# Investigating the disaggregation of stress-induced phase-separated poly(A)-binding protein (Pab1) by its cognate disaggregation system

> **NIH NIH F31** · UNIVERSITY OF CHICAGO · 2020 · $45,520

## Abstract

Project Summary/Abstract
Understanding how cells sense and respond to stressful environmental changes is a major goal in cell biology.
The two representative features of the cellular stress response that are conserved in all eukaryotes are 1)
upregulation of molecular chaperones and 2) aggregation of RNA and protein into stress granules. These
clusters had long been interpreted to be aggregates of misfolded proteins. However, we recently demonstrated
(Riback et al., 2017) that poly(A)-binding protein (Pab1), a universally conserved stress-granule marker, forms
clusters by phase separating without misfolding, and that disrupting phase separation impairs cellular growth
during stress, indicating that stress-triggered aggregation of Pab1 is a part of the adaptive stress response. We
refer to such fitness-promoting stress-triggered aggregates as adaptive protein assemblies.
The realization that stress triggers formation of two distinct class of aggregates, adaptive protein assemblies
and misfolded protein aggregates, motivated us to investigate whether and how molecular chaperones can
distinguish these different substrates. Pab1 phase-separates into a hydrogel under physiologically stressful
conditions. To investigate how Pab1 hydrogels are disaggregated by molecular chaperones—specifically the
stress-induced Hsp104 disaggregation system—we developed a fluorescence anisotropy assay that permits
both quantitative and kinetic measurement of Pab1 dispersal. This is the first in vitro system for studying
disaggregation of adaptive protein assemblies. The yeast Hsp104 consists of three molecular chaperones,
Hsp104/70/40. We will ask how Pab1 disaggregation differs from disaggregation of misfolded aggregates. Our
preliminary data show that disaggregation of Pab1 hydrogel is much faster than disaggregation of the model
substrate firefly luciferase. We will also ask what molecular features of Pab1 are recognized by molecular
chaperones. Hsp70 preferentially binds unfolded proteins with an extended hydrophobic sequence, and
hydrophobic residues in the proline-rich low-complexity domain of Pab1 modulate phase-separation of Pab1.
We thus hypothesize that features which alter phase-separation of Pab1 will also affect disaggregation. We will
test this hypothesis using our recently reported Pab1 mutants with altered phase-separation behavior. Finally,
we have a strong prediction based on both literature and preliminary data that phosphorylation of Hsp70/Ssa1
at T36 will increase the efficiency of the Hsp104 disaggregation system toward Pab1 hydrogels. We will test
this prediction using the fluorescence anisotropy assay. Additionally, we employ an evolutionary analysis
approach to identify other regulatory phosphorylation sites; we will test our findings biochemically using the
fluorescence anisotropy assay. The outcome of this research will advance our understanding of how the
Hsp104 disaggregation system disperses different stress-induced structures.

## Key facts

- **NIH application ID:** 9967802
- **Project number:** 5F31ES030697-02
- **Recipient organization:** UNIVERSITY OF CHICAGO
- **Principal Investigator:** Haneul Yoo
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $45,520
- **Award type:** 5
- **Project period:** 2019-07-01 → 2021-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9967802

## Citation

> US National Institutes of Health, RePORTER application 9967802, Investigating the disaggregation of stress-induced phase-separated poly(A)-binding protein (Pab1) by its cognate disaggregation system (5F31ES030697-02). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9967802. Licensed CC0.

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