Ubiquitin Modulates Liquid-Liquid Phase Separation of UBQLN2 via Disruption of Multivalent Interactions - PubMed (original) (raw)

Ubiquitin Modulates Liquid-Liquid Phase Separation of UBQLN2 via Disruption of Multivalent Interactions

Thuy P Dao et al. Mol Cell. 2018.

Abstract

Under stress, certain eukaryotic proteins and RNA assemble to form membraneless organelles known as stress granules. The most well-studied stress granule components are RNA-binding proteins that undergo liquid-liquid phase separation (LLPS) into protein-rich droplets mediated by intrinsically disordered low-complexity domains (LCDs). Here we show that stress granules include proteasomal shuttle factor UBQLN2, an LCD-containing protein structurally and functionally distinct from RNA-binding proteins. In vitro, UBQLN2 exhibits LLPS at physiological conditions. Deletion studies correlate oligomerization with UBQLN2's ability to phase-separate and form stress-induced cytoplasmic puncta in cells. Using nuclear magnetic resonance (NMR) spectroscopy, we mapped weak, multivalent interactions that promote UBQLN2 oligomerization and LLPS. Ubiquitin or polyubiquitin binding, obligatory for UBQLN2's biological functions, eliminates UBQLN2 LLPS, thus serving as a switch between droplet and disperse phases. We postulate that UBQLN2 LLPS enables its recruitment to stress granules, where its interactions with ubiquitinated substrates reverse LLPS to enable shuttling of clients out of stress granules.

Keywords: LLPS; NMR spectroscopy; Ubiquilin-2; amyotrophic lateral sclerosis; ligand-induced phase transition; liquid-liquid phase separation; multivalent interactions; protein quality control; stress granules; ubiquitin.

Copyright © 2018 Elsevier Inc. All rights reserved.

PubMed Disclaimer

Figures

Figure 1.

Figure 1.. UBQLN2 is recruited to SGs and undergoes LLPS.

(A) Immunostaining for endogenous UBQLN2 in U2OS cells shows that UBQLN2 is diffuse in cytoplasm, but form puncta under four stress conditions tested. UBQLN2 colocalizes with eIF4G1, a SG marker. DAPI is used to stain nuclei. (B) Quantitation of UBQLN2 colocalization in SGs with error bars reflecting standard deviation from data in triplicate. (C) DIC and fluorescence microscopy shows 50 μM protein in 20 mM NaPhosphate, 200 mM NaCl, pH 6.8 phase separating into micron-sized droplets at 30oC but not at 16oC. (D) DIC microscopy shows UBQLN2 LLPS at physiological protein concentrations at 37oC in pH 7.4 buffer consisting of 150 mM KCl, 20 mM NaPhosphate, 1 mM DTT and 150 mg/mL Ficoll. (E) UBQLN2 LLPS is observed by measuring A600 as a function of temperature. (F) FRAP of UBQLN2 droplets. (Upper) Fluorescence images of partial droplet photobleaching experiments. (Lower) Black curve is an average of FRAP recovery curves from 6 separate droplets. Error bars represent the standard deviation. Green curve is a single exponential fit to the data. See also Figure S2.

Figure 2.

Figure 2.. UBLQN2 LLPS is modulated by its different domains, oligomerization states, temperature, protein and salt concentrations.

(A) Domain architecture of UBQLN2. The proline-rich repeat (Pxx) region of UBQLN2 harbors most familial ALS mutations (red). (B) Turbidity assays as a function of temperature comparing LLPS of different UBQLN2 constructs using 50 µM protein in 20 mM NaPhosphate, 200 mM NaCl, pH 6.8. The last two assays monitored the turbidity of solution consisting either 50 or 250 µM of the UBL domain (1–107) and 50 µM of UBQLN2 379–624. (C) DIC microscopy shows solutions of 50 μM constructs in 20 mM NaPhosphate, 200 mM NaCl, pH 6.8 after incubation at 37oC for 10 minutes. *ΔUBA microscopy was obtained at 500 mM NaCl since no droplets were observed at 200 mM NaCl. (D,E) HeLa cells were transfected with mCherry or mCherry-tagged UBQLN2 as indicated. Twenty-four hours post-transfection, cells were stimulated with 0.5 mM sodium arsenite for 30 min, and immunostained with anti-eIF4G and DAPI. Arrows indicate UBQLN2-positive puncta. Cells at pre- (D) and 30 min post-arsenite treatment (E) are shown. Scale bar: 10 µm. (F) Western blot analysis of mCherry-tagged UBQLN2 constructs shows comparable expression. Actin was blotted as a loading control. (G) Quantification of (D) and (E). The percentage of transfected cells with UBQLN2-positive puncta is plotted. ***p<0.001 two- way ANOVA, Sidak’s multiple comparisons test, n=3 biological repeats. Error bars reflect standard deviation. (H) SEC of UBQLN2 Δ379–486 (blue), 379–624 (orange), 450–624 (black) and 487–624 (red) at 10 µM (thinnest line), 100 µM (medium-thick), and 500 µM (thickest) protein concentrations. For UBQLN2 Δ379–486 construct, highest concentration used was 200µM. See also Figures S1, S2, S3 and S4.

