Yeast Unfolded Protein Response Research Articles

Fundamental and Applicative Aspects of the Unfolded Protein Response in Yeasts.

Upon the dysfunction or functional shortage of the endoplasmic reticulum (ER), namely, ER stress, eukaryotic cells commonly provoke a protective gene expression program called the unfolded protein response (UPR). The molecular mechanism of UPR has been uncovered through frontier genetic studies using Saccharomyces cerevisiae as a model organism. Ire1 is an ER-located transmembrane protein that directly senses ER stress and is activated as an RNase. During ER stress, Ire1 promotes the splicing of HAC1 mRNA, which is then translated into a transcription factor that induces the expression of various genes, including those encoding ER-located molecular chaperones and protein modification enzymes. While this mainstream intracellular UPR signaling pathway was elucidated in the 1990s, new intriguing insights have been gained up to now. For instance, various additional factors allow UPR evocation strictly in response to ER stress. The UPR machineries in other yeasts and fungi, including pathogenic species, are another important research topic. Moreover, industrially beneficial yeast strains carrying an enforced and enlarged ER have been produced through the artificial and constitutive induction of the UPR. In this article, we review canonical and up-to-date insights concerning the yeast UPR, mainly from the viewpoint of the functions and regulation of Ire1 and HAC1.

Optogenetic closed-loop feedback control of the unfolded protein response optimizes protein production

In biotechnological protein production processes, the onset of protein unfolding at high gene expression levels leads to diminishing production yields and reduced efficiency. Here we show that in silico closed-loop optogenetic feedback control of the unfolded protein response (UPR) in S. cerevisiae clamps gene expression rates at intermediate near-optimal values, leading to significantly improved product titers. Specifically, in a fully-automated custom-built 1L-photobioreactor, we used a cybergenetic control system to steer the level of UPR in yeast to a desired set-point by optogenetically modulating the expression of α-amylase, a hard-to-fold protein, based on real-time feedback measurements of the UPR, resulting in 60% higher product titers. This proof-of-concept study paves the way for advanced optimal biotechnology production strategies that diverge from and complement current strategies employing constitutive overexpression or genetically hardwired circuits.

Detecting the Non-conventional mRNA Splicing and Translational Activation of HAC1 in Budding Yeast.

Protein-folding homeostasis in the endoplasmic reticulum (ER) is maintained by the unfolded protein response (UPR). UPR in Saccharomyces cerevisiae is regulated by a bZIP transcription factor, Hac1p. Under non-stress condition, HAC1 mRNA is translationally repressed. When un- or mis-folded proteins accumulate in the ER, HAC1 mRNA undergoes non-conventional mRNA splicing. The spliced HAC1 mRNA is translationally active and produces functional Hac1p, which initiates a transcriptional response that restores ER protein-folding homeostasis. Thus, the activation of yeast UPR is tightly regulated by HAC1 mRNA splicing. Here, we describe two methods that are used to monitor the splicing and translational status of HAC1 mRNA in budding yeast.

Phosphorylation of Pal2 by the protein kinases Kin1 and Kin2 modulates HAC1 mRNA splicing in the unfolded protein response in yeast.

During cellular stress in the budding yeast Saccharomyces cerevisiae, an endoplasmic reticulum (ER)-resident dual kinase and RNase Ire1 splices an intron from HAC1 mRNA in the cytosol, thereby releasing its translational block. Hac1 protein then activates an adaptive cellular stress response called the unfolded protein response (UPR) that maintains ER homeostasis. The polarity-inducing protein kinases Kin1 and Kin2 contribute to HAC1 mRNA processing. Here, we showed that an RNA-protein complex that included the endocytic proteins Pal1 and Pal2 mediated HAC1 mRNA splicing downstream of Kin1 and Kin2. We found that Pal1 and Pal2 bound to the 3' untranslated region (3'UTR) of HAC1 mRNA, and a yeast strain lacking both Pal1 and Pal2 was deficient in HAC1 mRNA processing. We also showed that Kin1 and Kin2 directly phosphorylated Pal2, and that a nonphosphorylatable Pal2 mutant could not rescue the UPR defect in a pal1Δ pal2Δ strain. Thus, our work uncovers a Kin1/2-Pal2 signaling pathway that coordinates HAC1 mRNA processing and ER homeostasis.

