The epigenetic calnexin-independent state is induced in response to environmental changes

Yeasts have evolved numerous responsive pathways to survive in fluctuating and stressful environments. The endoplasmic reticulum (ER) is sensitive to adverse conditions, which are detected by response pathways to ensure correct protein folding. Calnexin is an ER transmembrane chaperone acting in both quality control of folding and response to persistent stress. Calnexin is a key protein required for viability in certain organisms such as mammals and the fission yeast Schizosaccharomyces pombe . Nevertheless, S. pombe calnexin-independent (Cin) cells were obtained after transient expression of a particular calnexin mutant. The Cin state is dominant, is stably propagated by an epigenetic mechanism and segregates in a non-Mendelian fashion to the meiotic progeny. The nucleolar protein Cif1p was identified as an inducer of the Cin state in a previous genetic screen. Here, we report the identification of novel inducers isolated in an overexpression genetic screen: pyruvate kinase (Pyk1p) and phosphoglycerate kinase (Pgk1p). Addition of pyruvate, the end product of pyruvate kinase and glycolysis, also induced calnexin independence in a dose-dependent manner. Remarkably, growth in respiration media or cold temperatures induced the appearance of Cin cells at high frequencies. Taken together, our results indicate that the Cin state can be triggered by extracellular changes, suggesting that this state represents an epigenetic adaptative response to environmental modifications.     

Expression of bak in S. pombe results in a lethality mediated through interaction with the calnexin homologue Cnx1

Expression studies in the yeast S. pombe have been utilised to establish the basis for a genetic analysis designed to identify the lethal partners of the pro-apoptotic proteins bak and bax. Bak expression in S. pombe is lethal and this lethality is rescued by co-expression of bcl-2 or bcl-x(L). S. pombe cells expressing bak have a terminal phenotype in which the majority of cells are blocked in the G1 phase of the cell cycle while the remainder of cells, unable to complete M-phase, mis-coordinate the timing of subsequent events in the cell cycle. Although bax expression in S. pombe gives rise to a slow growth phenotype, not a lethality, bax expressing cells display the same cell cycle phenotypes described for bak. Electron microscopy of cells expressing bak reveals a dramatic accumulation of large vesicular structures. A two-hybrid screen designed to identify S. pombe proteins which interact with bak, isolated the S. pombe calnexin homologue cnx1. Genetic analysis demonstrates that the Cnx1 domain which binds to bak in two-hybrid experiments, is necessary for bak lethality in S. pombe. This report identifies a lethal interacting partner for bak and the observations suggest a model for bak mediated lethality which can be tested in higher cells.     

Isolation and characterisation of a calnexin homologue<Emphasis Type="Italic">, clxA</Emphasis>, from <Emphasis Type="Italic"> Aspergillus niger</Emphasis>

We describe the isolation of a gene (clxA) encoding calnexin from laboratory and industrial strains of Aspergillus niger. Calnexin is a chaperone, which specifically recognises monoglucosylated glycoproteins in the endoplasmic reticulum, and is thus an essential component of the process that assesses the folded state of nascent secreted glycoproteins. Manipulation of chaperones has previously been adopted in attempts to overcome some of the problems associated with the secretion of heterologous proteins from filamentous fungi. The A. niger clxA gene encodes a 562-residue protein with strong homology to the calnexin of Schizosaccharomyces pombe. The clxAgene product complements a S. pombe cnx1 mutant. Motifs associated with genes controlled via the Unfolded Protein Response (UPR) were identified by sequence homology in the promoter of clxA. Steady-state levels of clxA mRNA were elevated in a strain expressing bovine prochymosin fused to the catalytic domain of glucoamylase. The ORF is punctuated by four introns, and contains two sets of four repeated peptide motifs that are characteristic of the calnexin family, together with a putative membrane-spanning domain. Deletion studies indicate that clxA is not an essential gene in A. niger.     

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.     

