Quantification of Hsp90 availability reveals differential coupling to the heat shock response

Alford, Brian; Brandman, Onn

doi:10.1083/jcb.201803127

Cited by 21 publications

(23 citation statements)

References 39 publications

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“…Our results put previous observations from high-throughput studies on connections between Hgh1, Cns1, eEF2, and Hsp90 (Alford and Brandman, 2018;Brandman et al, 2012;McClellan et al, 2007) into context. Interestingly, Hgh1 was also shown to be involved in stress granule formation (Cherkasov et al, 2015), a process that is connected to translation elongation.…”

Section: Discussionsupporting

confidence: 79%

The Co-chaperone Cns1 and the Recruiter Protein Hgh1 Link Hsp90 to Translation Elongation via Chaperoning Elongation Factor 2

et al. 2019

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Graphical AbstractHighlights d Cns1 contains a disordered region, a TPR domain, and a domain with a novel fold d Cns1's essential function is associated with protein translation d Cns1, Hgh1, and Hsp90 form a complex with eEF2d The Cns1 complex including the co-factor Hgh1 chaperones eEF2 SUMMARYThe Hsp90 chaperone machinery in eukaryotes comprises a number of distinct accessory factors. Cns1 is one of the few essential co-chaperones in yeast, but its structure and function remained unknown.Here, we report the X-ray structure of the Cns1 fold and NMR studies on the partly disordered, essential segment of the protein. We demonstrate that Cns1 is important for maintaining translation elongation, specifically chaperoning the elongation factor eEF2. In this context, Cns1 interacts with the novel co-factor Hgh1 and forms a quaternary complex together with eEF2 and Hsp90. The in vivo folding and solubility of eEF2 depend on the presence of these proteins. Chaperoning of eEF2 by Cns1 is essential for yeast viability and requires a defined subset of the Hsp90 machinery as well as the identified eEF2 recruiting factor Hgh1.

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Section: Discussionsupporting

confidence: 79%

The Co-chaperone Cns1 and the Recruiter Protein Hgh1 Link Hsp90 to Translation Elongation via Chaperoning Elongation Factor 2

et al. 2019

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“…Several genes had strong interactions in some stressors but not others. For example, knockdown of the Hsp90 co-chaperone Sti1 (Chang et al, 1997;Nicolet and Craig, 1989) had aggravating interactions specifically with macbecin and canavaine, two stressors which have been demonstrated to reduce Hsp90 availability (Alford and Brandman, 2018) , and unexpectedly, bortezomib ( Figure 5C). Knockdown of GCN3, a gene involved in translation initiation regulation (Yang and Hinnebusch, 1996) , had the strongest aggravating interactions in the screen, hyperinducing the response to hydrogen peroxide, amino acid starvation, and arsenite stress.…”

Section: Reporterseq Reveals Stress-specific Hsr Regulatorsmentioning

confidence: 97%

Genome-wide, time-sensitive interrogation of the heat shock response under diverse stressors via ReporterSeq

Alford

Valiant

Brandman

2020

Preprint

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Interrogating cellular stress response pathways is challenging because of the complexity of regulatory mechanisms and response dynamics, which can vary with both time and the type of stress. We developed a reverse genetic method called ReporterSeq to comprehensively identify genes regulating a stress-induced transcription factor under multiple conditions in a time-resolved manner. ReporterSeq links RNA-encoded barcode levels to pathway-specific output under genetic perturbations, allowing pooled pathway activity measurements via DNA sequencing alone and without cell enrichment or single cell isolation. Here, we used ReporterSeq to identify regulators of the heat shock response (HSR), a conserved, poorly understood transcriptional program that protects cells from proteotoxicity and is misregulated in disease. We measured genome-wide HSR regulation in budding yeast across thirteen stress conditions, uncovering novel stress-specific, time-specific, and constitutive regulators. ReporterSeq can assess the genetic regulators of any transcriptional pathway with the scale of pooled genetic screens and the precision of pathway-specific readouts.

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“…Indeed, it has been previously reported that AZC can cause aggregation of endogenous proteins (Weids and Grant 2014), and that aggregation can be a mechanism for avoiding degradation (Wallace et al 2015). Because canavanine and AZC robustly activate the HSR, a response that is driven by a drop in protein chaperone availability (Zheng et al 2016;Alford and Brandman 2018), we reasoned that chaperones become limiting under folding stressors and this may drive protein aggregation. To test this, we boosted Hsf1 activity to increase chaperone levels and determined if this would increase the stability of our degron reporters during canavanine and AZC treatment.…”

Section: Folding Stressors Cause Aggregation and Results In Failure Tomentioning

confidence: 97%

“…an indirect proteasomal stress), transcriptional activation of RPN4, or both. Indeed, canavanine and AZC robustly activate Hsf1, a transcriptional activator of RPN4 (Hahn, Neef, and Thiele 2006;Yamamoto et al 2008;Alford and Brandman 2018). We therefore investigated the role of transcriptional regulation of RPN4 in activating the PSR during stressor treatment.…”

Section: Folding Stressors Activate the Psr Exclusively Via Transcripmentioning

confidence: 99%

Quantitative analysis of the ubiquitin-proteasome system under proteolytic and folding stressors

Jj¹,

Brandman²

2019

Preprint

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Aging, disease, and environmental stress are associated with failures in the ubiquitinproteasome system (UPS), yet a quantitative understanding of how the UPS responds to stress conditions is lacking. Here we assessed the performance and adaptability of the UPS in yeast under stress conditions using quantitative measurements of misfolded substrate stability and stress-dependent UPS regulation by the transcription factor Rpn4. We found that impairing degradation rates ("proteasomal stress") and generating misfolded proteins ("folding stress") stabilized UPS substrates through separate, non-overlapping mechanisms. Folding stress stabilized proteins by causing aggregation rather than targeting to the proteasome and increasing overall proteasomal load. Despite differences in underlying proteostasis defects, the UPS productively adapted to both proteasomal and folding stressors using separate mechanisms of Rpn4 activation, in some cases resulting in no loss in UPS substrate degradation. Our work reveals the distinct effects of proteotoxic stressors and the versatility of cells in productively adapting the UPS. ____________________________________________________________________________

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Quantification of Hsp90 availability reveals differential coupling to the heat shock response

Cited by 21 publications

References 39 publications

The Co-chaperone Cns1 and the Recruiter Protein Hgh1 Link Hsp90 to Translation Elongation via Chaperoning Elongation Factor 2

The Co-chaperone Cns1 and the Recruiter Protein Hgh1 Link Hsp90 to Translation Elongation via Chaperoning Elongation Factor 2

Genome-wide, time-sensitive interrogation of the heat shock response under diverse stressors via ReporterSeq

Quantitative analysis of the ubiquitin-proteasome system under proteolytic and folding stressors

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