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

A screen for genes involved in respiration control and longevity in Schizosaccharomyces pombe

We present results showing that glucose signaling has proaging effects in the yeast Schizosaccharomyces pombe. Deletion of the receptor that senses extracellular glucose (Git3) increases the life span of S. pombe, while constitutive activation of the Gα subunit acting downstream of this receptor (Gpa2) shortens its life span. The latter mutant is also impaired for growth under respiration conditions. We have used this phenotype in a selection strategy to identify genes that when overexpressed can rescue the respiratory defect of constitutively active Gα subunit mutants. Here, we report an extended version of the work we presented at the IABG meeting and the results of this screen. This strategy allowed us to isolate four genes: psp1 + /moc1 + , cka1 + , adh1 + , and rpb10 + . Interestingly, the overexpression of these genes was also capable of increasing the chronological life span of wild-type yeast cells

The Schizosaccharomyces pombe Hsp104 Disaggregase Is Unable to Propagate the [PSI+] Prion

The molecular chaperone Hsp104 is a crucial factor in the acquisition of thermotolerance in yeast. Under stress conditions, the disaggregase activity of Hsp104 facilitates the reactivation of misfolded proteins. Hsp104 is also involved in the propagation of fungal prions. For instance, the well-characterized [PSI+] prion of Saccharomyces cerevisiae does not propagate in Δhsp104 cells or in cells overexpressing Hsp104. In this study, we characterized the functional homolog of Hsp104 from Schizosaccharomyces pombe (Sp_Hsp104). As its S. cerevisiae counterpart, Sp_hsp104+ is heat-inducible and required for thermotolerance in S. pombe. Sp_Hsp104 displays low disaggregase activity and cannot propagate the [PSI+] prion in S. cerevisiae. When overexpressed in S. cerevisiae, Sp_Hsp104 confers thermotolerance to Δhsp104 cells and reactivates heat-aggregated proteins. However, overexpression of Sp_Hsp104 does not propagate nor eliminate [PSI+]. Strikingly, [PSI+] was cured by overexpression of a chimeric chaperone bearing the C-terminal domain (CTD) of the S. cerevisiae Hsp104 protein. Our study demonstrates that the ability to untangle aggregated proteins is conserved between the S. pombe and S. cerevisiae Hsp104 homologs, and points to a role of the CTD in the propagation of the S. cerevisiae [PSI+] prion

Inter-Species Complementation of the Translocon Beta Subunit Requires Only Its Transmembrane Domain

In eukaryotes, proteins enter the secretory pathway through the translocon pore of the endoplasmic reticulum. This protein translocation channel is composed of three major subunits, called Sec61α, β and γ in mammals. Unlike the other subunits, the β subunit is dispensable for translocation and cell viability in all organisms studied. Intriguingly, the knockout of the Sec61β encoding genes results in different phenotypes in different species. Nevertheless, the β subunit shows a high level of sequence homology across species, suggesting the conservation of a biological function that remains ill-defined. To address its cellular roles, we characterized the homolog of Sec61β in the fission yeast Schizosaccharomyces pombe (Sbh1p). Here, we show that the knockout of sbh1+ results in severe cold sensitivity, increased sensitivity to cell-wall stress, and reduced protein secretion at 23°C. Sec61β homologs from Saccharomyces cerevisiae and human complement the knockout of sbh1+ in S. pombe. As in S. cerevisiae, the transmembrane domain (TMD) of S. pombe Sec61β is sufficient to complement the phenotypes resulting from the knockout of the entire encoding gene. Remarkably, the TMD of Sec61β from S. cerevisiae and human also complement the gene knockouts in both yeasts. Together, these observations indicate that the TMD of Sec61β exerts a cellular function that is conserved across species

Inter-Species Complementation of the Translocon Beta Subunit Requires Only Its Transmembrane Domain