The yeast FIT2 homologs are necessary to maintain cellular proteostasis and membrane lipid homeostasis.

Lipid droplets (LDs) are implicated in conditions of lipid and protein dysregulation. The fat storage-inducing transmembrane (FIT; also known as FITM) family induces LD formation. Here, we establish a model system to study the role of the Saccharomyces cerevisiae FIT homologues (ScFIT), SCS3 and YFT2, in the proteostasis and stress response pathways. While LD biogenesis and basal endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR) remain unaltered in ScFIT mutants, SCS3 was found to be essential for proper stress-induced UPR activation and for viability in the absence of the sole yeast UPR transducer IRE1 Owing to not having a functional UPR, cells with mutated SCS3 exhibited an accumulation of triacylglycerol within the ER along with aberrant LD morphology, suggesting that there is a UPR-dependent compensatory mechanism that acts to mitigate lack of SCS3 Additionally, SCS3 was necessary to maintain phospholipid homeostasis. Strikingly, global protein ubiquitylation and the turnover of both ER and cytoplasmic misfolded proteins is impaired in ScFITΔ cells, while a screen for interacting partners of Scs3 identifies components of the proteostatic machinery as putative targets. Together, our data support a model where ScFITs play an important role in lipid metabolism and proteostasis beyond their defined roles in LD biogenesis.This article has an associated First Person interview with the first author of the paper.

Regulation of the unfolded protein response in yeast by oxidative stress.

In the unfolded protein response (UPR), Ire1 activates Hac1 to coordinate the transcription of hundreds of genes to mitigate ER stress. Recent work in Caenorhabditis elegans suggests that oxidative stress inhibits this canonical Ire1 signalling pathway, activating instead an antioxidant stress response. We sought to determine whether this novel mode of UPR function also existed in yeast, where Ire1 has been best characterized. We show that the yeast UPR is also subject to inhibition by oxidative stress. Inhibition is mediated by a single evolutionarily conserved cysteine, and affects both luminal and membrane pathways of Ire1 activation. In yeast, Ire1 appears dispensable for resistance to oxidative stress and, therefore, the physiological significance of this pathway remains to be demonstrated.

Multiple decay events target HAC1 mRNA during splicing to regulate the unfolded protein response.

In the unfolded protein response (UPR), stress in the endoplasmic reticulum (ER) activates a large transcriptional program to increase ER folding capacity. During the budding yeast UPR, Ire1 excises an intron from the HAC1 mRNA and the exon products of cleavage are ligated, and the translated protein induces hundreds of stress-response genes. Using cells with mutations in RNA repair and decay enzymes, we show that phosphorylation of two different HAC1 splicing intermediates is required for their degradation by the 5'→3' exonuclease Xrn1 to enact opposing effects on the UPR. We also found that ligated but 2'-phosphorylated HAC1 mRNA is cleaved, yielding a decay intermediate with both 5'- and 2'-phosphates at its 5'-end that inhibit 5'→3' decay and suggesting that Ire1 degrades incompletely processed HAC1. These decay events expand the scope of RNA-based regulation in the budding yeast UPR and have implications for the control of the metazoan UPR.

How to design an optimal sensor network for the unfolded protein response.

Cellular protein homeostasis requires continuous monitoring of stress in the endoplasmic reticulum (ER). Stress-detection networks control protein homeostasis by mitigating the deleterious effects of protein accumulation, such as aggregation and misfolding, with precise modulation of chaperone production. Here, we develop a coarse model of the unfolded protein response in yeast and use multi-objective optimization to determine which sensing and activation strategies optimally balance the trade-off between unfolded protein accumulation and chaperone production. By comparing a stress-sensing mechanism that responds directly to the level of unfolded protein in the ER to a mechanism that is negatively regulated by unbound chaperones, we show that chaperone-mediated sensors are more efficient than sensors that detect unfolded proteins directly. This results from the chaperone-mediated sensor having separate thresholds for activation and deactivation. Finally, we demonstrate that a sensor responsive to both unfolded protein and unbound chaperone does not further optimize homeostatic control. Our results suggest a strategy for designing stress sensors and may explain why BiP-mitigated ER stress-sensing networks have evolved.

