granules. These results demonstrate that Ste20 has an unexpected role in the modulation of mRNA decay and translation and that phosphorylation of Dcp2 is an important control point for mRNA decapping.     

Functional Expression and Characterization of Schizosaccharomyces pombe Avt3p as a Vacuolar Amino Acid Exporter in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, Avt3p and Avt4p mediate the extrusion of several amino acids from the vacuolar lumen into the cytosol. SpAvt3p of Schizosaccharomyces pombe, a homologue of these vacuolar amino acid transporters, has been indicated to be involved in spore formation. In this study, we confirmed that GFP-SpAvt3p localized to the vacuolar membrane in S. pombe. The amounts of various amino acids increased significantly in the vacuolar pool of avt3Δ cells, but decreased in that of avt3 ⁺-overexpressing avt3Δ cells. These results suggest that SpAvt3p participates in the vacuolar compartmentalization of amino acids in S. pombe. To examine the export activity of SpAvt3p, we expressed the avt3 ⁺ gene in S. cerevisiae cells. We found that the heterologously overproduced GFP-SpAvt3p localized to the vacuolar membrane in S. cerevisiae. Using the vacuolar membrane vesicles isolated from avt3 ⁺-overexpressing S. cerevisiae cells, we detected the export activities of alanine and tyrosine in an ATP-dependent manner. These activities were inhibited by the addition of a V-ATPase inhibitor, concanamycin A, thereby suggesting that the activity of SpAvt3p is dependent on a proton electrochemical gradient generated by the action of V-ATPase. In addition, the amounts of various amino acids in the vacuolar pools of S. cerevisiae cells were decreased by the overproduction of SpAvt3p, which indicated that SpAvt3p was functional in S. cerevisiae cells. Thus, SpAvt3p is a vacuolar transporter that is involved in the export of amino acids from S. pombe vacuoles.     

Localization to,and Effects of Pbp1, Pbp4, Lsm12, Dhh1, and Pab1 on Stress Granules in Saccharomyces cerevisiae

The regulation of translation and mRNA degradation in eukaryotic cells involves the formation of cytoplasmic mRNP granules referred to as P-bodies and stress granules. The yeast Pbp1 protein and its mammalian ortholog, Ataxin-2, localize to stress granules and promote their formation. In Saccharomyces cerevisiae, Pbp1 also interacts with the Pab1, Lsm12, Pbp4, and Dhh1 proteins. In this work, we determined whether these Pbp1 interacting proteins also accumulated in stress granules and/or could affect their formation. These experiments revealed the following observations. First, the Lsm12, Pbp4, and Dhh1 proteins all accumulate in stress granules, whereas only the Dhh1 protein is a constitutive P-body component. Second, deletion or over-expression of the Pbp4 and Lsm12 proteins did not dramatically affect the formation of stress granules or P-bodies. In contrast, Pbp1 and Dhh1 over-expression inhibits cell growth, and for Dhh1, leads to the accumulation of stress granules. Finally, a strain lacking the Pab1 protein was reduced at forming stress granules, although they could still be detected. This indicates that Pab1 affects, but is not absolutely required for, stress granule formation. These observations offer new insight into the function of stress granule components with roles in stress granule assembly and mRNP regulation.     

Oxidative stress in yeast

The mechanisms of production and elimination of reactive oxygen species in the cells of the budding yeast Saccharomyces cerevisiae are analyzed. Coordinative role of special regulatory proteins including Yap1p, Msn2/4p, and Skn7p (Pos9p) in regulation of defense mechanisms in S. cerevisiae is described. A special section is devoted to two other well-studied species from the point of view of oxidative stress — Schizosaccharomyces pombe and Candida albicans. Some examples demonstrating the use of yeast for investigation of apoptosis, aging, and some human diseases are given in the conclusion part.     

Isolation and characterization of the fission yeast gene rpa42 +, which encodes a subunit shared by RNA polymerases I and III

Eukaryotic RNA polymerases I and III share two distinct α-related subunits that show limited homology to the α subunit of Escherichia coli RNA polymerase, which forms a homodimer to nucleate the assembly of prokaryotic RNA polymerase. To gain insight into the functions of α-related subunits in eukaryotes, we have previously identified the α-related small subunit RPA17 of RNA polymerase I (and III) in Schizosaccharomyces pombe, and have shown that it is a functional homolog of Saccharomyces cerevisiae AC19. In an extension of that study, we have now isolated and characterized rpa42 ⁺, which encodes the α-related large subunit RPA42 of S. pombe RNA polymerase I, by virtue of the fact that its product interacts with RPA17 in the yeast two-hybrid system. We have found that rpa42 ⁺ encodes a polypeptide with an apparent molecular mass of 42?kDa, which shows 58% identity to the AC40 subunit shared by RNA polymerases I and III in S. cerevisiae. Furthermore, we have shown that rpa42 ⁺ complements a temperature-sensitive mutation in RPC40 the gene that encodes AC40 in S. cerevisiae and which is essential for cell growth. Finally, we have shown that neither RPA42 nor RPA17 can self-associate. These results provide evidence that the two distinct α-related subunits, RPA42 and RPA17, of RNA polymerases I and III are functionally conserved between S. pombe and S. cerevisiae, and suggest that heterodimer formation between them is essential for the assembly of RNA polymerases I and III in eukaryotes.