The promoter region of the yeast KAR2 (BiP) gene contains a regulatory domain that responds to the presence of unfolded proteins in the endoplasmic reticulum

The endoplasmic reticulum (ER) of eukaryotic cells contains an abundant 78,000-Da protein (BiP) that is involved in the translocation, folding, and assembly of secretory and transmembrane proteins. In the yeast Saccharomyces cerevisiae, as in mammalian cells, BiP mRNA is synthesized at a high basal rate and is further induced by the presence of increased amounts of unfolded proteins in the ER. However, unlike mammalian BiP, yeast BiP is also induced severalfold by heat shock, albeit in a transient fashion. To identify the regulatory sequences that respond to these stimuli in the yeast KAR2 gene that encodes BiP, we have cloned a 1.3-kb segment of DNA from the region upstream of the sequences coding for BiP and fused it to a reporter gene, the Escherichia coli beta-galactosidase gene. Analysis of a series of progressive 5' truncations as well as internal deletions of the upstream sequence showed that the information required for accurate transcriptional regulation of the KAR2 gene in S. cerevisiae is contained within a approximately 230-bp XhoI-DraI fragment (nucleotides -245 to -9) and that this fragment contains at least two cis-acting elements, one (heat shock element [HSE]) responding to heat shock and the other (unfolded protein response element [UPR]) responding to the presence of unfolded proteins in the ER. The HSE and UPR elements are functionally independent of each other but work additively for maximum induction of the yeast KAR2 gene. Lying between these two elements is a GC-rich region that is similar in sequence to the consensus element for binding of the mammalian transcription factor Sp1 and that is involved in the basal expression of the KAR2 gene. Finally, we provide evidence suggesting that yeast cells monitor the concentration of free BiP in the ER and adjust the level of transcription of the KAR2 gene accordingly; this effect is mediated via the UPR element in the KAR2 promoter.

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Heat shock factor can activate transcription while bound to nucleosomal DNA in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.14.1.189-199.1994 ◽

1994 ◽

Vol 14 (1) ◽

pp. 189-199

Author(s):

D S Pederson ◽

T Fidrych

Keyword(s):

Saccharomyces Cerevisiae ◽

Heat Shock ◽

Transcription Initiation ◽

Heat Shock Factor ◽

Micrococcal Nuclease ◽

Nucleosome Assembly ◽

Heat Shock Element ◽

Yeast Saccharomyces Cerevisiae ◽

Nucleosomal Dna

After each round of replication, new transcription initiation complexes must assemble on promoter DNA. This process may compete with packaging of the same promoter sequences into nucleosomes. To elucidate interactions between regulatory transcription factors and nucleosomes on newly replicated DNA, we asked whether heat shock factor (HSF) could be made to bind to nucleosomal DNA in vivo. A heat shock element (HSE) was embedded at either of two different sites within a DNA segment that directs the formation of a stable, positioned nucleosome. The resulting DNA segments were coupled to a reporter gene and transfected into the yeast Saccharomyces cerevisiae. Transcription from these two plasmid constructions after induction by heat shock was similar in amount to that from a control plasmid in which HSF binds to nucleosome-free DNA. High-resolution genomic footprint mapping of DNase I and micrococcal nuclease cleavage sites indicated that the HSE in these two plasmids was, nevertheless, packaged in a nucleosome. The inclusion of HSE sequences within (but relatively close to the edge of) the nucleosome did not alter the position of the nucleosome which formed with the parental DNA fragment. Genomic footprint analyses also suggested that the HSE-containing nucleosome was unchanged by the induction of transcription. Quantitative comparisons with control plasmids ruled out the possibility that HSF was bound only to a small fraction of molecules that might have escaped nucleosome assembly. Analysis of the helical orientation of HSE DNA in the nucleosome indicated that HSF contacted DNA residues that faced outward from the histone octamer. We discuss the significance of these results with regard to the role of nucleosomes in inhibiting transcription and the normal occurrence of nucleosome-free regions in promoters.

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Identification of cis and trans components of a novel heat shock stress regulatory pathway in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.13.1.248-256.1993 ◽

1993 ◽

Vol 13 (1) ◽

pp. 248-256

Author(s):

N Kobayashi ◽

K McEntee

Keyword(s):

