Transcription Factor Hac1 Research Articles

Microbial production of 5-epi-jinkoheremol, a plant-derived antifungal sesquiterpene.

Synthetic biology using microbial chassis is emerging as a powerful tool for the production of natural chemicals. In the present study, we constructed a microbial platform for the high-level production of a sesquiterpene from Catharanthus roseus, 5-epi-jinkoheremol, which exhibits strong fungicidal activity. First, the mevalonate and sterol biosynthesis pathways were optimized in engineered yeast to increase the metabolic flux toward the biosynthesis of the precursor farnesyl pyrophosphate. Then, the transcription factor Hac1- and m6A writer Ime4-based metabolic engineering strategies were implemented in yeast to increase 5-epi-jinkoheremol production further. Next, protein engineering was performed to improve the catalytic activity and enhance the stability of the 5-epi-jinkoheremol synthase TPS18, resulting in the variant TPS18I21P/T414S, with the most improved properties. Finally, the titer of 5-epi-jinkoheremol was elevated to 875.25 mg/L in a carbon source-optimized medium in shake flask cultivation. To the best of our knowledge, this is the first study to construct an efficient microbial cell factory for the sustainable production of this antifungal sesquiterpene.IMPORTANCEBiofungicides represent a new and sustainable tool for the control of crop fungal diseases. However, hindered by the high cost of biofungicide production, their use is not as popular as expected. Synthetic biology using microbial chassis is emerging as a powerful tool for the production of natural chemicals. We previously identified a promising sesquiterpenoid biofungicide, 5-epi-jinkoheremol. Here, we constructed a microbial platform for the high-level production of this chemical. The metabolic engineering of the terpene biosynthetic pathway was firstly employed to increase the metabolic flux toward 5-epi-jinkoheremol production. However, the limited catalytic activity of the key enzyme, TPS18, restricted the further yield of 5-epi-jinkoheremol. By using protein engineering, we improved its catalytic efficiency, and combined with the optimization of regulation factors, the highest production of 5-epi-jinkoheremol was achieved. Our work was useful for the larger-scale efficient production of this antifungal sesquiterpene.

Tailored UPRE2 variants for dynamic gene regulation in yeast

Genetic elements are foundational in synthetic biology serving as vital building blocks. They enable programming host cells for efficient production of valuable chemicals and recombinant proteins. The unfolded protein response (UPR) is a stress pathway in which the transcription factor Hac1 interacts with the upstream unfolded protein response element (UPRE) of the promoter to restore endoplasmic reticulum (ER) homeostasis. Here, we created a UPRE2 mutant (UPRE2m) library. Several rounds of screening identified many elements with enhanced responsiveness and a wider dynamic range. The most active element m84 displayed a response activity 3.72 times higher than the native UPRE2. These potent elements are versatile and compatible with various promoters. Overexpression of HAC1 enhanced stress signal transduction, expanding the signal output range of UPRE2m. Through molecular modeling and site-directed mutagenesis, we pinpointed the DNA-binding residue Lys60 in Hac1(Hac1-K60). We also confirmed that UPRE2m exhibited a higher binding affinity to Hac1. This shed light on the mechanism underlying the Hac1-UPRE2m interaction. Importantly, applying UPRE2m for target gene regulation effectively increased both recombinant protein production and natural product synthesis. These genetic elements provide valuable tools for dynamically regulating gene expression in yeast cell factories.

Endoplasmic stress sensor Ire1 is involved in cytosolic/nuclear protein quality control in Pichia pastoris cells independent of HAC1.

In eukaryotic species, dysfunction of the endoplasmic reticulum (ER), namely, ER stress, provokes a cytoprotective transcription program called the unfolded protein response (UPR). The UPR is triggered by transmembrane ER-stress sensors, including Ire1, which acts as an endoribonuclease to splice and mature the mRNA encoding the transcription factor Hac1 in many fungal species. Through analyses of the methylotrophic yeast Pichia pastoris (syn. Komagataella phaffii), we revealed a previously unknown function of Ire1. In P. pastoris cells, the IRE1 knockout mutation (ire1Δ) and HAC1 knockout mutation (hac1Δ) caused only partially overlapping gene expression changes. Protein aggregation and the heat shock response (HSR) were induced in ire1Δ cells but not in hac1Δ cells even under non-stress conditions. Moreover, Ire1 was further activated upon high-temperature culturing and conferred heat stress resistance to P. pastoris cells. Our findings cumulatively demonstrate an intriguing case in which the UPR machinery controls cytosolic protein folding status and the HSR, which is known to be activated upon the accumulation of unfolded proteins in the cytosol and/or nuclei.