The molybdenum cofactor biosynthetic protein Cnx1 complements molybdate-repairable mutants, transfers molybdenum to the metal binding pterin, and is associated with the cytoskeleton

Molybdenum (Mo) plays an essential role in the active site of all eukaryotic Mo-containing enzymes. In plants, Mo enzymes are important for nitrate assimilation, phytohormone synthesis, and purine catabolism. Mo is bound to a unique metal binding pterin (molybdopterin [MPT]), thereby forming the active Mo cofactor (Moco), which is highly conserved in eukaryotes, eubacteria, and archaebacteria. Here, we describe the function of the two-domain protein Cnx1 from Arabidopsis in the final step of Moco biosynthesis. Cnx1 is constitutively expressed in all organs and in plants grown on different nitrogen sources. Mo-repairable cnxA mutants from Nicotiana plumbaginifolia accumulate MPT and show altered Cnx1 expression. Transformation of cnxA mutants and the corresponding Arabidopsis chl-6 mutant with cnx1 cDNA resulted in functional reconstitution of their Moco deficiency. We also identified a point mutation in the Cnx1 E domain of Arabidopsis chl-6 that causes the molybdate-repairable phenotype. Recombinant Cnx1 protein is capable of synthesizing Moco. The G domain binds and activates MPT, whereas the E domain is essential for activating Mo. In addition, Cnx1 binds to the cytoskeleton in the same way that its mammalian homolog gephyrin does in neuronal cells, which suggests a hypothetical model for anchoring the Moco-synthetic machinery by Cnx1 in plant cells.     

Molybdenum co-factor biosynthesis: the Arabidopsis thaliana cDNA cnx1 encodes a multifunctional two-domain protein homologous to a mammalian neuroprotein, the insect protein Cinnamon and three Escherichia coli proteins

The molybdenum co-factor (Moco) is an essential part of all eukaryotic molybdoenzymes. It is a molybdopterin and reveals the same principal structure in eubacteria, archaebacteria and eukaryotes. This paper reports the isolation of cnx1 , a cDNA clone of Arabidopsis thaliana which complements the Escherichia coli Moco mutant mogA . The mapping data of this cDNA correlate well with the mapping position of the A. thaliana molybdenum cofactor locus chl6 . As mutants in chl6 are known to be repairable by high concentrations of molybdate, the defective gene is very likely to be involved in the last step of Moco biosynthesis, that is, the insertion of molybdenum into molybdopterin. The protein encoded by cnx1 shows a two-domain structure: the N-terminal domain is homologous to the E. coli Moco protein MoeA, the C-terminal domain is homologous to the E. coli Moco proteins MoaB and MogA, respectively. These homologies show that part of the prokaryotic Moco biosynthetic pathway accomplished by monofunctional proteins in E. coli , is performed by a single multifunctional protein in eukaryotes. In addition Cnx1 is homologous to the eukaryotic proteins Gephyrin, a rat neuroprotein, and Cinnamon, a Drosophila protein with a function in Moco biosynthesis. These proteins also show a two-domain structure but the order of the domains is inversed as compared with Cnx1. Southern analysis indicates the existence of at least one further member, in addition to the cnx1 gene, of this novel gene family in the Arabidopsis genome.     

The two Caenorhabditis elegans UDP-glucose:glycoprotein glucosyltransferase homologues have distinct biological functions

The UDP-Glc:glycoprotein glucosyltransferase (UGGT) is the sensor of glycoprotein conformations in the glycoprotein folding quality control as it exclusively glucosylates glycoproteins not displaying their native conformations. Monoglucosylated glycoproteins thus formed may interact with the lectin-chaperones calnexin (CNX) and calreticulin (CRT). This interaction prevents premature exit of folding intermediates to the Golgi and enhances folding efficiency. Bioinformatic analysis showed that in C. elegans there are two open reading frames (F48E3.3 and F26H9.8 to be referred as uggt-1 and uggt-2, respectively) coding for UGGT homologues. Expression of both genes in Schizosaccharomyces pombe mutants devoid of UGGT activity showed that uggt-1 codes for an active UGGT protein (CeUGGT-1). On the other hand, uggt-2 coded for a protein (CeUGGT-2) apparently not displaying a canonical UGGT activity. This protein was essential for viability, although cnx/crt null worms were viable. We constructed transgenic worms carrying the uggt-1 promoter linked to the green fluorescent protein (GFP) coding sequence and found that CeUGGT-1 is expressed in cells of the nervous system. uggt-1 is upregulated under ER stress through the ire-1 arm of the unfolded protein response (UPR). Real-time PCR analysis showed that both uggt-1 and uggt-2 genes are expressed during the entire C. elegans life cycle. RNAi-mediated depletion of CeUGGT-1 but not of CeUGGT-2 resulted in a reduced lifespan and that of CeUGGT-1 and CeUGGT-2 in a developmental delay. We found that both CeUGGT1 and CeUGGT2 play a protective role under ER stress conditions, since 10 μg/ml tunicamycin arrested development at the L2/L3 stage of both uggt-1(RNAi) and uggt-2(RNAi) but not of control worms. Furthermore, we found that the role of CeUGGT-2 but not CeUGGT-1 is significant in relieving low ER stress levels in the absence of the ire-1 unfolding protein response signaling pathway. Our results indicate that both C. elegans UGGT homologues have distinct biological functions.     

