Liquid–liquid phase separation (LLPS) of proteins containing intrinsically disordered regions (IDRs) has been proposed as a mechanism underlying the formation of membrane-less organelles. Tight regulation of IDR behavior is essential to ensure that LLPS only takes place when necessary. Here, we report that IDR acetylation/deacetylation regulates LLPS and assembly of stress granules (SGs), membrane-less organelles forming in response to stress. Acetylome analysis revealed that the RNA helicase DDX3X, an important component of SGs, is a novel substrate of the deacetylase HDAC6. The N-terminal IDR of DDX3X (IDR1) can undergo LLPS in vitro, and its acetylation at multiple lysine residues impairs the formation of liquid droplets. We also demonstrated that enhanced LLPS propensity through deacetylation of DDX3X-IDR1 by HDAC6 is necessary for SG maturation, but not initiation. Our analysis provides a mechanistic framework to understand how acetylation and deacetylation of IDRs regulate LLPS spatiotemporally, and impact membrane-less organelle formation in vivo.
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We are grateful to T. Hyman for use of the microscope with thermal stage on short notice and for comments on the manuscript, and R. Voit (German Cancer Research Center, Heidelberg) for HAT expression vectors. We thank L. Gelman and S. Bourke for help with microscopic analysis, H. Kohler for FACS analysis, J. Seebacher and V. Iesmantavicius for interpretation of mass spectrometry data, H. Gut for help with structure predictions, M.B. Stadler for acetylome-wide IDR analysis, J. Wilbertz for help with live-cell imaging, L. Giorgetti and Y. Zhan for help with mathematical modeling, W. Filipowicz and J. Chao for critical comments on the manuscript. We thank C. Schölz for valuable suggestions. We also thank L. Wang for advice on protein purification, G. Matthias and C. Cao for their helpful technical assistance, Y. Miyake for providing us with biological materials for experiments, R. Clerc for critical comments on the manuscript, and all the Matthias laboratory members for fruitful discussions. M. Saito is supported in part by a fellowship from the Nakajima Foundation. A.W. Fritsch is supported by the ELBE postdoctoral fellows program. The Novo Nordisk Foundation Center for Protein Research is supported financially by the Novo Nordisk Foundation (Grant agreement NNF14CC0001). This work was supported by the Novartis Research Foundation.
Supplementary Figures 1–27
Total DDX3X SG volume (related to Fig. 5)
DDX3X-interactome (related to Fig. 6)
Raw data used for mathematical modeling of SG growth (related to Fig. 6)
Fusion behavior of DDX3X-IDR1 droplet (related to Fig. 3)
DDX3X-IDR1 droplet disappearance by LLPS following a temperature increase (related to Fig. 3)
Liquid-like properties of mCherry-DDX3X SGs (related to Fig. 4)
SG formation of WT DDX3X and its mutants (related to Fig. 6)
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Nature Chemical Biology (2019)