Molecular background of HAC1-driven improvement in the secretion of recombinant protein in Yarrowia lipolytica based on comparative transcriptomics

While the unfolded protein response (UPR) and its major regulator – transcription factor Hac1 are well-conserved across Eukarya, species-specific variations are repeatedly reported. Here we investigated molecular mechanisms by which co-over-expression of HAC1 improves secretion of a recombinant protein (r-Prot) in Yarrowia lipolytica, using comparative transcriptomics. Co-over-expression of HAC1 caused an >2-fold increase in secreted r-Prot, but its intracellular levels were decreased. The unconventional splicing rate of the HAC1 mRNA was counted through transcript sequencing. Multiple biological processes were affected in the HAC1-and-r-Prot co-over-expressing strain, including ribosome biogenesis, nuclear and mitochondrial events, cell cycle arrest, attenuation of gene expression by RNA polymerase III and II, as well as modulation of proteolysis and RNA metabolism; but whether the HAC1 co-over-expression/induction was the actual causative agent for these changes, was not always clear. We settled that the expression of the “conventional” HAC1 targets (KAR2 and PDI1) is not affected by its over-expression.

Functional genomic analysis of Candida albicans protein kinases reveals modulators of morphogenesis in diverse environments

Candida albicans is a leading cause of mycotic infection. The ability to transition between yeast and filamentous forms is critical to C.albicans virulence and complex signaling pathways regulate this process. Here, we screened a C.albicans protein kinase mutant library in six environmental conditions to identify regulators of morphogenesis. We identified the uncharacterized gene orf19.3751 as a negative regulator of filamentation and follow-up investigations implicated a role for orf19.3751 in cell cycle regulation. We also uncovered a dual role for the kinases Ire1 and protein kinase A (Tpk1 and Tpk2) in C.albicans morphogenesis, specifically as negative regulators of wrinkly colony formation on solid medium but positive regulators of filamentation in liquid medium. Further analyses suggested Ire1 modulates morphogenesis in both media states in part through the transcription factor Hac1 and in part through independent mechanisms. Overall, this work provides insights into the signaling governing morphogenesis in C.albicans.

Multifarious Translational Regulation during Replicative Aging in Yeast.

Protein synthesis is strictly regulated during replicative aging in yeast, but global translational regulation during replicative aging is poorly characterized. To conduct ribosome profiling during replicative aging, we collected a large number of dividing aged cells using a miniature chemostat aging device. Translational efficiency, defined as the number of ribosome footprints normalized to transcript abundance, was compared between young and aged cells for each gene. We identified more than 700 genes with changes greater than twofold during replicative aging. Increased translational efficiency was observed in genes involved in DNA repair and chromosome organization. Decreased translational efficiency was observed in genes encoding ribosome components, transposon Ty1 and Ty2 genes, transcription factor HAC1 gene associated with the unfolded protein response, genes involved in cell wall synthesis and assembly, and ammonium permease genes. Our results provide a global view of translational regulation during replicative aging, in which the pathways involved in various cell functions are translationally regulated and cause diverse phenotypic changes.

Intragenic complementation in the protein kinase domain in Ire1

In eukaryotic cells, secretory and transmembrane proteins fold in the endoplasmic reticulum (ER) before they exit the ER. Accumulation of unfolded proteins activatesIre1 to promote the unfolded protein response (UPR). In the yeast Saccharomyces cerevisiae, activation of Ire1 leads tosplicing of the mRNA for the transcription factor Hac1. Wehave usedb-galactosidase reporter assaysto demonstrateexpression of a UPRE-lacZreporter genethat is positively regulated by the Ire1–Hac1 signalling pathway. In this study, the protein kinase domain was subjected to mutations to alter the catalytic aspartate D797 and lysine K799, which interacts with the terminal phosphate group of ATP, to alanine. Also, point mutations in the Mg+2coordinating loop converted asparagine N802 and aspartic acid D828 to alanine. The expression of theUPRE-lacZreporter gene in all single mutants K799, N802 and D797 was decreased compared to WT-Ire1. Under conditions of various ER stress inducing drugs the ability of the D797A mutant strain to survive was reduced in growth assays compared to other mutants in theMg+2 coordinating loop and catalytic domain. D797-Ire1 mutant has less inb-galactosidase activity, survival of ER stress and HAC1 splicing. Expression of Ire1 carrying double mutations D797A-N802A increasedb-galactosidase activity significantly and restored growth compared to the single mutant D797A-Ire1. WT and protein kinase mutants express to the same level. This study suggeststhat introducing a second mutation such as K799A-Ire1 or N802A-Ire1 to the single mutant D797A-Ire1, restores signaling activity of Ire1.