Comparison and contrast of plant, yeast, and mammalian ER stress and UPR

The endoplasmic reticulum (ER) is a well-characterized protein folding mechanism in eukaryotic organisms. Many secretory and membrane proteins are folded in the ER before they are translocated to their functional destination. Various conditions, such as biotic, abiotic, or physiological stresses, lead to the accumulation of unfolded and misfolded proteins in the ER, resulting in ER stress. In response to ER stress, cells initiate a protective response called the unfolded protein response (UPR) to maintain cellular homeostasis. Previous studies suggest that inositol-requiring kinase 1 (IRE1) is a universal ER stress sensor in yeast, mammals, and plants. IRE1-mediated splicing of UPR transducers, such as HAC1, XBP1, and bZIP60, triggers the UPR in yeast, mammals, and plants, respectively. In mammals, activated transcription factor 6 and double stranded RNA-activated protein kinase-like ER kinases are involved in the UPR. In plants, the additional UPR transducers bZIP28 and bZIP17 are activated by Golgi-localized S1P and S2P proteases. Subsequently, these UPR transducers are exported to the nucleus and upregulate the expression of UPR-responsive genes encoding BiP, calreticulin, calnexin, protein disulfide isomerase, and glucose-regulated protein 94 to decrease the amount of misfolded proteins and induce endoplasmic reticulum-associated degradation. In plants, the UPR signaling pathway plays an important role in ER homeostasis and normal biological processes; however, the molecular mechanisms of the UPR in plants remain poorly understood. This paper provides an overview of the regulatory and signaling mechanisms of the UPR across kingdoms. In addition, the emerging role of the UPR in plant physiology and defense response will be discussed.

Unfolded protein response is required for Aspergillus oryzae growth under conditions inducing secretory hydrolytic enzyme production

Unfolded protein response (UPR) is an intracellular signaling pathway for adaptation to endoplasmic reticulum (ER) stress. In yeast UPR, Ire1 cleaves the unconventional intron of HAC1 mRNA, and the functional Hac1 protein translated from the spliced HAC1 mRNA induces the expression of ER chaperone genes and ER-associated degradation genes for the refolding or degradation of unfolded proteins. In this study, we constructed an ireA (IRE1 ortholog) conditionally expressing strain of Aspergillus oryzae, a filamentous fungus producing a large amount of amylolytic enzymes, and examined the contribution of UPR to ER stress adaptation under physiological conditions. Repression of ireA completely blocked A. oryzae growth under conditions inducing the production of hydrolytic enzymes, such as amylases and proteases. This growth defect was restored by the introduction of unconventional intronless hacA (hacA-i). Furthermore, UPR was observed to be induced by amylolytic gene expression, and the disruption of the transcriptional activator for amylolytic genes resulted in partial growth restoration of the ireA-repressing strain. In addition, a homokaryotic ireA disruption mutant was successfully generated using the strain harboring hacA-i as a parental host. These results indicated that UPR is required for A. oryzae growth to alleviate ER stress induced by excessive production of hydrolytic enzymes.