Cell Cycle-dependent Degradation of the Saccharomyces cerevisiae Spindle Motor Cin8p Requires APCCdh1 and a Bipartite Destruction Sequence

Saccharomyces cerevisiae Cin8p belongs to the BimC family of kinesin-related motor proteins that are essential for spindle assembly. Cin8p levels were found to oscillate in the cell cycle due in part to a high rate of degradation imposed from the end of mitosis through the G1 phase. Cin8p degradation required the anaphase-promoting complex ubiquitin ligase and its late mitosis regulator Cdh1p but not the early mitosis regulator Cdc20p. Cin8p lacks a functional destruction box sequence that is found in the majority of anaphase-promoting complex substrates. We carried out an extensive mutagenesis study to define the cis-acting sequence required for Cin8p degradation in vivo. The C terminus of Cin8p contains two elements required for its degradation: 1) a bipartite destruction sequence composed of a KEN-box plus essential residues within the downstream 22 amino acids and 2) a nuclear localization signal. The bipartite destruction sequence appears in other BimC kinesins as well. Expression of nondegradable Cin8p showed very mild phenotypic effects, with an increase in the fraction of mitotic cells with broken spindles.     

Calnexin Improves the Folding Efficiency of Mutant Rhodopsin in the Presence of Pharmacological Chaperone 11-cis-Retinal

The lectin chaperone calnexin (Cnx) is important for quality control of glycoproteins, and the chances of correct folding of a protein increase the longer the protein interacts with Cnx. Mutations in glycoproteins increase their association with Cnx, and these mutant proteins are retained in the endoplasmic reticulum. However, until now, the increased interaction with Cnx was not known to increase the folding of mutant glycoproteins. Because many human diseases result from glycoprotein misfolding, a Cnx-assisted folding of mutant glycoproteins could be beneficial. Mutations of rhodopsin, the glycoprotein pigment of rod photoreceptors, cause misfolding resulting in retinitis pigmentosa. Despite the critical role of Cnx in glycoprotein folding, surprisingly little is known about its interaction with rhodopsin or whether this interaction could be modulated to increase the folding of mutant rhodopsin. Here, we demonstrate that Cnx preferentially associates with misfolded mutant opsins associated with retinitis pigmentosa. Furthermore, the overexpression of Cnx leads to an increased accumulation of misfolded P23H opsin but not the correctly folded protein. Finally, we demonstrate that increased levels of Cnx in the presence of the pharmacological chaperone 11-cis-retinal increase the folding efficiency and result in an increase in correct folding of mutant rhodopsin. These results demonstrate that misfolded rather than correctly folded rhodopsin is a substrate for Cnx and that the interaction between Cnx and mutant, misfolded rhodopsin, can be targeted to increase the yield of folded mutant protein.     

Function of tubulin binding proteins in vivo

Overexpression of the beta-tubulin binding protein Rbl2p/cofactor A is lethal in yeast cells expressing a mutant alpha-tubulin, tub1-724, that produces unstable heterodimer. Here we use RBL2 overexpression to identify mutations in other genes that affect formation or stability of heterodimer. This approach identifies four genes-CIN1, CIN2, CIN4, and PAC2-as affecting heterodimer formation in vivo. The vertebrate homologues of two of these gene products-Cin1p/cofactor D and Pac2p/cofactor E-can catalyze exchange of tubulin polypeptides into preexisting heterodimer in vitro. Previous work suggests that both Cin2p or Cin4p act in concert with Cin1p in yeast, but no role for vertebrate homologues of either has been reported in the in vitro reaction. Results presented here demonstrate that these proteins can promote heterodimer formation in vivo. RBL2 overexpression in cin1 and pac2 mutant cells causes microtubule disassembly and enhanced formation of Rbl2p-beta-tubulin complex, as it does in the alpha-tubulin mutant that produces weakened heterodimer. Significantly, excess Cin1p/cofactor D suppresses the conditional phenotypes of that mutant alpha-tubulin. Although none of the four genes is essential for viability under normal conditions, they become essential under conditions where the levels of dissociated tubulin polypeptides increase. Therefore, these proteins may provide a salvage pathway for dissociated tubulin heterodimers and so rescue cells from the deleterious effects of free beta-tubulin.     

Mitotic spindle function in Saccharomyces cerevisiae requires a balance between different types of kinesin-related motors.

Two Saccharomyces cerevisiae kinesin-related motors, Cin8p and Kip1p, perform an essential role in the separation of spindle poles during spindle assembly and a major role in spindle elongation. Cin8p and Kip1p are also required to prevent an inward spindle collapse prior to anaphase. A third kinesin-related motor, Kar3p, may act antagonistically to Cin8p and Kip1p since loss of Kar3p partially suppresses the spindle collapse in cin8 kip1 mutants. We have tested the relationship between Cin8p and Kar3p by overexpressing both motors using the inducible GAL1 promoter. Overexpression of KAR3 results in a shrinkage of spindle size and a temperature-dependent inhibition of the growth of wild-type cells. Excess Kar3p has a stronger inhibitory effect on the growth of cin8 kip1 mutants and can completely block anaphase spindle elongation in these cells. In contrast, overexpression of CIN8 leads to premature spindle elongation in all cells tested. This is the first direct demonstration of antagonistic motors acting on the intact spindle and suggests that spindle length is determined by the relative activity of Kar3p-like and Cin8p/Kip1p-like motors.     

Calnexin overexpression sensitizes recombinant CHO cells to apoptosis induced by sodium butyrate treatment

Sodium butyrate (NaBu) can enhance the expression of foreign genes in recombinant Chinese hamster ovary (rCHO) cells, but it can also inhibit cell growth and induce cellular apoptosis. In this study, the potential role of calnexin (Cnx) expression in rCHO cells treated with 5 mM NaBu was investigated for rCHO cells producing tumor necrosis factor receptor FC. To regulate the Cnx expression level, a tetracycline-inducible system was used. Clones with different Cnx expression levels were selected and investigated. With regard to productivity per cell (q_p), NaBu enhanced the q_p by over twofold. Under NaBu treatment, Cnx overexpression further enhanced the q_p by about 1.7-fold. However, under NaBu stress, the cells overexpressing Cnx showed a poorer viability profile with a consistent difference of over 25% in the viability when compared to the Cnx-repressed condition. This drop in the viability was attributed to increased apoptosis seen in these cells as evidenced by enhanced poly (ADP-ribose) polymerase cleavage and cytochrome C release. Ca²⁺ localization staining and subsequent confocal imaging revealed elevated cytosolic Ca²⁺ ([Ca²⁺]_c) in the Cnx-overexpressing cells when compared to the Cnx-repressed condition, thus endorsing the increased apoptosis observed in these cells. Taken together, Cnx overexpression not only improved the q_p of cells treated with NaBu, but it also sensitized cells to apoptosis.     