Saccharomyces Cerevisiae ◽

Stress Response ◽

Heat Shock ◽

Reporter Gene ◽

Heat Shock Element ◽

Cis Acting ◽

Heat Shock Stress ◽

Specific Complex ◽

Cis And Trans ◽

Shock Stress

The stress-responsive DDR2 gene (previously called DDRA2) of Saccharomyces cerevisiae is transcribed at elevated levels following stress caused by heat shock or DNA damage. Previously, we identified a 51-bp promoter fragment, oligo31/32, which conferred heat shock inducibility on the heterologous CYC1-lacZ reporter gene in S. cerevisiae (N. Kobayashi and K. McEntee, Proc. Natl. Acad. Sci. USA 87:6550-6554, 1990). Using a series of synthetic oligonucleotides, we have identified a pentanucleotide, CCCCT (C4T), as an essential component of this stress response sequence. This element is not a binding site for the well-characterized heat shock transcription factor which recognizes a distinct cis-acting heat shock element in the promoters of many heat shock genes. Here we demonstrate the ability of oligonucleotides containing the C4T sequence to confer heat shock inducibility on the reporter gene and show that the presence of two such elements produces more than additive effects on induction. Gel retardation experiments have been used to demonstrate specific complex formation between C4T-containing fragments and one or more yeast proteins. Formation of these complexes was not competed by fragments containing mutations in the C4T sequence nor by heat shock element-containing competitor DNAs. Fragments containing the C4T element bound to a single 140-kDa polypeptide, distinct from heat shock transcription factors in yeast crude extracts. These experiments identify key cis- and trans-acting components of a novel heat shock stress response pathway in S. cerevisiae.

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Transcriptional regulation of an hsp70 heat shock gene in the yeast Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.7.5.1906-1916.1987 ◽

1987 ◽

Vol 7 (5) ◽

pp. 1906-1916

Author(s):

M R Slater ◽

E A Craig

Keyword(s):

Saccharomyces Cerevisiae ◽

Heat Shock ◽

Promoter Region ◽

Inducible Expression ◽

Proximal Promoter ◽

Heat Shock Gene ◽

Heat Shock Element ◽

Yeast Saccharomyces Cerevisiae ◽

Hybrid Promoter ◽

Shock Gene

The yeast Saccharomyces cerevisiae contains three heat-inducible hsp70 genes. We have characterized the promoter region of the hsp70 heat shock gene YG100, that also displays a basal level of expression. Deletion of the distal region of the promoter resulted in an 80% drop in the basal level of expression without affecting expression after heat shock. Progressive-deletion analysis suggested that sequences necessary for heat-inducible expression are more proximal, within 233 base pairs of the initiation region. The promoter region of YG100 contains multiple elements related to the Drosophila melanogaster heat shock element (HSE; CnnGAAnnT TCnnG). Deletion of a proximal promoter region containing one element, HSE2, eliminated most of the heat-inducible expression of YG100. The upstream activation site (UAS) of the yeast cytochrome c gene (CYC1) can be substituted by a single copy of HSE2 plus its adjoining nucleotides (UASHS). This hybrid promoter displayed a substantial level of expression before heat shock, and the level of expression was elevated eightfold by heat shock. YG100 sequences that flank UASHS inhibited basal expression of UASHS in the hybrid promoter but not its heat-inducible expression. This inhibition of basal UASHS activity suggests that negative regulation is involved in modulating expression of this yeast heat shock gene.

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A bipartite operator interacts with a heat shock element to mediate early meiotic induction of Saccharomyces cerevisiae HSP82.

Molecular and Cellular Biology ◽

10.1128/mcb.15.12.6754 ◽

1995 ◽

Vol 15 (12) ◽

pp. 6754-6769 ◽

Cited By ~ 23

Author(s):

C Szent-Gyorgyi

Keyword(s):

Saccharomyces Cerevisiae ◽

Heat Shock ◽

Point Mutations ◽

Early Step ◽

Heat Shock Element ◽

Cis Acting ◽

Major Mode ◽

Dna Elements ◽

Insight Into ◽

Activation Sequence

Although key genetic regulators of early meiotic transcription in Saccharomyces cerevisiae have been well characterized, the activation of meiotic genes is still poorly understood in terms of cis-acting DNA elements and their associated factors. I report here that induction of HSP82 is regulated by the early meiotic IME1-IME2 transcriptional cascade. Vegetative repression and meiotic induction depend on interactions of the promoter-proximal heat shock element (HSE) with a nearby bipartite repression element, composed of the ubiquitous early meiotic motif, URS1 (upstream repression sequence 1), and a novel ancillary repression element. The ancillary repression element is required for efficient vegetative repression, is spatially separable from URS1, and continues to facilitate repression during sporulation. In contrast, URS1 also functions as a vegetative repression element but is converted early in meiosis into an HSE-dependent activation element. An early step in this transformation may be the antagonism of URS1-mediated repression by IME1. The HSE also nonspecifically supports a second major mode of meiotic activation that does not require URS1 but does require expression of IME2 and concurrent starvation. Interestingly, increased rather than decreased URS1-mediated vegetative transcription can be artificially achieved by introducing rare point mutations into URS1 or by deleting the UME6 gene. These lesions offer insight into mechanisms of URS-dependent repression and activation. Experiments suggest that URS1-bound factors functionally modulate heat shock factor during vegetative transcription and early meiotic induction but not during heat shock. The loss of repression and activation observed when the IME2 activation element, T4C, is substituted for the HSE suggests specific requirements for URS1-upstream activation sequence interactions.

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Transcriptional regulation of an hsp70 heat shock gene in the yeast Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.7.5.1906 ◽

1987 ◽

Vol 7 (5) ◽

pp. 1906-1916 ◽

Cited By ~ 67

Author(s):

M R Slater ◽

E A Craig

Keyword(s):

Saccharomyces Cerevisiae ◽

Heat Shock ◽

Promoter Region ◽

Inducible Expression ◽

Proximal Promoter ◽

Heat Shock Gene ◽

Heat Shock Element ◽

Yeast Saccharomyces Cerevisiae ◽

Hybrid Promoter ◽

Shock Gene

The yeast Saccharomyces cerevisiae contains three heat-inducible hsp70 genes. We have characterized the promoter region of the hsp70 heat shock gene YG100, that also displays a basal level of expression. Deletion of the distal region of the promoter resulted in an 80% drop in the basal level of expression without affecting expression after heat shock. Progressive-deletion analysis suggested that sequences necessary for heat-inducible expression are more proximal, within 233 base pairs of the initiation region. The promoter region of YG100 contains multiple elements related to the Drosophila melanogaster heat shock element (HSE; CnnGAAnnT TCnnG). Deletion of a proximal promoter region containing one element, HSE2, eliminated most of the heat-inducible expression of YG100. The upstream activation site (UAS) of the yeast cytochrome c gene (CYC1) can be substituted by a single copy of HSE2 plus its adjoining nucleotides (UASHS). This hybrid promoter displayed a substantial level of expression before heat shock, and the level of expression was elevated eightfold by heat shock. YG100 sequences that flank UASHS inhibited basal expression of UASHS in the hybrid promoter but not its heat-inducible expression. This inhibition of basal UASHS activity suggests that negative regulation is involved in modulating expression of this yeast heat shock gene.

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Characterization of constitutive ER-phagy of excess membrane proteins

PLoS Genetics ◽

10.1371/journal.pgen.1009255 ◽

2020 ◽

Vol 16 (12) ◽

pp. e1009255

Author(s):

Zhanna Lipatova ◽

Valeriya Gyurkovska ◽

Sarah F. Zhao ◽

Nava Segev

Keyword(s):

Quality Control ◽

Endoplasmic Reticulum ◽

Membrane Proteins ◽

Human Health ◽

Mammalian Cells ◽

Normal Growth ◽

Integral Membrane Proteins ◽

Yeast Cells ◽

Cellular Proteins

Thirty percent of all cellular proteins are inserted into the endoplasmic reticulum (ER), which spans throughout the cytoplasm. Two well-established stress-induced pathways ensure quality control (QC) at the ER: ER-phagy and ER-associated degradation (ERAD), which shuttle cargo for degradation to the lysosome and proteasome, respectively. In contrast, not much is known about constitutive ER-phagy. We have previously reported that excess of integral-membrane proteins is delivered from the ER to the lysosome via autophagy during normal growth of yeast cells. Whereas endogenously expressed ER resident proteins serve as cargos at a basal level, this level can be induced by overexpression of membrane proteins that are not ER residents. Here, we characterize this pathway as constitutive ER-phagy. Constitutive and stress-induced ER-phagy share the basic macro-autophagy machinery including the conserved Atgs and Ypt1 GTPase. However, induction of stress-induced autophagy is not needed for constitutive ER-phagy to occur. Moreover, the selective receptors needed for starvation-induced ER-phagy, Atg39 and Atg40, are not required for constitutive ER-phagy and neither these receptors nor their cargos are delivered through it to the vacuole. As for ERAD, while constitutive ER-phagy recognizes cargo different from that recognized by ERAD, these two ER-QC pathways can partially substitute for each other. Because accumulation of membrane proteins is associated with disease, and constitutive ER-phagy players are conserved from yeast to mammalian cells, this process could be critical for human health.

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Visualizing the intracellular membrane system of yeast cells using a laser scanning confocal microscope

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100147211 ◽

1993 ◽

Vol 51 ◽

pp. 274-275

Author(s):

Karen L. McCoy ◽

Andrew G. Dillin ◽

Ardythe A. McCracken

Keyword(s):