Ribosomal Protein L5 Plays a Moonlighting Role in HAC1 mRNA

The unfolded protein response (UPR) pathway is a conserved pathway from yeast to human, which maintains cellular protein homeostasis. An endoplasmic reticulum (ER)‐resident RNase Ire1 initiates the UPR in yeast Saccharomyces cerevisiae by cleaving out an inhibitory intron from the HAC1 mRNA, thus generating a new open reading frame that yields Hac1 protein. Hac1 is a transcription factor that activates the expression of the UPR genes. For effective cleavage and processing, HAC1 mRNA assembles around the Ire1 protein, which requires a 3'‐bipartite element (3'BE) located at the 3'‐untranslated leader. To understand the inner mechanisms of the 3'BE‐mediated HAC1‐mRNAIre1 protein assembly, we expressed in vivo a 3'BE‐RMB mini RNA consisting of 48 nucleotides of the BE mRNA and 43 nucleotides of an RNA aptamer mimicking biotin (RNA mimic of biotin, RMB). Uracils of the 3'BE‐RMB mini RNA were labeled with a thiol group while growing yeast cells in the presence of 4‐thiouridine (4sU). The 4sU‐labeled RNA and bound proteins were photo‐crosslinked and immobilized on streptavidin‐agarose. The 3'BE‐RMB mini RNA bound proteins were identified by SDS‐PAGE followed by LC/MALDI mass spectrophotometry. We identified two S1 RNA‐binding domain‐containing proteins: the ribosomal protein L5 (RpL5) and the a‐subunit of initiation factor eIF2 (eIF2a). An in vitro electrophoretic mobility shift assay showed that RpL5, but not eIF2a, specifically bound to the synthetic 3'BE RNA. Furthermore, we found that targeted mutations in the RPL5 gene reduced the normal yeast growth, but increased the ER stress resistance. These results suggest that, apart from ribosome biogenesis, RpL5 plays a moonlighting role in the UPR signaling while regulating HAC1 mRNA processing.

Kinases Kin1 and Kin2 pleiotropically modulate splicing and translation of HAC1 mRNA (746.2)

Most nascent proteins fold and mature in the endoplasmic reticulum (ER). From the ER, matured proteins are packed inside a membrane‐bound vesicle that buds from the ER, travels and fuses with their target destination, thereby delivering the cargo protein. Stresses that disrupt ER function cause an over‐accumulation of un‐folded proteins in the ER, a state referred to as ER stress. To adapt to ER stress, cells activate a network of signaling pathways collectively called the unfolded protein response (UPR). The UPR in yeast Saccharomyces cerevisiae is initiated by a dual kinase/RNase Ire1 that removes an inhibitory intron from HAC1 pre‐mRNA, resulting in expression of Hac1 protein, a transcriptional activator of several UPR genes involved in adapting to ER stress. We have discovered that protein kinases Kin1 and Kin2 are dosage suppressors of a HAC1 allele that is devoid of a consensus element in the 3’ un‐translated region of pre‐mRNA. We have further discovered that both splicing and translation of HAC1 mRNA are reduced in a strain lacking kin1 and kin2 kinases, suggesting that the Ire1‐Hac1 signaling pathway is pleotropically regulated by Kin kinases. These Kin kinases have been reported to regulate polarized exocytosis and vesicular transport of proteins in yeast cells. Thus, our results revealed a novel role of Kin kinases in the yeast UPR.

The yeast Rab GTPase Ypt1 modulates unfolded protein response dynamics by regulating the stability of HAC1 RNA.

The unfolded protein response (UPR) is a conserved mechanism that mitigates accumulation of unfolded proteins in the ER. The yeast UPR is subject to intricate post-transcriptional regulation, involving recruitment of the RNA encoding the Hac1 transcription factor to the ER and its unconventional splicing. To investigate the mechanisms underlying regulation of the UPR, we screened the yeast proteome for proteins that specifically interact with HAC1 RNA. Protein microarray experiments revealed that HAC1 interacts specifically with small ras GTPases of the Ypt family. We characterized the interaction of HAC1 RNA with one of these proteins, the yeast Rab1 homolog Ypt1. We found that Ypt1 protein specifically associated in vivo with unspliced HAC1 RNA. This association was disrupted by conditions that impaired protein folding in the ER and induced the UPR. Also, the Ypt1-HAC1 interaction depended on IRE1 and ADA5, the two genes critical for UPR activation. Decreasing expression of the Ypt1 protein resulted in a reduced rate of HAC1 RNA decay, leading to significantly increased levels of both unspliced and spliced HAC1 RNA, and delayed attenuation of the UPR, when ER stress was relieved. Our findings establish that Ypt1 contributes to regulation of UPR signaling dynamics by promoting the decay of HAC1 RNA, suggesting a potential regulatory mechanism for linking vesicle trafficking to the UPR and ER homeostasis.