Homotetrameric form of Cin8p, a Saccharomyces cerevisiae kinesin-5 motor, is essential for its in vivo function

Kinesin-5 motor proteins are evolutionarily conserved and perform essential roles in mitotic spindle assembly and spindle elongation during anaphase. Previous studies demonstrated a specialized homotetrameric structure with two pairs of catalytic domains, one at each end of a dumbbell-shaped molecule. This suggests that they perform their spindle roles by cross-linking and sliding antiparallel spindle microtubules. However, the exact kinesin-5 sequence elements that are important for formation of the tetrameric complexes have not yet been identified. In addition, it has not been demonstrated that the homotetrameric form of these proteins is essential for their biological functions. Thus, we investigated a series of Saccharomyces cerevisiae Cin8p truncations and internal deletions, in order to identify structural elements in the Cin8p sequence that are required for Cin8p functionality, spindle localization, and multimerization. We found that all variants of Cin8p that are functional in vivo form tetrameric complexes. The first coiled-coil domain in the stalk of Cin8p, a feature that is shared by all kinesin-5 homologues, is required for its dimerization, and sequences in the last part of the stalk, specifically those likely involved in coiled-coil formation, are required for Cin8p tetramerization. We also found that dimeric forms of Cin8p that are nonfunctional in vivo can nonetheless bind to microtubules. These findings suggest that binding of microtubules is not sufficient for the functionality of Cin8p and that microtubule cross-linking by the tetrameric complex is essential for Cin8p mitotic functions.     

The maize Viviparous10/Viviparous13 locus encodes the Cnx1 gene required for molybdenum cofactor biosynthesis

Abscisic acid (ABA), auxin and nitrate are important signaling molecules that affect plant growth responses to the environment. The synthesis or metabolism of these compounds depends on the molybdenum cofactor (MoCo). We show that maize (Zea mays) viviparous10 (vp10) mutants have strong precocious germination and seedling lethal phenotypes that cannot be rescued with tissue culture. We devised a novel PCR-based method to clone a transposon-tagged allele of vp10, and show that Vp10 encodes the ortholog of Cnx1, which catalyzes the final common step of MoCo synthesis. The seedling phenotype of vp10 mutants is consistent with disruptions in ABA and auxin biosynthesis, as well as a disruption in nitrate metabolism. Levels of ABA and auxin are reduced in vp10 mutants, and vp10 seedlings lack MoCo-dependent enzyme activities that are repairable with exogenous molybdenum. vp10 and an Arabidopsis cnx1 mutant, chlorate6 (chl6), have similar defects in aldehyde oxidase (AO) enzyme activity, which is required for ABA synthesis. Surprisingly, chl6 mutants do not show defects in abiotic stress responses. These observations confirm an orthologous function for Cnx1 and Vp10, as well as defining a characteristic viviparous phenotype to identify other maize cnx mutants. Finally, the vp10 mutant phenotype suggests that cnx mutants can have auxin- as well as ABA-biosynthesis defects, while the chl6 mutant phenotype suggests that low levels of AO activity are sufficient for normal abiotic stress responses.     

Immunological Characterization of cdc2 and wee1 Proteins during the Growth and Differentiation of Dictyostelium discoideum

Two different antibody preparations, raised independently against the conserved EGVPSTAIREISLLKE sequence of the protein kinases encoded by the Schizosaccharomyces pombe cdc2 gene and its species homologs, immunoblotted a Dictyostelium protein of 32 kDa (p32). This polypeptide bound to p13^suc1-agarose beads, suggesting that it represents the Dictyostelium cdc2 and / or cdk2 products. The amounts of p32 detectable in cell free extracts and bound to p13^suc1-agarose were unaltered during the growth of cells synchronized for division. Although there was also essentially no change in the level of p32 during differentiation, the protein from the pseudoplasmodial stage of development did not bind to p13^suc1-agarose, implicating a developmentally regulated modification of the kinase. One of the EGVPSTAIREISLLKE antibodies also recognized a protein of 49 kDa (p49) that increased in amount dramatically during aggregation and then remained at elevated levels throughout the remainder of the differentiation process. This protein was present in low amounts throughout the growth of cells synchronized for division and was not absorbed by p13^suc1-agarose.
A 103 kDa protein (p103) was detected by Western blot analysis using antibodies raised against two different peptides corresponding to sequences in the S. pombe protein kinase p105^wee1, which is a putative upstream negative regulator of p34^cdc2 in fission yeast. The levels of p103 were constant during differentiation and during the growth of cells synchronized for division.     

