Hsf1 and Hsp70 constitute a two-component feedback loop that regulates the yeast heat shock response
Abstract
Models for regulation of the eukaryotic heat shock response typically invoke a negative feedback loop consisting of the transcriptional activator Hsf1 and a molecular chaperone. Previously we identified Hsp70 as the chaperone responsible for Hsf1 repression and constructed a mathematical model that recapitulated the yeast heat shock response (Zheng et al., 2016). The model was based on two assumptions: dissociation of Hsp70 activates Hsf1, and transcriptional induction of Hsp70 deactivates Hsf1. Here we validate these assumptions. First, we severed the feedback loop by uncoupling Hsp70 expression from Hsf1 regulation. As predicted by the model, Hsf1 was unable to efficiently deactivate in the absence of Hsp70 transcriptional induction. Next, we mapped a discrete Hsp70 binding site on Hsf1 to a C-terminal segment known as conserved element 2 (CE2). In vitro, CE2 binds to Hsp70 with low affinity (9 µM), in agreement with model requirements. In cells, removal of CE2 resulted in increased basal Hsf1 activity and delayed deactivation during heat shock, while tandem repeats of CE2 sped up Hsf1 deactivation. Finally, we uncovered a role for the N-terminal domain of Hsf1 in negatively regulating DNA binding. These results reveal the quantitative control mechanisms underlying the heat shock response.
Article and author information
Author details
Funding
National Institutes of Health (DP5 OD017941-01)
- David Pincus
National Science Foundation (MCB-1350949)
- Ahmad S Khalil
National Science Foundation (MCB-1518345)
- David S Gross
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2018, Krakowiak et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 5,360
- views
-
- 738
- downloads
-
- 117
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Cell Biology
- Computational and Systems Biology
Heat shock factor (Hsf1) regulates the expression of molecular chaperones to maintain protein homeostasis. Despite its central role in stress resistance, disease and aging, the mechanisms that control Hsf1 activity remain unresolved. Here we show that in budding yeast, Hsf1 basally associates with the chaperone Hsp70 and this association is transiently disrupted by heat shock, providing the first evidence that a chaperone repressor directly regulates Hsf1 activity. We develop and experimentally validate a mathematical model of Hsf1 activation by heat shock in which unfolded proteins compete with Hsf1 for binding to Hsp70. Surprisingly, we find that Hsf1 phosphorylation, previously thought to be required for activation, in fact only positively tunes Hsf1 and does so without affecting Hsp70 binding. Our work reveals two uncoupled forms of regulation - an ON/OFF chaperone switch and a tunable phosphorylation gain - that allow Hsf1 to flexibly integrate signals from the proteostasis network and cell signaling pathways.
-
- Biochemistry and Chemical Biology
- Microbiology and Infectious Disease
Birnaviruses are a group of double-stranded RNA (dsRNA) viruses infecting birds, fish, and insects. Early endosomes (EE) constitute the platform for viral replication. Here, we study the mechanism of birnaviral targeting of EE membranes. Using the Infectious Bursal Disease Virus (IBDV) as a model, we validate that the viral protein 3 (VP3) binds to phosphatidylinositol-3-phosphate (PI3P) present in EE membranes. We identify the domain of VP3 involved in PI3P-binding, named P2 and localized in the core of VP3, and establish the critical role of the arginine at position 200 (R200), conserved among all known birnaviruses. Mutating R200 abolishes viral replication. Moreover, we propose a two-stage modular mechanism for VP3 association with EE. Firstly, the carboxy-terminal region of VP3 adsorbs on the membrane, and then the VP3 core reinforces the membrane engagement by specifically binding PI3P through its P2 domain, additionally promoting PI3P accumulation.