In eukaryotes, proteins enter the secretory pathway through the translocon pore of the endoplasmic reticulum. This protein translocation channel is composed of three major subunits, called Sec61α, β and γ in mammals. Unlike the other subunits, the β subunit is dispensable for translocation and cell viability in all organisms studied. Intriguingly, the knockout of the Sec61β encoding genes results in different phenotypes in different species. Nevertheless, the β subunit shows a high level of sequence homology across species, suggesting the conservation of a biological function that remains ill-defined. To address its cellular roles, we characterized the homolog of Sec61β in the fission yeast Schizosaccharomyces pombe (Sbh1p). Here, we show that the knockout of sbh1+ results in severe cold sensitivity, increased sensitivity to cell-wall stress, and reduced protein secretion at 23°C. Sec61β homologs from Saccharomyces cerevisiae and human complement the knockout of sbh1+ in S. pombe. As in S. cerevisiae, the transmembrane domain (TMD) of S. pombe Sec61β is sufficient to complement the phenotypes resulting from the knockout of the entire encoding gene. Remarkably, the TMD of Sec61β from S. cerevisiae and human also complement the gene knockouts in both yeasts. Together, these observations indicate that the TMD of Sec61β exerts a cellular function that is conserved across species

The translocon β subunit is conserved from yeast to human.

<p>Amino-acid sequence comparison between translocon beta subunits of <i>S. pombe</i> (SP_Sec61β), <i>S. cerevisiae</i> (SC_Sec61β1 and SC_Sec61β2) and human (HS_Sec61β). Identical amino acids are shaded in black, similar amino acids are shaded in grey. The predicted conserved transmembrane domain is underlined.</p

Yeast strains used in this study.

<p>Yeast strains used in this study.</p

Plasmids used in this study.

<p>Plasmids used in this study.</p

The transmembrane domain (TMD) of different Sec61β homologs is sufficient to complement the knockout of the whole gene in fission and budding yeast.

<p>(A) Cold sensitivity of <i>S. pombe</i> Δ<i>sbh1</i> cells (SP15039) and (B) heat sensitivity of <i>S. cerevisiae sbh1</i>Δ<i>sbh2</i>Δ cells (SC3232) are rescued by the TMD of different Sec61β homologs. Δ<i>sbh1</i> and <i>sbh1</i>Δ<i>sbh2</i>Δ cells expressing the 26 amino acids of the TMD of Sec61β homologs from <i>S. pombe</i> (SP), <i>S. cerevisiae</i> (SC) and human (HS) were serial-diluted (10⁻¹–10⁻⁴) and spotted on MM+AL plates or SD-L plates. Growth was monitored during 5 days for <i>S. cerevisiae</i> and 7 days for <i>S. pombe</i> at the indicated temperatures. Sec61γ from <i>S. pombe</i> (SP) or <i>S. cerevisiae</i> (SC) were used as negative controls. Results are representative of three or more independent experiments. (C) Suppression of <i>S. pombe</i> Δ<i>sbh1</i> SDS sensitivity by the TMD of different Sec61β homologs. SDS-sensitivity halo was measured after 3–5 days of incubation at the indicated temperatures for cultures of the strains presented in (A). Data shown are mean±standard deviation of three or more independent experiments. *** indicates <i>p</i><0.001 for Student's t-test versus WT.</p

Effects of SP_Sec61β levels on protein secretion.

<p>(A) Cellulase secretion efficiency at different temperatures. WT (SP15073) and Δ<i>sbh1</i> (SP15074) cells expressing <i>A. aculeatus</i> cellulase from a genomic cassette under the control of the <i>adh1p</i> promoter, and bearing the empty vector or a plasmid overexpressing (↑) SP_Sec61β were analyzed for secretion. Cells were grown to exponential phase and spotted on EMM+ALH plates supplemented with 0.1% AZCL-HE-cellulose as substrate. The area of the blue halo created after cleavage of the chromogenic substrate by secreted cellulase was monitored for 7 days at 30°C or at 23°C. Time zero represents the moment when area of the halo exceeds the area under the colony. Each point is the mean±standard deviation of three independent cultures. (B) Rate of cellulase secretion at different temperatures. Secretion rates for strains presented in (A) were calculated as the mean slope of three independent cultures, using the WT value (left panel) or the complemented knockout (right panel) at the corresponding temperature as 100% (± standard deviation). WT cells overexpressing SP_Sec61γ and Δ<i>sbh1</i> cells bearing a plasmid encoding SP_Sec61β or its transmembrane domain only (TMD) are shown as controls. *** indicates <i>p</i><0.001 for Student's t-test.</p