Saccharomyces cerevisiae genes required in the absence of the CIN8-encoded spindle motor act in functionally diverse mitotic pathways.

Kinesin-related Cin8p is the most important spindle-pole-separating motor in Saccharomyces cerevisiae but is not essential for cell viability. We identified 20 genes whose products are specifically required by cell deficient for Cin8p. All are associated with mitotic roles and represent at least four different functional pathways. These include genes whose products act in two spindle motor pathways that overlap in function with Cin8p, the kinesin-related Kip1p pathway and the cytoplasmic dynein pathway. In addition, genes required for mitotic spindle checkpoint function and for normal microtubule stability were recovered. Mutant alleles of eight genes caused phenotypes similar to dyn1 (encodes the dynein heavy chain), including a spindle-positioning defect. We provide evidence that the products of these genes function in concept with dynein. Among the dynein pathway gene products, we found homologues of the cytoplasmic dynein intermediate chain, the p150Glued subunit of the dynactin complex, and human LIS-1, required for normal brain development. These findings illustrate the complex cellular interactions exhibited by Cin8p, a member of a conserved spindle motor family.     

Cinnamaldehyde-induced apoptosis in human PLC/PRF/5 cells through activation of the proapoptotic Bcl-2 family proteins and MAPK pathway

Cinnamaldehyde (Cin) has been shown to be effective in inducing apoptotic cell death in a number of human cancer cells. However, the intracellular death signaling mechanisms by which Cin inhibits tumor cell growth are poorly understood. In this study, we investigated the effect of mitogen-activated protein kinases (MAPKs) inhibitors [namely SP600125 (a specific JNK inhibitor), SB203580 (a specific p38 inhibitor) and PD98059 (a specific ERK inhibitor)] on the stress-responsive MAPK pathway induced by Cin in PLC/PRF/5 cells. Trypan blue staining assay indicated that Cin was cytotoxic to PLC/PRF/5 cells. Cin caused cell cycle perturbation (S-phase arrest) and triggered apoptosis as revealed by the externalization of annexin V-targeted phosphatidylserine and accumulation of sub-G1 peak. It down-regulated the Bcl-2 and Mcl-1 expression, and up-regulated Bax protein in a time-response manner. Treatment with 1 microM Cin resulted in an activation of caspase-8 and cleavage of Bid to its truncated form in a time-dependent pattern. JNK, ERK and p38 kinases in cells were activated and phosphorylated after Cin treatment. Pre-incubation with SP600125 and SB203580 markedly suppressed the effect of Cin-induced apoptosis, but not PD98059. Both SP600125 and SB203580 significantly prevented the phosphorylation of JNK and p38 proteins, but not ERK. These results conclude that Cin triggers apoptosis in PLC/PRF/5 cells could be through the activation of pro-apoptotic Bcl-2 family (Bax and Bid) proteins and MAPK signaling pathway.     

Calnexin Regulates Apoptosis Induced by Inositol Starvation in Fission Yeast

Inositol is a precursor of numerous phospholipids and signalling molecules essential for the cell. Schizosaccharomyces pombe is naturally auxotroph for inositol as its genome does not have a homologue of the INO1 gene encoding inositol-1-phosphate synthase, the enzyme responsible for inositol biosynthesis. In this work, we demonstrate that inositol starvation in S. pombe causes cell death with apoptotic features. This apoptotic death is dependent on the metacaspase Pca1p and is affected by the UPR transducer Ire1p. Previously, we demonstrated that calnexin is involved in apoptosis induced by ER stress. Here, we show that cells expressing a lumenal version of calnexin exhibit a 2-fold increase in the levels of apoptosis provoked by inositol starvation. This increase is reversed by co-expression of a calnexin mutant spanning the transmembrane domain and C-terminal cytosolic tail. Coherently, calnexin is physiologically cleaved at the end of its lumenal domain, under normal growth conditions when cells approach stationary phase. This cleavage suggests that the two naturally produced calnexin fragments are needed to continue growth into stationary phase and to prevent cell death. Collectively, our observations indicate that calnexin takes part in at least two apoptotic pathways in S. pombe, and suggest that the cleavage of calnexin has regulatory roles in apoptotic processes involving calnexin.