RtcB, a Novel RNA Ligase, Can Catalyze tRNA Splicing and HAC1 mRNA Splicing in Vivo

RtcB enzymes are novel RNA ligases that join 2',3'-cyclic phosphate and 5'-OH ends. The phylogenetic distribution of RtcB points to its candidacy as a tRNA splicing/repair enzyme. Here we show that Escherichia coli RtcB is competent and sufficient for tRNA splicing in vivo by virtue of its ability to complement growth of yeast cells that lack the endogenous "healing/sealing-type" tRNA ligase Trl1. RtcB also protects yeast trl1Δ cells against a fungal ribotoxin that incises the anticodon loop of cellular tRNAs. Moreover, RtcB can replace Trl1 as the catalyst of HAC1 mRNA splicing during the unfolded protein response. Thus, RtcB is a bona fide RNA repair enzyme with broad physiological actions. Biochemical analysis of RtcB highlights the uniqueness of its active site and catalytic mechanism. Our findings draw attention to tRNA ligase as a promising drug target.

Attenuation of yeast UPR is essential for survival and is mediated by IRE1 kinase

The unfolded protein response (UPR) activates Ire1, an endoplasmic reticulum (ER) resident transmembrane kinase and ribonuclease (RNase), in response to ER stress. We used an in vivo assay, in which disappearance of the UPR-induced spliced HAC1 messenger ribonucleic acid (mRNA) correlates with the recovery of the ER protein-folding capacity, to investigate the attenuation of the UPR in yeast. We find that, once activated, spliced HAC1 mRNA is sustained in cells expressing Ire1 carrying phosphomimetic mutations within the kinase activation loop, suggesting that dephosphorylation of Ire1 is an important step in RNase deactivation. Additionally, spliced HAC1 mRNA is also sustained after UPR induction in cells expressing Ire1 with mutations in the conserved DFG kinase motif (D828A) or a conserved residue (F842) within the activation loop. The importance of proper Ire1 RNase attenuation is demonstrated by the inability of cells expressing Ire1-D828A to grow under ER stress. We propose that the activity of the Ire1 kinase domain plays a role in attenuating its RNase activity when ER function is recovered.

Misfolded proteins impose a dosage-dependent fitness cost and trigger a cytosolic unfolded protein response in yeast

Evolving lineages face a constant intracellular threat: most new coding sequence mutations destabilize the folding of the encoded protein. Misfolded proteins form insoluble aggregates and are hypothesized to be intrinsically cytotoxic. Here, we experimentally isolate a fitness cost caused by toxicity of misfolded proteins. We exclude other costs of protein misfolding, such as loss of functional protein or attenuation of growth-limiting protein synthesis resources, by comparing growth rates of budding yeast expressing folded or misfolded variants of a gratuitous protein, YFP, at equal levels. We quantify a fitness cost that increases with misfolded protein abundance, up to as much as a 3.2% growth rate reduction when misfolded YFP represents less than 0.1% of total cellular protein. Comparable experiments on variants of the yeast gene orotidine-5'-phosphate decarboxylase (URA3) produce similar results. Quantitative proteomic measurements reveal that, within the cell, misfolded YFP induces coordinated synthesis of interacting cytosolic chaperone proteins in the absence of a wider stress response, providing evidence for an evolved modular response to misfolded proteins in the cytosol. These results underscore the distinct and evolutionarily relevant molecular threat of protein misfolding, independent of protein function. Assuming that most misfolded proteins impose similar costs, yeast cells express almost all proteins at steady-state levels sufficient to expose their encoding genes to selection against misfolding, lending credibility to the recent suggestion that such selection imposes a global constraint on molecular evolution.

Dual functions of yeast tRNA ligase in the unfolded protein response: unconventional cytoplasmic splicing of HAC1 pre-mRNA is not sufficient to release translational attenuation.

The unfolded protein response (UPR) is an essential signal transduction to cope with protein-folding stress in the endoplasmic reticulum. In the yeast UPR, the unconventional splicing of HAC1 mRNA is a key step. Translation of HAC1 pre-mRNA (HAC1(u) mRNA) is attenuated on polysomes and restarted only after splicing upon the UPR. However, the precise mechanism of this restart remained unclear. Here we show that yeast tRNA ligase (Rlg1p/Trl1p) acting on HAC1 ligation has an unexpected role in HAC1 translation. An RLG1 homologue from Arabidopsis thaliana (AtRLG1) substitutes for yeast RLG1 in tRNA splicing but not in the UPR. Surprisingly, AtRlg1p ligates HAC1 exons, but the spliced mRNA (HAC1(i) mRNA) is not translated efficiently. In the AtRLG1 cells, the HAC1 intron is circularized after splicing and remains associated on polysomes, impairing relief of the translational repression of HAC1(i) mRNA. Furthermore, the HAC1 5' UTR itself enables yeast Rlg1p to regulate translation of the following ORF. RNA IP revealed that yeast Rlg1p is integrated in HAC1 mRNP, before Ire1p cleaves HAC1(u) mRNA. These results indicate that the splicing and the release of translational attenuation of HAC1 mRNA are separable steps and that Rlg1p has pivotal roles in both of these steps.

The cell wall and endoplasmic reticulum stress responses are coordinately regulated in Saccharomyces cerevisiae

The unfolded protein response (UPR) is an intracellular signaling pathway that regulates the cellular response to the accumulation of misfolded proteins in eukaryotes. Our group has demonstrated that cell wall stress activates UPR in yeast through signals transmitted by the cell wall integrity (CWI) mitogen-activated protein (MAP) kinase cascade. The UPR is required to maintain cell wall integrity; mutants lacking a functional UPR have defects in cell wall biosynthesis and are hypersensitive to cell wall-directed antifungal drugs. Since ER stress also activates CWI signaling, we propose that ER and cell wall stress responses are coordinated by CWI and UPR signaling pathways in order to protect cells against these related stressors. Further investigation of the mechanisms of this coordinate regulation may lead to improved cell wall-directed antifungal therapies.

The ER Chaperone LHS1 Is Involved in Asexual Development and Rice Infection by the Blast FungusMagnaporthe oryzae

In planta secretion of fungal pathogen proteins, including effectors destined for the plant cell cytoplasm, is critical for disease progression. However, little is known about the endoplasmic reticulum (ER) secretion mechanisms used by these pathogens. To determine if normal ER function is crucial for fungal pathogenicity, Magnaporthe oryzae genes encoding proteins homologous to yeast Lhs1p and Kar2p, members of the heat shock protein 70 family in Saccharomyces cerevisiae, were cloned and characterized. Like their yeast counterparts, both LHS1 and KAR2 proteins localized in the ER and functioned in an unfolded protein response (UPR) similar to the yeast UPR. Mutants produced by disruption of LHS1 were viable but showed a defect in the translocation of proteins across the ER membrane and reduced activities of extracellular enzymes. The Deltalhs1 mutant was severely impaired not only in conidiation, but also in both penetration and biotrophic invasion in susceptible rice (Oryza sativa) plants. This mutant also had defects in the induction of the Pi-ta resistance gene-mediated hypersensitive response and in the accumulation of fluorescently-labeled secreted effector proteins in biotrophic interfacial complexes. Our results suggest that proper processing of secreted proteins, including effectors, by chaperones in the ER is requisite for successful disease development and for determining host-pathogen compatibility via the gene-for-gene interaction.

Cellular response to unfolded proteins in the endoplasmic reticulum of plants

Secretory and transmembrane proteins are synthesized in the endoplasmic reticulum (ER) in eukaryotic cells. Nascent polypeptide chains, which are translated on the rough ER, are translocated to the ER lumen and folded into their native conformation. When protein folding is inhibited because of mutations or unbalanced ratios of subunits of hetero-oligomeric proteins, unfolded or misfolded proteins accumulate in the ER in an event called ER stress. As ER stress often disturbs normal cellular functions, signal-transduction pathways are activated in an attempt to maintain the homeostasis of the ER. These pathways are collectively referred to as the unfolded protein response (UPR). There have been great advances in our understanding of the molecular mechanisms underlying the UPR in yeast and mammals over the past two decades. In plants, a UPR analogous to those in yeast and mammals has been recognized and has recently attracted considerable attention. This review will summarize recent advances in the plant UPR and highlight the remaining questions that have yet to be addressed.