296 | Nature | Vol 578 | 13 February 2020
Article
Stress- and ubiquitylation-dependent phase
separation of the proteasome
Sayaka Yasuda1,8, Hikaru Tsuchiya1,8, Ai Kaiho1,8, Qiang Guo^2 , Ken Ikeuchi3,4, Akinori Endo^1 ,
Naoko Arai^1 , Fumiaki Ohtake^1 , Shigeo Murata^5 , Toshifumi Inada^3 , Wolfgang Baumeister^2 ,
Rubén Fernández-Busnadiego2,6,7, Keiji Tanaka^1 * & Yasushi Saeki^1 *The proteasome is a major proteolytic machine that regulates cellular proteostasis
through selective degradation of ubiquitylated proteins^1 ,^2. A number of ubiquitin-
related molecules have recently been found to be involved in the regulation of
biomolecular condensates or membraneless organelles, which arise by liquid–liquid
phase separation of specific biomolecules, including stress granules, nuclear speckles
and autophagosomes^3 –^8 , but it remains unclear whether the proteasome also
participates in such regulation. Here we reveal that proteasome-containing nuclear
foci form under acute hyperosmotic stress. These foci are transient structures that
contain ubiquitylated proteins, p97 (also known as valosin-containing protein (VCP))
and multiple proteasome-interacting proteins, which collectively constitute a
proteolytic centre. The major substrates for degradation by these foci were ribosomal
proteins that failed to properly assemble. Notably, the proteasome foci exhibited
properties of liquid droplets. RAD23B, a substrate-shuttling factor for the
proteasome, and ubiquitylated proteins were necessary for formation of proteasome
foci. In mechanistic terms, a liquid–liquid phase separation was triggered by
multivalent interactions of two ubiquitin-associated domains of RAD23B and
ubiquitin chains consisting of four or more ubiquitin molecules. Collectively, our
results suggest that ubiquitin-chain-dependent phase separation induces the
formation of a nuclear proteolytic compartment that promotes proteasomal
degradation.To enable visualization of proteasomes in live cells, we generated
derivatives of the HCT116 colon cancer cell line in which endogenous
proteasome subunits, the core particle subunit proteasome subunit
β type-2 (PSMB2, also known as β4), and regulatory particle subunit
26S proteasome non-ATPase regulatory subunit 6 (PSMD6, also known
as RPN7), were labelled with an eGFP or FusionRed fluorescent tag
(Extended Data Fig. 1). Consistent with previous studies^9 ,^10 , the protea-
somes were primarily observed in the nucleoplasm and cytoplasm of
highly proliferating cells. Over the course of a series of experiments, we
unexpectedly observed that under hyperosmotic stress, proteasomes
rapidly formed multiple foci in the nucleus (Fig. 1a, Supplementary
Video 1). We confirmed that foci formed in wild-type (WT) HCT116
cells using an endogenous antibody against the proteasome (Fig. 1a,
Extended Data Fig. 2a). Various osmolytes, sucrose, glucose and NaCl
stimulated foci formation, and the osmolarity required to induce
the response was 100 mOsmol l−1, close to the value observed during
physiological changes associated with type II diabetes^11 (Extended
Data Fig. 2b). PSMD6–eGFP also formed foci, and the foci were stained
with an activity-based probe, suggesting the involvement of active 26S
proteasomes (Fig. 1a, Extended Data Fig. 2c). Consistently, a snapshot
obtained by cryo-electron tomography revealed the clustering of
26S proteasomes in the nucleus upon osmotic stimulation (Fig. 1b,
Extended Data Fig. 2d). We also observed these foci in immortalized
retinal pigment epithelial (RPE-1) cells and mouse embryonic stem
(ES) cells, suggesting that hyperosmotic-stress-induced formation of
proteasome foci is a universal phenomenon (Extended Data Fig. 2e).Proteasome foci are sites of proteolysis
Several nuclear bodies are related to the proteasome, such as promyelo-
cytic leukemia protein (PML) nuclear bodies and Cajal bodies^12 –^14 , but these
bodies did not colocalize with proteasome foci (Extended Data Fig. 3a, b).
Instead, proteasome foci colocalized almost completely with lysine 48
(K48)-linked ubiquitin chains, a major proteolytic signal for the protea-
some, but not with non-proteolytic K63-linked chains (Fig. 1c, Extended
Data Fig. 3c). To obtain further functional insights, we performed time-
lapse imaging of PSMB2–eGFP cells (Fig. 1d). Five minutes after the addi-
tion of 0.2 M sucrose, the number of foci increased to a maximum of around
30 per nucleus, and the diameter of the foci increased to approximately
500 nm within 30 min. Subsequently, the foci gradually disappeared overhttps://doi.org/10.1038/s41586-020-1982-9
Received: 23 September 2018
Accepted: 9 December 2019
Published online: 5 February 2020
(^1) Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan. (^2) Department of Molecular Structural Biology, Max Planck Institute of Biochemistry,
Martinsried, Germany.^3 Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan.^4 Gene Center and Center for Integrated Protein Science Munich, Department of
Biochemistry, University of Munich, Munich, Germany.^5 Laboratory of Protein Metabolism, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan.^6 Institute of
Neuropathology, University Medical Center Göttingen, Göttingen, Germany.^7 Cluster of Excellence ‘Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells’ (MBExC),
University of Göttingen, Göttingen, Germany.^8 These authors contributed equally: Sayaka Yasuda, Hikaru Tsuchiya, Ai Kaiho. *e-mail: [email protected]; [email protected]