The protein kinase Ire1 has a Hac1-independent essential role in iron uptake and virulence of Candida albicans.

Protein kinases play central roles in virtually all signaling pathways that enable organisms to adapt to their environment. Microbial pathogens must cope with severely restricted iron availability in mammalian hosts to invade and establish themselves within infected tissues. To uncover protein kinase signaling pathways that are involved in the adaptation of the pathogenic yeast Candida albicans to iron limitation, we generated a comprehensive protein kinase deletion mutant library of a wild-type strain. Screening of this library revealed that the protein kinase Ire1, which has a conserved role in the response of eukaryotic cells to endoplasmic reticulum stress, is essential for growth of C. albicans under iron-limiting conditions. Ire1 was not necessary for the activity of the transcription factor Sef1, which regulates the response of the fungus to iron limitation, and Sef1 target genes that are induced by iron depletion were normally upregulated in ire1Δ mutants. Instead, Ire1 was required for proper localization of the high-affinity iron permease Ftr1 to the cell membrane. Intriguingly, iron limitation did not cause increased endoplasmic reticulum stress, and the transcription factor Hac1, which is activated by Ire1-mediated removal of the non-canonical intron in the HAC1 mRNA, was dispensable for Ftr1 localization to the cell membrane and growth under iron-limiting conditions. Nevertheless, expression of a pre-spliced HAC1 copy in ire1Δ mutants restored Ftr1 localization and rescued the growth defects of the mutants. Both ire1Δ and hac1Δ mutants were avirulent in a mouse model of systemic candidiasis, indicating that an appropriate response to endoplasmic reticulum stress is important for the virulence of C. albicans. However, the specific requirement of Ire1 for the functionality of the high-affinity iron permease Ftr1, a well-established virulence factor, even in the absence of endoplasmic reticulum stress uncovers a novel Hac1-independent essential role of Ire1 in iron acquisition and virulence of C. albicans.

The acyltransferase, Gpc1, is regulated by the unfolded protein response in Saccharomyces cerevisiae

The unfolded protein response (UPR) is a proteostatic process is in which unfolded and misfolded proteins are degraded or modified. The UPR is initiated by abnormal proteins binding to the membrane protein Ire1, which dimerizes to become active and then splices the transcription factor Hac1. Spliced Hac1 produces a transcriptional response in downstream genes related to protein folding, secretion, and degradation. In addition, studies show that the UPR can be activated by endoplasmic reticulum (ER) membrane stress and can impact the expression of genes involved in lipid metabolism. Membrane abberancies that can activate the UPR include altered lipid composition and membrane fluidity. We have identified a glycerophosphocholine acyltransferase, Gpc1, that contributes to phosphatidylcholine (PC) synthesis in Saccharomyces cerevisiae and impacts PC species saturation. Gpc1 participates in the PC Deacylation-Reacylation Pathway (PC-DRP). In PC-DRP, PC is first completely deacylated by type B phospholipases, yielding glycerophosphocholine, which is then reacylated by Gpc1 to form lysophosphotidylcholine. Finally, a second acylation by Ale1 generates a PC molecule. Notably, our data demonstrate that Gpc1 produces more saturated PC species, potentially impacting membrane fluidity. Here, we report that GPC1 expressionis upregulated by UPR inducers such as tunicamycin and that loss of Gpc1 leads to increased sensitivity to tunicamycin. Additionally, loss of GPC1 is shown to activate the UPR based on expression of KAR2, a known UPR target. Ongoing studies include an examination of other constituents of PC-DRP are their relationship to the UPR.

Unfolded Protein Response and Scaffold Independent Pheromone MAP Kinase Signaling Control Verticillium dahliae Growth, Development, and Plant Pathogenesis.

Differentiation, growth, and virulence of the vascular plant pathogen Verticillium dahliae depend on a network of interconnected cellular signaling cascades. The transcription factor Hac1 of the endoplasmic reticulum-associated unfolded protein response (UPR) is required for initial root colonization, fungal growth, and vascular propagation by conidiation. Hac1 is essential for the formation of microsclerotia as long-time survival resting structures in the field. Single endoplasmic reticulum-associated enzymes for linoleic acid production as precursors for oxylipin signal molecules support fungal growth but not pathogenicity. Microsclerotia development, growth, and virulence further require the pheromone response mitogen-activated protein kinase (MAPK) pathway, but without the Ham5 scaffold function. The MAPK phosphatase Rok1 limits resting structure development of V. dahliae, but promotes growth, conidiation, and virulence. The interplay between UPR and MAPK signaling cascades includes several potential targets for fungal growth control for supporting disease management of the vascular pathogen V. dahliae.

Overexpression of the transcription factor HAC1 improves nerolidol production in engineered yeast

Increasing the metabolic flux of the mevalonate pathway, reducing the metabolic flux of competing pathway and utilizing the diauxie-inducible system constructed by GAL promoters are strategies commonly used in yeast metabolic engineering for the production of terpenoids. Using these strategies, we constructed a series of yeast strains with a strengthened mevalonate pathway and finally produced 336.5 mg/L nerolidol in a shake flask. The spliced HAC1 mRNA assay indicated that the unfolded protein response (UPR) occurred in the strains that we constructed. UPR strains exhibited the low transcriptional activities of GAL1 promoter. HAC1-overexpressing strain exhibited dramatically enhanced transcriptional activity of GAL1 promoter at 72 h of fermentation in flasks. HAC1 overexpression also increased the nerolidol titer by 47.7 %, reaching 497.0 mg/L and increased cell vitality. RNA-seq showed that the genes whose transcription responded to HAC1-overexpression were involved in the regulation of monocarboxylic acid metabolic processes and cellular amino acid biosynthetic process, indicating that the metabolic regulation may be part of the reason of the improved nerolidol synthesis. Our findings enrich the knowledge of the relationship between the construction of sesquiterpene-producing cell factories and UPR regulation. This study provides an effective strategy for sesquiterpene production in yeast.

Bypass of Activation Loop Phosphorylation by Aspartate 836 in Activation of the Endoribonuclease Activity of Ire1

ABSTRACTThe bifunctional protein kinase-endoribonuclease Ire1 initiates splicing of the mRNA for the transcription factor Hac1 when unfolded proteins accumulate in the endoplasmic reticulum. Activation of Saccharomyces cerevisiae Ire1 coincides with autophosphorylation of its activation loop at S840, S841, T844, and S850. Mass spectrometric analysis of Ire1 expressed in Escherichia coli identified S837 as another potential phosphorylation site in vivo. Mutation of all five potential phosphorylation sites in the activation loop decreased, but did not completely abolish, splicing of HAC1 mRNA, induction of KAR2 and PDI1 mRNAs, and expression of a β-galactosidase reporter activated by Hac1i. Phosphorylation site mutants survive low levels of endoplasmic reticulum stress better than IRE1 deletions strains. In vivo clustering and inactivation of Ire1 are not affected by phosphorylation site mutants. Mutation of D836 to alanine in the activation loop of phosphorylation site mutants nearly completely abolished HAC1 splicing, induction of KAR2, PDI1, and β-galactosidase reporters, and survival of ER stress, but it had no effect on clustering of Ire1. By itself, the D836A mutation does not confer a phenotype. These data argue that D836 can partially substitute for activation loop phosphorylation in activation of the endoribonuclease domain of Ire1.

The bZIP Transcription Factor HAC-1 Is Involved in the Unfolded Protein Response and Is Necessary for Growth on Cellulose in Neurospora crassa.

High protein secretion capacity in filamentous fungi requires an extremely efficient system for protein synthesis, folding and transport. When the folding capacity of the endoplasmic reticulum (ER) is exceeded, a pathway known as the unfolded protein response (UPR) is triggered, allowing cells to mitigate and cope with this stress. In yeast, this pathway relies on the transcription factor Hac1, which mediates the up-regulation of several genes required under these stressful conditions. In this work, we identified and characterized the ortholog of the yeast HAC1 gene in the filamentous fungus Neurospora crassa. We show that its mRNA undergoes an ER stress-dependent splicing reaction, which in N. crassa removes a 23 nt intron and leads to a change in the open reading frame. By disrupting the N. crassa hac-1 gene, we determined it to be crucial for activating UPR and for proper growth in the presence of ER stress-inducing chemical agents. Neurospora is naturally found growing on dead plant material, composed primarily by lignocellulose, and is a model organism for the study of plant cell wall deconstruction. Notably, we found that growth on cellulose, a substrate that requires secretion of numerous enzymes, imposes major demands on ER function and is dramatically impaired in the absence of hac-1, thus broadening the range of physiological functions of the UPR in filamentous fungi. Growth on hemicellulose however, another carbon source that necessitates the secretion of various enzymes for its deconstruction, is not impaired in the mutant nor is the amount of proteins secreted on this substrate, suggesting that secretion, as a whole, is unaltered in the absence of hac-1. The characterization of this signaling pathway in N. crassa will help in the study of plant cell wall deconstruction by fungi and its manipulation may result in important industrial biotechnological applications.

The Monoterpene Carvacrol Generates Endoplasmic Reticulum Stress in the Pathogenic Fungus Candida albicans

The monoterpene carvacrol, the major component of oregano and thyme oils, is known to exert potent antifungal activity against the pathogenic yeast Candida albicans. This monoterpene has been the subject of a considerable number of investigations that uncovered extensive pharmacological properties, including antifungal and antibacterial effects. However, its mechanism of action remains elusive. Here, we used integrative chemogenomic approaches, including genome-scale chemical-genetic and transcriptional profiling, to uncover the mechanism of action of carvacrol associated with its antifungal property. Our results clearly demonstrated that fungal cells require the unfolded protein response (UPR) signaling pathway to resist carvacrol. The mutants most sensitive to carvacrol in our genome-wide competitive fitness assay in the yeast Saccharomyces cerevisiae expressed mutations of the transcription factor Hac1 and the endonuclease Ire1, which is required for Hac1 activation by removing a nonconventional intron from the 3' region of HAC1 mRNA. Confocal fluorescence live-cell imaging revealed that carvacrol affects the morphology and the integrity of the endoplasmic reticulum (ER). Transcriptional profiling of pathogenic yeast C. albicans cells treated with carvacrol demonstrated a bona fide UPR transcriptional signature. Ire1 activity detected by the splicing of HAC1 mRNA in C. albicans was activated by carvacrol. Furthermore, carvacrol was found to potentiate antifungal activity of the echinocandin antifungal caspofungin and UPR inducers dithiothreitol and tunicamycin against C. albicans. This comprehensive chemogenomic investigation demonstrated that carvacrol exerts its antifungal activity by altering ER integrity, leading to ER stress and the activation of the UPR to restore protein-folding homeostasis.

Sir2 links the unfolded protein response and the heat shock response in a stress response network

The Heat Shock Response (HSR) in the cytosol and the Unfolded Protein Response (UPR) in the endoplasmic reticulum are major pathways of the cellular proteostasis network. In Saccharomyces cerevisiae, HSR is regulated by transcription factor Hsf1, and UPR Ire1 branch activates transcription factor Hac1. Here we demonstrate systemic regulation of proteostasis through a direct link between UPR and HSR. Hsf1 is activated by UPR and its HSR depends on intact UPR. This link is mediated by Sir2, which is not only essential for Hsf1 HSR but also required for Hsf1 activation by UPR. Excess Sir2 augments Hsf1 activation by UPR and can compensate for its impairment in UPR-defective strains. Sir2 is upregulated by UPR but, in turn, it also attenuates this pathway, ensuring that UPR functions only transiently.

Comprehensive clone screening and evaluation of fed-batch strategies in a microbioreactor and lab scale stirred tank bioreactor system: application on Pichia pastoris producing Rhizopus oryzae lipase

BackgroundIn Pichia pastoris bioprocess engineering, classic approaches for clone selection and bioprocess optimization at small/micro scale using the promoter of the alcohol oxidase 1 gene (PAOX1), induced by methanol, present low reproducibility leading to high time and resource consumption.ResultsAn automated microfermentation platform (RoboLector) was successfully tested to overcome the chronic problems of clone selection and optimization of fed-batch strategies. Different clones from Mut+P. pastoris phenotype strains expressing heterologous Rhizopus oryzae lipase (ROL), including a subset also overexpressing the transcription factor HAC1, were tested to select the most promising clones.The RoboLector showed high performance for the selection and optimization of cultivation media with minimal cost and time. Syn6 medium was better than conventional YNB medium in terms of production of heterologous protein.The RoboLector microbioreactor was also tested for different fed-batch strategies with three clones producing different lipase levels. Two mixed substrates fed-batch strategies were evaluated. The first strategy was the enzymatic release of glucose from a soluble glucose polymer by a glucosidase, and methanol addition every 24 hours. The second strategy used glycerol as co-substrate jointly with methanol at two different feeding rates. The implementation of these simple fed-batch strategies increased the levels of lipolytic activity 80-fold compared to classical batch strategies used in clone selection. Thus, these strategies minimize the risk of errors in the clone selection and increase the detection level of the desired product.Finally, the performance of two fed-batch strategies was compared for lipase production between the RoboLector microbioreactor and 5 liter stirred tank bioreactor for three selected clones. In both scales, the same clone ranking was achieved.ConclusionThe RoboLector showed excellent performance in clone selection of P. pastoris Mut+ phenotype. The use of fed-batch strategies using mixed substrate feeds resulted in increased biomass and lipolytic activity. The automated processing of fed-batch strategies by the RoboLector considerably facilitates the operation of fermentation processes, while reducing error-prone clone selection by increasing product titers.The scale-up from microbioreactor to lab scale stirred tank bioreactor showed an excellent correlation, validating the use of microbioreactor as a powerful tool for evaluating fed-batch operational strategies.

Quantitative iTRAQ LC–MS/MS proteomics reveals the cellular response to heterologous protein overexpression and the regulation of HAC1 in Pichia pastoris

The methylotrophic yeast Pichia pastoris is an attractive platform for a plethora of recombinant proteins. There is growing evidence that host cells producing recombinant proteins are exposed to a variety of cellular stresses resulting in the induction of the unfolded protein response (UPR) pathway. At present, there is only limited information about the cellular reactions of the host cells at the level of the proteome, especially with regard to recombinant protein secretion. Here we monitored xylanase A secretion from Bacillus halodurans C-125 (xynA) in P. pastoris, using strains containing different copy numbers of the gene encoding xylanase A and co-overexpressing the gene encoding the UPR-regulating transcription factor HAC1 by applying a quantitative proteomics approach (iTRAQ-LC-MS/MS). Many important cellular processes, including carbon metabolism, stress response and protein folding are affected in the investigated conditions. Notably, the analysis revealed that strong over-expression of xynA can efficiently improve protein production but simultaneously cause an unfolded protein burden with a subsequent induction of the UPR. This limits the further improvement of protein production levels. Remarkably, constitutive expression of the gene encoding HAC1 lessens the unfolded protein burden by attenuating protein synthesis and increasing ER protein folding efficiency which is beneficial for protein secretion. Pichia pastoris expression systems have been successfully used for over 20years in basic research and in the biotechnology industry for the production and secretion of a wide range of recombinant proteins. In particular, secretion of recombinant proteins is still one of the main reasons for using P. pastoris. It has become obvious that many protein products can lead to severe stress on the host cell when being over-expressed, thus limiting the potential yield. Detailed understanding of the physiological responses to such stresses gives rise to engineering of host cells that can better cope with the stress factors. Therefore, the regulatory mechanism of heterologous protein secretion by quantitative mass-spectrometry (MS) proteomics is a growing field and an important endeavor in improving protein annotation. Many important cellular processes, including carbon and amino acid metabolism, stress response and protein folding are affected in the over-expression strains. This data represent a first step towards a systems wide approach to assess the response with recombinant protein induced stress in P. pastoris.

Dissection of Ire1 functions reveals stress response mechanisms uniquely evolved in Candida glabrata.

Proper protein folding in the endoplasmic reticulum (ER) is vital in all eukaryotes. When misfolded proteins accumulate in the ER lumen, the transmembrane kinase/endoribonuclease Ire1 initiates splicing of HAC1 mRNA to generate the bZIP transcription factor Hac1, which subsequently activates its target genes to increase the protein-folding capacity of the ER. This cellular machinery, called the unfolded protein response (UPR), is believed to be an evolutionarily conserved mechanism in eukaryotes. In this study, we comprehensively characterized mutant phenotypes of IRE1 and other related genes in the human fungal pathogen Candida glabrata. Unexpectedly, Ire1 was required for the ER stress response independently of Hac1 in this fungus. C. glabrata Ire1 did not cleave mRNAs encoding Hac1 and other bZIP transcription factors identified in the C. glabrata genome. Microarray analysis revealed that the transcriptional response to ER stress is not mediated by Ire1, but instead is dependent largely on calcineurin signaling and partially on the Slt2 MAPK pathway. The loss of Ire1 alone did not confer increased antifungal susceptibility in C. glabrata contrary to UPR-defective mutants in other fungi. Taken together, our results suggest that the canonical Ire1-Hac1 UPR is not conserved in C. glabrata. It is known in metazoans that active Ire1 nonspecifically cleaves and degrades a subset of ER-localized mRNAs to reduce the ER load. Intriguingly, this cellular response could occur in an Ire1 nuclease-dependent fashion in C. glabrata. We also uncovered the attenuated virulence of the C. glabrata Δire1 mutant in a mouse model of disseminated candidiasis. This study has unveiled the unique evolution of ER stress response mechanisms in C. glabrata.

Basic leucine zipper transcription factor Hac1 binds DNA in two distinct modes as revealed by microfluidic analyses

A quantitative understanding of how transcription factors interact with genomic target sites is crucial for reconstructing transcriptional networks in vivo. Here, we use Hac1, a well-characterized basic leucine zipper (bZIP) transcription factor involved in the unfolded protein response (UPR) as a model to investigate interactions between bZIP transcription factors and their target sites. During the UPR, the accumulation of unfolded proteins leads to unconventional splicing and subsequent translation of HAC1 mRNA, followed by transcription of UPR target genes. Initial candidate-based approaches identified a canonical cis-acting unfolded protein response element (UPRE-1) within target gene promoters; however, subsequent studies identified a large set of Hac1 target genes lacking this UPRE-1 and containing a different motif (UPRE-2). Using a combination of unbiased and directed microfluidic DNA binding assays, we established that Hac1 binds in two distinct modes: (i) to short (6-7bp) UPRE-2-like motifs and (ii) to significantly longer (11-13bp) extended UPRE-1-like motifs. Using a genetic screen, we demonstrate that a region of extended homology N-terminal to the basic DNA binding domain is required for this dual site recognition. These results establish Hac1 as the first bZIP transcription factor known to adopt more than one binding mode and unify previously conflicting and discrepant observations of Hac1 function into a cohesive model of UPR target gene activation. Our results also suggest that even structurally simple transcription factors can recognize multiple divergent target sites of very different lengths, potentially enriching their downstream target repertoire.

Ribosome Deficiency Protects Against ER Stress in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, 59 of the 78 ribosomal proteins are encoded by duplicated genes that, in most cases, encode identical or very similar protein products. However, different sets of ribosomal protein genes have been identified in screens for various phenotypes, including life span, budding pattern, and drug sensitivities. Due to potential suppressors of growth rate defects among this set of strains in the ORF deletion collection, we regenerated the entire set of haploid ribosomal protein gene deletion strains in a clean genetic background. The new strains were used to create double deletions lacking both paralogs, allowing us to define a set of 14 nonessential ribosomal proteins. Replicative life-span analysis of new strains corresponding to ORF deletion collection strains that likely carried suppressors of growth defects identified 11 new yeast replicative aging genes. Treatment of the collection of ribosomal protein gene deletion strains with tunicamycin revealed a significant correlation between slow growth and resistance to ER stress that was recapitulated by reducing translation of wild-type yeast with cycloheximide. Interestingly, enhanced tunicamycin resistance in ribosomal protein gene deletion mutants was independent of the unfolded protein response transcription factor Hac1. These data support a model in which reduced translation is protective against ER stress by a mechanism distinct from the canonical ER stress response pathway and further add to the diverse yet specific phenotypes associated with ribosomal protein gene deletions.