The use of high-throughput microscopy in the characterisation of phenotypes associated with the Unfolded Protein Response in Saccharomyces cerevisiae

<p>Proteins traversing the secretory pathway begin their passage in the endoplasmic reticulum (ER) where they must be correctly folded and processed to pass quality control measures. Complications with this process can result in the accumulation of misfolded proteins, commonly referred to as ER-stress, which has been associated with a number of diseases. The unfolded protein response (UPR) is the cell’s mechanism of dealing with ER-stress and is activated via the IRE1-HAC1 pathway in yeast. Ire1p is the ER-stress sensor and upon recognising misfolded proteins Ire1 oligomerises and forms local clusters. Activated Ire1p then splices out an inhibitory intron from the UPR specific transcription factor Hac1p which goes on to initiate downstream responses to alleviate ER-stress. Here we utilise high-throughput microscopy and UPR-specific GFP reporter systems to characterise the UPR in the yeast Saccharomyces cerevisiae. High-throughput microscopy and automated image analysis is increasingly being used as a screening tool for investigating genome-wide collections of yeast strains, including the yeast deletion mutant array and the yeast GFP collection. We describe the use of GFP labelled Ire1p to visualise cluster formation as a reporter for early UPR recognition of misfolded proteins, as well as a GFP controlled by a Hac1p responsive promoter to measure downstream UPR activation. These UPR-specific GFP reporter systems were used to screen a collection of non-essential gene deletion strains, identifying gene deletions that induce UPR activation and thus are likely to function in the early secretory pathway. This included well known components such as the ALG members of the glycosylation pathway and various ER chaperones such as LHS1 and SCJ1. Additionally this analysis revealed 44 previously uncharacterised genes, suggesting there are still processes related to the secretory pathway that are yet to be described. Moreover, by inducing ER-stress in this screening system we revealed genes required for the normal activation of the UPR including ribosomal/translation and chromatin/transcriptionally related genes, as well as various genes from throughout the secretory pathway. Furthermore, we screened a collection of ~4000 strains, each expressing a different GFP fusion protein, under ER-stress conditions to identify protein expression and localisation changes induced by the UPR. Comparison to UPR deficient Δhac1 cells uncovered a set of UPR specific targets including 26 novel UPR targets that had not been identified in previous studies measuring changes at the transcript level. As part of this work, we developed a dual red fluorescent protein system to label cells for automated image segmentation to enable single cell phenotype measurements. Here we describe the use of texture analysis as a means of increasing automation in the identification of phenotypic changes across the proteome. These novel techniques may be more widely applied to screening GFP collections to increase automation of image analysis, particularly as manual annotation of phenotypic changes is a major bottleneck in high-throughput screening. The results presented here from microscopy based screening compare well with other techniques in the literature, but also provide new information highlighting the synergistic effects of integrating high-throughput imaging into traditional screening methodologies.</p>

The use of high-throughput microscopy in the characterisation of phenotypes associated with the Unfolded Protein Response in Saccharomyces cerevisiae

10.26686/wgtn.17008045 ◽

2021 ◽

Author(s):

◽

Peter William Bircham

Keyword(s):

Er Stress ◽

High Throughput ◽

Secretory Pathway ◽

Misfolded Proteins ◽

Phenotypic Changes ◽

Unfolded Protein ◽

Gfp Reporter ◽

Reporter Systems ◽

ER stress causes widespread protein aggregation and prion formation

The Journal of Cell Biology ◽

10.1083/jcb.201612165 ◽

2017 ◽

Vol 216 (8) ◽

pp. 2295-2304 ◽

Cited By ~ 28

Author(s):

Norfadilah Hamdan ◽

Paraskevi Kritsiligkou ◽

Chris M. Grant

Keyword(s):

Protein Aggregation ◽

Er Stress ◽

Secretory Pathway ◽

Cellular Protein ◽

Protein Homeostasis ◽

Er Quality Control ◽

Unfolded Protein ◽

Protein Response ◽

Er Homeostasis

Disturbances in endoplasmic reticulum (ER) homeostasis create a condition termed ER stress. This activates the unfolded protein response (UPR), which alters the expression of many genes involved in ER quality control. We show here that ER stress causes the aggregation of proteins, most of which are not ER or secretory pathway proteins. Proteomic analysis of the aggregated proteins revealed enrichment for intrinsically aggregation-prone proteins rather than proteins which are affected in a stress-specific manner. Aggregation does not arise because of overwhelming proteasome-mediated degradation but because of a general disruption of cellular protein homeostasis. We further show that overexpression of certain chaperones abrogates protein aggregation and protects a UPR mutant against ER stress conditions. The onset of ER stress is known to correlate with various disease processes, and our data indicate that widespread amorphous and amyloid protein aggregation is an unanticipated outcome of such stress.