Figure 3.

Figure 3.. NMR spectra for UBQLN2 450–624.

(A) 1H-15N TROSY-HSQC and (B) 15N-13CO (HACA)CON spectra of UBQLN2 450–624 at 10oC, pH 6.8, 20 mM NaPhosphate. (C) Residue-level secondary structure determination using Cα and Cβ secondary chemical shifts at 10oC. (D) Secondary structure prediction from δ2D calculations using all backbone chemical shift data. See also Figure S3 and S5.

Figure 4.

Figure 4.. NMR identifies putative multivalent interactions in UBQLN2 450–624.

(A) Comparison of 1H-15N TROSY-HSQC spectra of 200 µM UBQLN2 450–624 and UBQLN2 487–624. Significant broadening was observed for residues identified in domain map above spectrum. (B) 15N R1 and R2 relaxation rates, and {1H}−15N hetNOE values for UBQLN2 450–624. Error bars represent standard errors of the experimental values. (C) Comparison of 1H-15N TROSY-HSQC spectra of UBQLN2 450–624 at 45 µM (green) and 600 µM (blue) protein concentrations. Contours are identical although 45 µM sample was collected with 4x scans as 600 µM sample. (D) CSPs represent residue-specific chemical shift differences between low (45µM) and high (600 µM) protein concentrations. Green bars mark resonances only visible at 45µM. Domain map marks residues that exhibit concentration-dependent peak broadening or significant CSPs. All spectra were collected at 25oC in pH 6.8 buffer under non-LLPS conditions. (E) UBQLN2 sequence whose amide resonances (red) exhibit elevated 15N R2 rates or concentration-dependent broadening or CSPs. See also Figure S6.

Figure 5.

Figure 5.. Ubiquitin binding eliminates UBQLN2 LLPS.

(A) DIC and fluorescence images showing disassembly of UBQLN2-protein droplets after Ub addition. (B) Turbidity assay as a function of temperature for mixtures containing both Ub and UBQLN2 in different molar ratios. For assays containing FL UBQLN2, solution consisted of 50 µM protein and 0–50 µM Ub (or diUb, tetraUb, L8AI44A Ub variant) in pH 6.8 buffer with 300 mM NaCl. For assay containing UBQLN2 ΔUBA, solution consisted of 75 µM protein and 0 or 75 µM Ub in a pH 6.8 buffer with 500 mM NaCl.

Figure 6.

Figure 6.. Ub binds specifically to UBQLN2 UBA.

(A) CSPs for residues in UBQLN2 450–624 at the titration endpoint with Ub (black) or L8AI44A Ub (red). Inset contains sample titration curves; line represents fit to single-site binding model. (B) CSPs were mapped onto UBA (gray, residues with CSPs ≥ 0.1 ppm in red, and Ub (yellow) using PDB 2JY6. (C) 15N R1 and R2 relaxation rates for UBQLN2 450–624 in the absence (black) and presence of Ub (blue). (D) Peak intensities decreased after salt-induced LLPS (black). Peak intensities are partially recovered upon Ub-mediated elimination of UBQLN2 LLPS (blue). (E) Peak intensities increase across UBQLN2 after Ub was added except for the UBA, where Ub binds. See also Figure S7.

Figure 7.

Figure 7.. Model for Ub-mediated elimination of UBQLN2 LLPS.

UBQLN2 undergoes LLPS under physiological conditions. Ub binding disrupts LLPS by interfering with multivalent interactions (dotted lines) involving the UBA domain. Inside cells, UBQLN2 LLPS promotes colocalization with SGs, whereby interaction with Ub or ubiquitinated substrates reverses LLPS and may shuttle clients out of stress granules.

Comment in

References

    1. Aumiller WM, Pir Cakmak F, Davis BW, and Keating CD (2016). RNA-Based Coacervates as a Model for Membraneless Organelles: Formation, Properties, and Interfacial Liposome Assembly. Langmuir ACS J. Surf. Colloids 32, 10042–10053. - PubMed
    1. Banani SF, Rice AM, Peeples WB, Lin Y, Jain S, Parker R, and Rosen MK (2016). Compositional Control of Phase-Separated Cellular Bodies. Cell 166, 651–663. - PMC - PubMed
    1. Bastidas M, Gibbs EB, Sahu D, and Showalter SA (2015). A primer for carbon-detected NMR applications to intrinsically disordered proteins in solution. Concepts Magn. Reson Part A 44, 54–66.
    1. Beal R, Deveraux Q, Xia G, Rechsteiner M, and Pickart C (1996). Surface hydrophobic residues of multiubiquitin chains essential for proteolytic targeting. Proc. Natl. Acad. Sci. U. S. A 93, 861–866. - PMC - PubMed
    1. Brangwynne CP, Eckmann CR, Courson DS, Rybarska A, Hoege C, Gharakhani J, Jülicher F, and Hyman AA (2009). Germline P granules are liquid droplets that localize by controlled dissolution/condensation. Science 324, 1729–1732. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources