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Kwang-Woo Jung, Yee-Seul So, Yong-Sun Bahn
Cryptococcus neoformans, a global fungal meningitis pathogen, employs the unfolded protein response pathway. This pathway, which consists of an evolutionarily conserved Ire1 kinase/endoribonuclease and a unique transcription factor (Hxl1), modulates the endoplasmic reticulum stress response and pathogenicity. Here, we report that the unfolded protein response pathway governs sexual and unisexual differentiation of C. neoformans in an Ire1-dependent but Hxl1-independent manner. The ire1∆ mutants showed defects in sexual mating, with reduced cell fusion and pheromone-mediated formation of the conjugation tube...
2016: Scientific Reports
Mengni Xu, Heather M Marsh, Carolyn S Sevier
Among the amino acids, cysteine stands apart based on its highly reactive sulfur group. In general, cysteine is underrepresented in proteins. Yet, when present, the features of cysteine often afford unique function. We have shown previously that a cysteine within the ATPase domain of yeast BiP (Kar2) serves as a sensor of the endoplasmic reticulum (ER) redox environment [1, 2]. Under conditions of increased oxidant (oxidative stress), this cysteine becomes oxidized, changing Kar2 from an ATP-dependent foldase to an ATP-independent holdase...
October 9, 2016: Journal of Molecular Biology
Bo Guan, Fengxiang Chen, Shuai Su, Zuoying Duan, Yun Chen, Huazhong Li, Jian Jin
Pichia pastoris is generally considered as an expression host for heterologous proteins with the coding gene under control of the alcohol oxidase 1 (AOX1) promoter. The secretion of heterologous proteins in P. pastoris can be potentially affected by many factors. Based on our previous results, the secretion levels of human albumin (HSA) fusion protein IL2-HSA were only around 500 mg/L or less in fermentor cultures, which decreased more than 50% comparing to that of HSA (>1 g/L). In this study, we selected five potential secretion helper factors, in which Ero1, Pdi1 and Kar2 involved in protein folding and Sec1 and Sly1 involved in vesicle trafficking...
August 17, 2016: Yeast
Jorg C de Ruijter, Essi V Koskela, Alexander D Frey
BACKGROUND: The yeast Saccharomyces cerevisiae provides intriguing possibilities for synthetic biology and bioprocess applications, but its use is still constrained by cellular characteristics that limit the product yields. Considering the production of advanced biopharmaceuticals, a major hindrance lies in the yeast endoplasmic reticulum (ER), as it is not equipped for efficient and large scale folding of complex proteins, such as human antibodies. RESULTS: Following the example of professional secretory cells, we show that inducing an ER expansion in yeast by deleting the lipid-regulator gene OPI1 can improve the secretion capacity of full-length antibodies up to fourfold...
2016: Microbial Cell Factories
Francisco Javier Piña, Tinya Fleming, Kit Pogliano, Maho Niwa
Segregation of functional organelles during the cell cycle is crucial to generate healthy daughter cells. In Saccharomyces cerevisiae, ER stress causes an ER inheritance block to ensure cells inherit a functional ER. Here, we report that formation of tubular ER in the mother cell, the first step in ER inheritance, depends on functional symmetry between the cortical ER (cER) and perinuclear ER (pnER). ER stress induces functional asymmetry, blocking tubular ER formation and ER inheritance. Using fluorescence recovery after photobleaching, we show that the ER chaperone Kar2/BiP fused to GFP and an ER membrane reporter, Hmg1-GFP, behave differently in the cER and pnER...
May 9, 2016: Developmental Cell
Rūta Zinkevičiūtė, Edita Bakūnaitė, Evaldas Čiplys, Raimundas Ražanskas, Jurgita Raškevičiūtė, Rimantas Slibinskas
The yield of heterologous proteins is often limited by several bottlenecks in the secretory pathway of yeast Saccharomyces cerevisiae. It was shown earlier that synthesis of measles virus hemagglutinin (MeH) is inefficient mostly due to a bottleneck in the translocation of viral protein precursors into the endoplasmic reticulum (ER) of yeast cells. Here we report that heat shock with subsequent induction of MeH expression at 37°C improved translocation of MeH precursors when applied at higher cell densities...
December 25, 2015: New Biotechnology
Jofre Ferrer-Dalmau, Francisca Randez-Gil, Maribel Marquina, José A Prieto, Antonio Casamayor
Glc7 is the only catalytic subunit of the protein phosphatase type 1 in the yeast S. cerevisiae and, together with its regulatory subunits, is involved in many essential processes. Analysis of the non-essential mutants in the regulatory subunits of Glc7 revealed that the lack of Reg1, and no other subunit, causes hypersensitivity to unfolded protein response (UPR)-inducers, which was concomitant with an augmented UPR element-dependent transcriptional response. The Glc7-Reg1 complex takes part in the regulation of the yeast AMP-activated serine/threonine protein kinase Snf1 in response to glucose...
May 15, 2015: Biochemical Journal
Tatiana Q Aguiar, Orquídea Ribeiro, Mikko Arvas, Marilyn G Wiebe, Merja Penttilä, Lucília Domingues
BACKGROUND: Ashbya gossypii is a filamentous Saccharomycete used for the industrial production of riboflavin that has been recently explored as a host system for recombinant protein production. To gain insight into the protein secretory pathway of this biotechnologically relevant fungus, we undertook genome-wide analyses to explore its secretome and its transcriptional responses to protein secretion stress. RESULTS: A computational pipeline was used to predict the inventory of proteins putatively secreted by A...
2014: BMC Genomics
Martin Mehnert, Franziska Sommermeyer, Maren Berger, Sathish Kumar Lakshmipathy, Robert Gauss, Markus Aebi, Ernst Jarosch, Thomas Sommer
Misfolded proteins of the secretory pathway are extracted from the endoplasmic reticulum (ER), polyubiquitylated by a protein complex termed the Hmg-CoA reductase degradation ligase (HRD-ligase), and degraded by cytosolic 26S proteasomes. This process is termed ER-associated protein degradation (ERAD). We previously showed that the membrane protein Der1, which is a subunit of the HRD-ligase, is involved in the export of aberrant polypeptides from the ER. Unexpectedly, we also uncovered a close spatial proximity of Der1 and the substrate receptor Hrd3 in the ER lumen...
January 15, 2015: Molecular Biology of the Cell
Seon Ah Cheon, Kwang-Woo Jung, Yong-Sun Bahn, Hyun Ah Kang
Unique and evolutionarily conserved signaling pathways allow an organism to sense, respond to, and adapt to internal and external environmental cues at its biological niche. In eukaryotic cells, the unfolded protein response (UPR) pathway regulates endoplasmic reticulum (ER) homeostasis upon exposure to environmental changes causing ER stress. The UPR pathway of Cryptococcus neoformans, an opportunistic fungal pathogen, which causes life-threatening meningoencephalitis in immunocompromised individuals, consists of the evolutionarily conserved Ire1 kinase, a unique bZIP transcription factor, Hxl1, and the ER-resident molecular chaperone Kar2/BiP...
February 15, 2014: Virulence
Hailong Liu, Yufeng Qin, Yuankai Huang, Yaosheng Chen, Peiqing Cong, Zuyong He
Increasing the gene copy number has been commonly used to enhance the protein expression level in the yeast Pichia pastoris. However, this method has been shown to be effective up to a certain gene copy number, and a further increase of gene dosage can result in a decrease of expression level. Evidences indicate the gene dosage effect is product-dependent, which needs to be determined when expressing a new protein. Here, we describe a direct detection of the gene dosage effect on protein secretion through expressing the enhanced green fluorescent protein (EGFP) gene under the direction of the α-factor preprosequence in a panel of yeast clones carrying increasing copies of the EGFP gene (from one to six copies)...
February 28, 2014: Journal of Microbiology and Biotechnology
John P Stanga, Steven M Smith, Winslow R Briggs, David C Nelson
Abiotic chemical signals discovered in smoke that are known as karrikins (KARs) and the endogenous hormone strigolactone (SL) control plant growth through a shared MORE AXILLARY GROWTH2 (MAX2)-dependent pathway. A SL biosynthetic pathway and candidate KAR/SL receptors have been characterized, but signaling downstream of MAX2 is poorly defined. A screen for genetic suppressors of the enhanced seed dormancy phenotype of max2 in Arabidopsis (Arabidopsis thaliana) led to identification of a suppressor of max2 1 (smax1) mutant...
September 2013: Plant Physiology
Chengchao Xu, Songyu Wang, Guillaume Thibault, Davis T W Ng
Newly synthesized polypeptides fold and assemble with assistance from protein chaperones. Full maturation can take multiple attempts, exchanging chaperones at each round. Improperly folded molecules must exit folding cycles and be degraded. In the endoplasmic reticulum (ER), prolonged substrate cycling is detrimental because it expends chaperone and energy resources and increases toxic reactive oxygen species. In budding yeast, we found that unfolded protein O-mannosylation terminated failed folding attempts through the Pmt1/Pmt2 complex...
May 24, 2013: Science
Kwang-Woo Jung, Hyun Ah Kang, Yong-Sun Bahn
The endoplasmic reticulum (ER) is a central hub where secreted or membrane-bound proteins are maturated and folded properly in eukaryotes. Maintenance of ER homeostasis is particularly important for human fungal pathogens, such as Cryptococcus neoformans, which encounter a plethora of host-mediated stresses during infection. Our previous study demonstrated that the unfolded protein response (UPR) pathway, composed of the evolutionarily conserved Ire1 kinase and the unique Hxl1 transcription factor, has pleiotropic roles in ER stress response, thermotolerance, antifungal drug resistance, and virulence in C...
2013: PloS One
Birsen Cakir
Bax, a multidomain pro-apoptotic Bcl-2 protein, localizes to the endoplasmic reticulum (ER), where it regulates ER stress-induced apoptosis. Adaptation to ER stress depends on the activation of an integrated signal transduction pathway known as the unfolded protein response (UPR). This study examined the death-inducing activity of Bax and its ability to induce UPR signalling pathways in yeast. We observed that inhibition of global translation in yeast cells expressing Bax correlated with Bax-induced cell death...
September 2012: Yeast
Patrick Lajoie, Robyn D Moir, Ian M Willis, Erik L Snapp
Accumulation of misfolded secretory proteins in the endoplasmic reticulum (ER) activates the unfolded protein response (UPR) stress pathway. To enhance secretory protein folding and promote adaptation to stress, the UPR upregulates ER chaperone levels, including BiP. Here we describe chromosomal tagging of KAR2, the yeast homologue of BiP, with superfolder green fluorescent protein (sfGFP) to create a multifunctional endogenous reporter of the ER folding environment. Changes in Kar2p-sfGFP fluorescence levels directly correlate with UPR activity and represent a robust reporter for high-throughput analysis...
March 2012: Molecular Biology of the Cell
Chia-Ling Hsu, Rupali Prasad, Christie Blackman, Davis T W Ng
The unfolded protein response (UPR) monitors and maintains protein homeostasis in the endoplasmic reticulum (ER). In budding yeast, the UPR is a transcriptional regulatory pathway that is quiescent under normal conditions. Under conditions of acute ER stress, activation of UPR targets is essential for cell viability. How individual target genes contribute to stress tolerance is unclear. Uncovering these roles is hampered because most targets also play important functions in the absence of stress. To differentiate stress-specific roles from everyday functions, a single target gene was uncoupled from UPR control by eliminating its UPR-specific regulatory element...
February 2012: Molecular Biology of the Cell
Lei Ji, Yu Shen, Lili Xu, Bingyin Peng, Yazhong Xiao, Xiaoming Bao
Saccharomyces cerevisiae is affected by the presence of certain phenolic compounds such as vanillin during fermentation of pretreated lignocellulosic hydrolysates. Since vanillin can be polymerized in the presence of laccase into compounds with lower toxicity, the laccase gene, lacA, from Trametes sp. AH28-2 was fused to the α-factor signal sequence and transferred into S. cerevisiae CEN.PK strains for secretory expression. Furthermore, the chaperone gene, KAR2, was overexpressed to promote the translocation of laccase...
September 2011: Bioresource Technology
Narendar K Khatri, Dörte Gocke, Oliver Trentmann, Peter Neubauer, Frank Hoffmann
Secretory production of a single-chain antibody fragment (scFv) by recombinant Pichia pastoris using the methanol inducible AOX1 promoter is limited biochemically by retarded secretion, and economically by the high demand for pure oxygen. To address the problem, the adaptation phase with growth-limiting feeding of glycerol before the production phase was optimized. In a standard procedure with a short glycerol-feeding phase before induction, scFv accumulated in the supernatant only after 15 h. Conversely, scFv started to appear immediately in the medium upon methanol induction when the glycerol-feeding phase was extended to 18 h...
April 2011: Biotechnology Journal
Tie Yin, Li-Li Miao, Fei-Fei Guan, Gui-Li Wang, Qing Peng, Bing-Xue Li, Guo-Hua Guan, Ying Li
An extremely thermostable xylanase gene, xynB, from hyperthermophilic bacterium Thermotoga maritima MSB8 was successful expressed in Kluyveromyces lactis. Response surface methodology (RSM) was applied to optimize medium components for production of XynB secreted by the recombinant K. lactis. Secretion level (102 mg/L) and enzyme activity (49 U/ml) of XynB in the optimized medium (yeast extract, lactose, and urea; YLU) were much higher than those (56 mg/L, 16 U/ml) in original medium (yeast extract, lactose, and peptone; YLP)...
November 2010: Journal of Microbiology and Biotechnology
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