Endoplasmic Reticulum ◽

Yeast Cell ◽

Mammalian Cells ◽

Laser Scanning ◽

Intracellular Localization ◽

Fluorescent Microscopy ◽

Confocal Microscope ◽

Yeast Cells ◽

Intracellular Membrane ◽

Laser Scanning Confocal Microscope

Progress has been made in developing new preparative techniques for electron microscopic visualization of the intracellular structures of yeast. In addition, development of the laser scanning confocal microscope (LSCM) has provided improved resolution for fluorescent microscopy. We asked whether the LSCM in combination with new preparative techniques could be used for comparable investigative research of the intracellular organizaton of the yeast cell.To investigate this possibility, a BioRad MRC600 LSCM equipped with a krypton/argon laser and integrated computer imaging capabilities, was used to study various dipliod strains of the yeast Saccharomyces cerevisiae. Cells were treated with the lipophilic, cationic fluorescent dye DiOC6 (3,3’-dihexyloxacarbocyanine iodide), which has been used to visualize intracellular membrane structures, and in particular the endoplasmic reticulum of mammalian cells and living yeast cells. Since one of our interests is the intracellular localization of proteins in the yeast cell, we utilized transformed yeast cells expressing a human gene encoding a protein that inappropriately accumulates in the endoplasmic reticulum (ER).

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HSEAT: A Tool for Plant Heat Shock Element Analysis, Motif Identification and Analysis

Current Bioinformatics ◽

10.2174/1574893614666190102151956 ◽

2020 ◽

Vol 15 (3) ◽

pp. 196-203 ◽

Cited By ~ 1

Author(s):

Sarah Rizwan Qazi ◽

Noor ul Haq ◽

Shakeel Ahmad ◽

Samina N. Shakeel

Keyword(s):

Heat Shock ◽

Differential Expression ◽

Promoter Region ◽

Analysis Tool ◽

Promoter Regions ◽

Heat Shock Element ◽

Element Analysis ◽

Cis Acting ◽

Plant Genes ◽

Hsp Genes

Background: Previous methods used to discover cis-regulatory motifs in promoter region of plant genes possess very limited performance, especially for analysis of novel and rare motifs. Different plant genes have differential expression under different environmental or experimental conditions and modular regulation of cis-regulatory sequences in promoter regions of the same or different genes. It has previously been revealed that Heat Shock Proteins (HSPs) creation is correlated with plant tolerance under heat and other stress conditions. Regulation of these HSP genes is controlled by interactions between heat shock factors (HSFs) with cis-acting motifs present in the promoter region of the genes. Differential expression of these HSP genes is because of their unique promoter architecture, cis-acting sequences and their interaction with HSFs. Objective: A versatile promoter analysis tool was proposed for identification and analysis of promoters of HSPs. Methods: Heat Shock Element Analysis Tool (HSEAT) has been implemented in java programming language using pattern recognition approach. This tool has build-in MS access database for storing different motifs. Results: HSEAT has been designed to detect different types of Heat Shock Elements (HSEs) in promoter regions of plant HSPs with integration of complete analysis of plant promoters to the tool. HSEAT is user-friendly, interactive application to discover various types of HSEs e.g. TTC Rich Types, Gap Types and Prefect HSE as well as STRE in HSPs. Here we examined and evaluated some known HSP promoters from different plants using this tool with already available tools. Conclusion: HSEAT has extensive potential to explore conserved or semi-conserved motifs or potential binding sites of different transcription factors for other stress regulating genes. This tool can be found at https://sourceforge.net/projects/heast/.

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Transcriptional derepression of the Saccharomyces cerevisiae HSP26 gene during heat shock

Molecular and Cellular Biology ◽

10.1128/mcb.10.12.6362-6373.1990 ◽

1990 ◽

Vol 10 (12) ◽

pp. 6362-6373

Author(s):

R E Susek ◽

S Lindquist

Keyword(s):

Saccharomyces Cerevisiae ◽

Heat Shock ◽

Transcriptional Control ◽

Critical Role ◽

Constitutive Expression ◽

Small Heat Shock Protein ◽

Yeast Cells ◽

Heat Shock Gene ◽

General Mechanism ◽

Regulatory Sequences

hsp26, the small heat shock protein of Saccharomyces cerevisiae, accumulates in response to heat and other types of stress. It also accumulates during the normal course of development, as cells enter stationary phase growth or begin to sporulate (S. Kurtz, J. Rossi, L. Petko, and S. Lindquist, Science 231:1154-1157, 1986). Analysis of deletion and insertion mutations demonstrated that transcriptional control plays a critical role in regulating HSP26 expression. The HSP26 promoter was found to be complex and appears to contain repressing elements as well as activating elements. Several upstream deletion mutations resulted in strong constitutive expression of HSP26. Furthermore, upstream sequences from the HSP26 gene repressed the constitutive expression of a heterologous heat shock gene. We propose that basal repression and heat-induced depression of transcription play major roles in regulating the expression of HSP26. None of the recombinant constructs that we analyzed separated cis-regulatory sequences responsible for heat shock regulation from those responsible for developmental regulation of HSP26. Depression of HSP26 transcription may be the general mechanism of HSP26 induction in yeast cells. This regulatory scheme is very different from that described for the regulation of most other heat shock genes.

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