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HDX-MS finds that partial unfolding with sequential domain activation controls condensation of a cellular stress marker

ABSTRACT: Poly(A)-binding protein (Pab1), a canonical stress granule marker, condenses upon heat shock or starvation, promoting adaptation, The molecular basis of condensation has remained elusive due to a dearth of techniques to probe structure directly in condensates. Here we apply hydrogendeuterium exchange/mass spectrometry (HDX-MS) to investigate the molecular mechanism of Pab1’s condensation. We find that Pab1’s four RNA recognition motifs (RRMs) undergo different levels of partial unfolding upon condensation, and the changes are similar for thermal and pH stresses. Although structural heterogeneity is observed, the ability of MS to describe individual subpopulations allows us to identify which regions become partially unfolded and contribute to the condensate’s interaction network. Our data yield a clear molecular picture of Pab1’s stresstriggered condensation, which we term sequential activation, wherein each RRM becomes activated at a temperature where it partially unfolds and associates with other likewise activated RRMs to form the condensate. This model thus implies that sequential activation is dictated by the underlying free energy surface, an effect we refer to as thermodynamic specificity. Our study represents a methodological advance for elucidating the interactions that drive biomolecular condensation that we anticipate will be widely applicable. Furthermore, our findings demonstrate how condensation can use thermodynamic specificity to perform an acute response to multiple stresses, a potentially general mechanism for stress-responsive proteins.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Escherichia Coli

SUBMITTER: Ruofan Chen

LAB HEAD: Tobin R.

PROVIDER: PXD049463 | Pride | 2024-03-22

REPOSITORIES: Pride

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			Action	DRS
	121020_d05_1uMPab1HeatPelletHX_6000s_2p33MU_0_20201210_141509.raw	Raw
	121020_d08_1uMPab1HeatPelletHX_100s_2p83MU_0_20201210_162555.raw	Raw
	121020_d10_1uMPab1HeatPelletHX_368min_2p33MU_0_20201210_184408.raw	Raw
	121020_d14_1uMPab1HeatPelletHX_1500s_2p33MU_0_20201210_220229.raw	Raw
	121120_d03_1uMPab1pHtPelletHX_100s_2p33MU_pD6_0_20201211_120108.raw	Raw

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Project description:Cellular responses to maladaptive environmental changes—stresses—allow for organismal adaptation to diverse and dynamic conditions. Across the tree of life, cells upregulate a highly conserved transcriptional program in response to so-called proteotoxic stresses such as heat shock. Correspondingly, in eukaryotes, these stresses induce the formation of biomolecular condensates, clusters of mRNA and protein which are referred to as stress granules under severe stress. However, major questions remain about this stress-induced response. How conserved is the condensation response relative to the transcriptional response? How does it vary across environmental niches, and to what extent does it correspond with the conserved transcriptional response? To answer these fundamental questions, we studied the growth, transcriptional, and condensation heat-induced stress responses in three fungal species adapted to thrive in different thermal environments: cryophilic S. kudriavzevii, mesophilic S. cerevisiae, and thermotolerant K. marxianus. Here we show that transcriptional heat shock responses track each species’ evolved temperature range of growth. Further, orthologous proteins—including poly(A)-binding protein, Pab1, a core marker of stress granules—form condensates in vivo at temperatures systematically tuned to the temperature at which the organisms activate the transcriptional heat shock response and slow their growth. In vitro, purified Pab1 from each species condenses autonomously at niche-specific temperatures. Homologous mutations in Pab1 cause similar shifts in relative condensation temperature across species, and crucially, mutations which suppress condensation in vitro also reduce fitness during heat stress. Our findings indicate that stress-induced protein condensation is adaptive, conserved, integrated with the growth and transcriptional responses, and tuned to features of the cellular and organismal environment to initiate at niche-specific levels.

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HDX-MS finds that partial unfolding with sequential domain activation controls condensation of a cellular stress marker

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