Endoplasmic Reticulum Stress Signaling Pathways: Activation and Diseases

Current Protein and Peptide Science ◽

10.2174/1389203720666190621103145 ◽

2019 ◽

Vol 20 (9) ◽

pp. 935-943 ◽

Cited By ~ 6

Author(s):

Zhi Zheng ◽

Yuxi Shang ◽

Jiahui Tao ◽

Jun Zhang ◽

Bingdong Sha

Keyword(s):

Endoplasmic Reticulum ◽

Er Stress ◽

Misfolded Proteins ◽

Endogenous Factors ◽

Unfolded Protein ◽

Dependent Protein ◽

Activating Transcription Factor ◽

Protein Response ◽

Sensor Molecules

Secretory and membrane proteins are folded in the endoplasmic reticulum (ER) prior to their exit. When ER function is disturbed by exogenous and endogenous factors, such as heat shock, ultraviolet radiation, hypoxia, or hypoglycemia, the misfolded proteins may accumulate, promoting ER stress. To rescue this unfavorable situation, the unfolded protein response is activated to reduce misfolded proteins within the ER. Upon ER stress, the ER transmembrane sensor molecules inositol-requiring enzyme 1 (IRE1), RNA-dependent protein kinase (PKR)-like ER kinase (PERK), and activating transcription factor 6, are activated. Here, we discuss the mechanisms of PERK and IRE1 activation and describe two working models for ER stress initiation: the BiP-dependent model and the ligand-driven model. ER stress activation has been linked to multiple diseases, including cancers, Alzheimer’s disease, and diabetes. Thus, the regulation of ER stress may provide potential therapeutic targets for these diseases.

Protein quality control in the secretory pathway

The Journal of Cell Biology ◽

10.1083/jcb.201906047 ◽

2019 ◽

Vol 218 (10) ◽

pp. 3171-3187 ◽

Cited By ~ 59

Author(s):

Zhihao Sun ◽

Jeffrey L. Brodsky

Keyword(s):

Quality Control ◽

Protein Folding ◽

Secretory Pathway ◽

Protein Quality ◽

Protein Quality Control ◽

Misfolded Proteins ◽

Unfolded Protein ◽

Protein Biogenesis ◽

Protein folding is inherently error prone, especially in the endoplasmic reticulum (ER). Even with an elaborate network of molecular chaperones and protein folding facilitators, misfolding can occur quite frequently. To maintain protein homeostasis, eukaryotes have evolved a series of protein quality-control checkpoints. When secretory pathway quality-control pathways fail, stress response pathways, such as the unfolded protein response (UPR), are induced. In addition, the ER, which is the initial hub of protein biogenesis in the secretory pathway, triages misfolded proteins by delivering substrates to the proteasome or to the lysosome/vacuole through ER-associated degradation (ERAD) or ER-phagy. Some misfolded proteins escape the ER and are instead selected for Golgi quality control. These substrates are targeted for degradation after retrieval to the ER or delivery to the lysosome/vacuole. Here, we discuss how these guardian pathways function, how their activities intersect upon induction of the UPR, and how decisions are made to dispose of misfolded proteins in the secretory pathway.

The proteasome biogenesis regulator Rpn4 cooperates with the unfolded protein response to promote resistance to endoplasmic reticulum stress

10.1101/457077 ◽

2018 ◽

Author(s):

Rolf M. Schmidt ◽

Sebastian Schuck

Keyword(s):

Endoplasmic Reticulum ◽

Er Stress ◽

Unfolded Protein Response ◽

Protein Misfolding ◽

Misfolded Proteins ◽

Secretory Proteins ◽

Unfolded Protein ◽

Protein Response ◽

Multiple Signaling

ABSTRACTMisfolded proteins in the endoplasmic reticulum (ER) activate the unfolded protein response (UPR), which enhances protein folding to restore homeostasis. Additional pathways respond to ER stress, but how they help counteract protein misfolding is incompletely understood. Here, we develop a titratable system for the induction of ER stress in yeast to enable a genetic screen for factors that augment stress resistance independently of the UPR. We identify the proteasome biogenesis regulator Rpn4 and show that it cooperates with the UPR. Rpn4 abundance increases during ER stress, first by a post-transcriptional, then by a transcriptional mechanism. Induction of RPN4 transcription is triggered by cytosolic mislocalization of secretory proteins, is mediated by multiple signaling pathways and accelerates clearance of misfolded proteins from the cytosol. Thus, Rpn4 and the UPR are complementary elements of a modular cross-compartment response to ER stress.

The proteasome biogenesis regulator Rpn4 cooperates with the unfolded protein response to promote ER stress resistance

eLife ◽

10.7554/elife.43244 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 13

Author(s):

Rolf M Schmidt ◽

Julia P Schessner ◽

Georg HH Borner ◽

Sebastian Schuck

Keyword(s):

Er Stress ◽

Unfolded Protein Response ◽

Stress Resistance ◽

Protein Misfolding ◽

Misfolded Proteins ◽

Secretory Proteins ◽

Unfolded Protein ◽

Protein Response ◽

Multiple Signaling

Misfolded proteins in the endoplasmic reticulum (ER) activate the unfolded protein response (UPR), which enhances protein folding to restore homeostasis. Additional pathways respond to ER stress, but how they help counteract protein misfolding is incompletely understood. Here, we develop a titratable system for the induction of ER stress in yeast to enable a genetic screen for factors that augment stress resistance independently of the UPR. We identify the proteasome biogenesis regulator Rpn4 and show that it cooperates with the UPR. Rpn4 abundance increases during ER stress, first by a post-transcriptional, then by a transcriptional mechanism. Induction of RPN4 transcription is triggered by cytosolic mislocalization of secretory proteins, is mediated by multiple signaling pathways and accelerates clearance of misfolded proteins from the cytosol. Thus, Rpn4 and the UPR are complementary elements of a modular cross-compartment response to ER stress.

Functional Coupling between the Unfolded Protein Response and Endoplasmic Reticulum/Golgi Ca2+-ATPases Promotes Stress Tolerance, Cell Wall Biosynthesis, and Virulence of Aspergillus fumigatus

mBio ◽

10.1128/mbio.01060-20 ◽

2020 ◽

Vol 11 (3) ◽

Cited By ~ 1

Author(s):

Martin Weichert ◽

José Guirao-Abad ◽

Vishukumar Aimanianda ◽

Karthik Krishnan ◽

Christina Grisham ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Cell Wall ◽

Er Stress ◽

Aspergillus Fumigatus ◽

Secretory Pathway ◽

Functional Coupling ◽

Content Type ◽

Unfolded Protein ◽

ABSTRACT Many species of pathogenic fungi deploy the unfolded protein response (UPR) to expand the folding capacity of the endoplasmic reticulum (ER) in proportion to the demand for virulence-related proteins that traffic through the secretory pathway. Although Ca2+ plays a pivotal role in ER function, the mechanism by which transcriptional upregulation of the protein folding machinery is coordinated with Ca2+ homeostasis is incompletely understood. In this study, we investigated the link between the UPR and genes encoding P-type Ca2+-ATPases in the human-pathogenic mold Aspergillus fumigatus. We demonstrate that acute ER stress increases transcription of the srcA gene, encoding a member of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) family, as well as that of pmrA, encoding a secretory pathway Ca2+-ATPase (SPCA) in the Golgi membrane. Loss of the UPR transcription factor HacA prevented the induction of srcA and pmrA transcription during ER stress, defining these ER/Golgi Ca2+ pumps as novel downstream targets of this pathway. While deletion of srcA alone caused no major deficiencies, a ΔsrcA/ΔpmrA mutant displayed a severe polarity defect, was hypersensitive to ER stress, and showed attenuated virulence. In addition, cell wall analyses revealed a striking reduction in mannose levels in the absence of both Ca2+ pumps. The ΔhacA mutant was hypersensitive to agents that block calcineurin-dependent signaling, consistent with a functional coupling between the UPR and Ca2+ homeostasis. Together, these findings demonstrate that the UPR integrates the need for increased levels of chaperone and folding enzymes with an influx of Ca2+ into the secretory pathway to support fungal growth, stress adaptation, and pathogenicity. IMPORTANCE The UPR is an intracellular signal transduction pathway that maintains homeostasis of the ER. The pathway is also tightly linked to the expression of virulence-related traits in diverse species of human-pathogenic and plant-pathogenic fungal species, including the predominant mold pathogen infecting humans, Aspergillus fumigatus. Despite advances in the understanding of UPR signaling, the linkages and networks that are governed by this pathway are not well defined. In this study, we revealed that the UPR is a major driving force for stimulating Ca2+ influx at the ER and Golgi membranes and that the coupling between the UPR and Ca2+ import is important for virulence, cell wall biosynthesis, and resistance to antifungal compounds that inhibit Ca2+ signaling.

Noncanonical binding of BiP ATPase domain to Ire1 and Perk is dissociated by unfolded protein CH1 to initiate ER stress signaling

eLife ◽

10.7554/elife.03522 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 83

Author(s):

Marta Carrara ◽

Filippo Prischi ◽

Piotr R Nowak ◽

Megan C Kopp ◽

Maruf MU Ali

Keyword(s):

Er Stress ◽

Cellular Response ◽

Misfolded Proteins ◽

Atpase Domain ◽

Unfolded Proteins ◽

Unfolded Protein ◽

Dual Functional ◽

Protein Response ◽

Substrate Binding Domain

The unfolded protein response (UPR) is an essential cell signaling system that detects the accumulation of misfolded proteins within the endoplasmic reticulum (ER) and initiates a cellular response in order to maintain homeostasis. How cells detect the accumulation of misfolded proteins remains unclear. In this study, we identify a noncanonical interaction between the ATPase domain of the ER chaperone BiP and the luminal domains of the UPR sensors Ire1 and Perk that dissociates when authentic ER unfolded protein CH1 binds to the canonical substrate binding domain of BiP. Unlike the interaction between chaperone and substrates, we found that the interaction between BiP and UPR sensors was unaffected by nucleotides. Thus, we discover that BiP is dual functional UPR sensor, sensing unfolded proteins by canonical binding to substrates and transducing this event to noncanonical, signaling interaction to Ire1 and Perk. Our observations implicate BiP as the key component for detecting ER stress and suggest an allosteric mechanism for UPR induction.