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Nuclear and cytoplasmic spatial protein quality control is coordinated by nuclear–vacuolar junctions and perinuclear ESCRT

Nature Cell Biology volume 25, pages 699–713 (2023)Cite this article

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Abstract

Effective protein quality control (PQC), essential for cellular health, relies on spatial sequestration of misfolded proteins into defined inclusions. Here we reveal the coordination of nuclear and cytoplasmic spatial PQC. Cytoplasmic misfolded proteins concentrate in a cytoplasmic juxtanuclear quality control compartment, while nuclear misfolded proteins sequester into an intranuclear quality control compartment (INQ). Particle tracking reveals that INQ and the juxtanuclear quality control compartment converge to face each other across the nuclear envelope at a site proximal to the nuclear–vacuolar junction marked by perinuclear ESCRT-II/III protein Chm7. Strikingly, convergence at nuclear–vacuolar junction contacts facilitates VPS4-dependent vacuolar clearance of misfolded cytoplasmic and nuclear proteins, the latter entailing extrusion of nuclear INQ into the vacuole. Finding that nuclear–vacuolar contact sites are cellular hubs of spatial PQC to facilitate vacuolar clearance of nuclear and cytoplasmic inclusions highlights the role of cellular architecture in proteostasis maintenance.

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Fig. 1: The INQ and JUNQ are separate nuclear and cytoplasmic PQC compartments.
Fig. 2: Nuclear entry of misfolded proteins is not required for clearance.
Fig. 3: INQ and JUNQ home to similar location on each side of the nuclear envelope via a cytoplasmic signal linked to nuclear pores.
Fig. 4: INQ resides near the nucleolus, JUNQ is surrounded by mitochondria and both compartments home into the NVJ.
Fig. 5: JUNQ and INQ converge at the NVJ to facilitate clearance.
Fig. 6: ESCRT-mediated extrusion from the nucleus and clearance.
Fig. 7: Vacuolar clearance of the INQ and JUNQ.
Fig. 8: Nuclear and cytoplasmic spatial PQC.

Data availability

Source data are provided with this paper. All other data that support the findings of this study are available from J.F. upon reasonable request.

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Acknowledgements

Funded by NIH (GM05643319 and AG054407 to J.F.; F32NS086253 to E.M.S.); Way Klingler Startup Funds from Marquette University (E.M.S.); The Pew Trusts postdoctoral Award 00034104 to F.M.-P. and Gordon and Betty Moore Foundation Award #3497 to C.L. and M.A.L.G. Cryo-SXT data were acquired at National Center for X-ray Tomography (NIH P41GM103445and DOE DE-AC02-5CH11231). We thank J. Mulholland and Y. Lim from the CSIF for training on the SIM and M. Rosbash (Brandeis University), S. Wente (Vanderbilt University), K. Madura (Rutgers University) and P. Lusk (Yale School of Medicine) for yeast strains and K. Weis (ETH Zurich), J. Nunnari (University of California, Davis) and M. P. Rout (Rockefeller University) for plasmids. We are grateful to C. Trail for support in microscopy data analysis and M. Wangeline (Stanford University) for assisting with the 2xKeima cloning. We thank K. Ullman (University of Utah), A. Frost (Altos Lab), J. Steffan (UC Irvine) and L. Veenhoff (University of Groningen) for discussions and advice, F. Serrano for assisting on model figure and the Frydman lab for advice and discussions.

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Author notes

  1. Emily M. Sontag

    Present address: Department of Biological Sciences, Marquette University, Milwaukee, WI, USA

  2. Patrick T. Dolan

    Present address: Quantitative Virology and Evolution Unit, National Institute of Allergy and Infectious Diseases, Bethesda, MD, USA

  3. These authors contributed equally: Emily M. Sontag, Fabián Morales-Polanco.

Authors and Affiliations

  1. Department of Biology, Stanford University, Stanford, CA, USA

    Emily M. Sontag, Fabián Morales-Polanco, Patrick T. Dolan, Daniel Gestaut & Judith Frydman

  2. Department of Anatomy, School of Medicine, University of California San Francisco, San Francisco, CA, USA

    Jian-Hua Chen, Gerry McDermott, Mark A. Le Gros & Carolyn Larabell

  3. Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    Mark A. Le Gros & Carolyn Larabell

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Contributions

E.M.S., F.M.-P. and J.F. designed all experiments. E.M.S. and F.M.-P. carried out all experiments. J.-H.C. collected and processed cryo-SXT data; C.L. assisted with planning and execution of cryo-SXT experiments; G.M. carried out cryo-SXT data analysis and modelling; M.A.L.G. performed cryo-fluorescence data acquisition and correlation with cryo-SXT data and built the microscope used for these experiments. P.T.D. analysed particle tracking data and assisted on statistical analyses. D.G. cloned the NLS- and NES-luciferase and VHL plasmids. F.M.-P. and D.G. generated, purified and labelled the GFP and RFP nanobodies. E.M.S., F.M.-P. and J.F. wrote the manuscript. All authors commented on the final version. J.F. and E.M.S. conceived the project; J.F. directed the project.

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Correspondence to Emily M. Sontag or Judith Frydman.

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Extended data

Extended Data Fig. 1 Spatial sequestration occurs during different types of stress with different client proteins.

(a) Western blot analyses of Gal Shut-off assays showing the clearance of NLS-LuciTs (top) and NES-LuciTs (bottom) with and without proteasome impairment by 50μM Bortezomib. Blot is representative of 3 biologically independent experiments. (b-c) Representative Structured Illumination super-resolution microscopy images taken of cells expressing NLS-VHL (b) or NES-VHL (c) after 120 minutes at 37 °C and treated with 100μM MG132. NLS-LuciTs is shown in green, NES-LuciTs in purple, nuclear pores in gold and Hoechst counterstain in blue. Scale bars are 1μm. (d) Drop test of W303 yeast expressing model proteins without heat shock at 30C (left), with heat shock at 37 °C (middle), and without expression of the plasmids (right). Unprocessed blots are available in source data.

Source data

Extended Data Fig. 2 The effect of blocking nucleocytoplasmic transport on Ubc9Ts clearance.

(a) Quantitation of the percentage of cells containing nuclear or cytoplasmic inclusions in WT yeast expressing Ubc9Ts-EGFP after 120 minutes at 37 °C with and without treatment with 100μM MG132. A minimum of 500 cells per condition from 3 biologically independent experiments were counted and two-tailed Student’s t-tests were performed comparing the WT yeast without MG132 treatment to WT yeast with MG132 treatment using Prism software. P values were adjusted using two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli with a Q of 5%. Adjusted P value for nuclear no MG132 vs. +MG132 is 0.0035 and cytoplasmic no MG132 vs. +MG132 is 0.0011. Data are shown as mean values ± S.E.M. (b) Representative Structured Illumination super-resolution microscopy images taken of cells expressing EGFP-VHL after 2 hr at 37 °C with DMSO (left) or with 100μM MG132 (right) treatment. VHL is shown in green, nuclear pores in gold, and Hoechst counterstain in blue. Scale bars are 1μm. Numerical source data are available in source data.

Source data

Extended Data Fig. 3 INQ-JUNQ homing does not occur at the LINC, nucleolus, or involve FG repeats of the nuclear pore central channel.

(a) Graph of the X-Y positions of the INQ and JUNQ compartments by particle tracking of inclusions from cell shown in Figure 2a over the time course of the experiment. (b) Representative confocal image taken of cells co-expressing NLS-EGFP-VHL and NES-DsRed-VHL after 2 hr at 37 °C and treated with 100μM MG132. NLS-fusion proteins are shown in green, NES-fusion proteins in purple, nuclear pores in gold, and Hoechst counterstain in blue. Scale bar is 1μm. (c) Representative confocal fluorescence microscopy images taken of cells co-expressing NLS-EGFP-LuciTs and NES-DsRed-LuciTs (left) after 2 hr at 37 °C and treated with 100μM MG132. NLS-LuciTs is shown in green, NES-LuciTs in purple, nucleolus (Nsr1) in gold and Hoechst counterstain in blue. (right) Line intensity profile showing distance between nucleolus and homed INQ/JUNQ. Scale bars are 1μm. (d) (left) schematic of Mps3 component of LINC complex linking inner and outer nuclear membranes. (right) Representative widefield fluorescence microscopy images taken of cells co-expressing endogenously tagged Mps3-EGFP and NES-DsRed-LuciTs after 120 minutes at 37 °C with and without treatment with 100μM MG132. White arrowheads indicate locations of Mps3 puncta while yellow arrowheads indicate NES-LuciTs puncta. Scale bars are 1μm. (e) WT (top) and nupΔFG (bottom) cells co-expressing NLS-LuciTs and NES-LuciTs were shifted to 37 °C and monitored by live cell time-lapse fluorescence microscopy for the times shown. Scale bars are 1μm.

Extended Data Fig. 4 Detailed representation of the cryo-SXT workflow and interactions between mitochondria and cytoplasmic PQC compartments.

(a) Optical path through the specimen. Key: COL, cryogenic objective lens; SS, specimen stage; SP, specimen port; MG, motorized goniometer; CIM, cryogenic immersion fluid; CCL, low magnification cryogenic objective; CS, cryogenic specimen; CIE, cryogenic imaging environment; AP, adapter port; AW, a heated, angled anti-reflection window. (b) Alignment of fluorescence and soft x-ray tomographic data using fiducial markers. (c) A representative confocal image of the spatial relationship between the INQ and nucleolus. NLS-LuciTs (INQ) is shown in green, nucleolus in gold, and Hoechst counterstain in blue. Scale bar is 1μm. (d) The interaction between mitochondria and cytoplasmic inclusions is also seen by fluorescence confocal microscopy in a representative image of a cell co-expressing mito-GFP and NES-RFP-LuciTs. NES-LuciTs is shown in purple, mitochondria in cyan, and Hoechst counterstain in blue. Scale bar is 1μm. (e) Representative confocal fluorescence microscopy images taken of WT, fission mutants (dnm1Δ and fis1Δ) and fusion mutant (fzo1Δ and ugo1Δ) cells expressing mito-GFP and NES-DsRed-LuciTs after 120 minutes at 37 °C and treated with 100μM MG132. Mito-GFP is shown in cyan, NES-LuciTs in purple, and Hoechst counterstain in blue. Scale bars are 1μm.

Extended Data Fig. 5 NVJ -mediated clearance of misfolded proteins.

(a) Endogenously tagged Nvj1-GFP yeast expressing Ubc9Ts-ChFP were shifted to 37 °C and monitored by live cell time-lapse fluorescence microscopy for the times shown. White arrowheads indicate locations of Nvj1 puncta while yellow arrowheads indicate Ubc9Ts-ChFP puncta. Scale bar is 1μm. (b) WT (top) and nvj1Δ (bottom) cells co-expressing NLS-LuciTs and NES-LuciTs were treated with 100μM MG132 and shifted to 37 °C for 30 mins to preform inclusions. Cells were then placed in media containing 50mg/ml cycloheximide (CHX) and 100μM MG132 at 37 °C and monitored by live cell time-lapse fluorescence microscopy for the times shown. Scale bars are 1μm. (c,d) Quantitation of the percentage of cells containing cytoplasmic inclusions in WT, nvj1Δ, and vac8Δ yeast co-expressing NLS-EGFP-LuciTs (c) and NES-DsRed-LuciTs (d) after 2 hr at 37 °C with and without treatment with 100μM MG132. Data are presented as mean values +/- SEM. Numerical source data are available in source data.

Source data

Extended Data Fig. 6 ESCRT involvement in the clearance of misfolded proteins.

(a) Representative confocal images of WT yeast co-expressing Chm7-EGFP and either NLS-EGFP-LuciTs (left) or NES-DsRed-LuciTs (right) after 120 minutes at 37 °C and treated with 100μM MG132. Chm7 is shown in teal and remains diffuse throughout the cell, NLS-EGFP-LuciTs in green, NES-DsRed-LuciTs in purple, nuclear pores in gold and Hoechst counterstain in blue. Scale bar is 1μm. (b) Representative confocal images of WT and vps23Δ, vps34Δ, and vps15Δ yeast co-expressing NLS-EGFP-LuciTs and NES-DsRed-LuciTs after 2 hr at 37 °C and treated with 100μM MG132. NLS-EGFP-LuciTs is shown in green, NES-DsRed-LuciTs in purple, nuclear pores in gold, and Hoechst counterstain in blue. Insets show the budding INQ encapsulated by nuclear pores. Scale bars are 1μm. Same data as shown in Fig. 6c, but with the green channel separated to clearly detail the colocalization with the cytoplasmic protein.

Extended Data Fig. 7 Vacuole-mediated clearance of INQ and JUNQ.

Representative images of WT cells expressing NES-2xKeima-LuciTs after 2 hr incubation at 37 °C with 100μM MG132. Over time, fluorescence is seen with excitation in the 558 nm channel indicating the NLS-LuciTs has encountered an acidic environment. Insets show the transition from green to red and a structure leaving the inclusion that is fully red. Scale bars on large images are 5 mm. Scale bars on magnifications are 1 μm. Same data shown in Fig. 7d, but with more time points and a larger field of view in the images. Scale bars on large images are 5 μm. Scale bars on magnifications are 1 μm. (b) WT cells expressing NES-2xKeima-LuciTs after 85 min incubation at 37 °C with 100μM MG132. (c) Longer exposure of the blot shown in Fig. 7f to highlight the difference in the number and pattern of the EGFP bands in the WT vs pep4Δ cells. (d) Levels of EGFP at time 0 were measured from Quantitative Western blots such as those shown in Fig. 7e, f (mean ± S.E.M. from three biologically independent experiments). WT and pep4Δ yeast were compared using a two-tailed paired Student’s t-test without reaching statistical significance. (e) WT yeast expressing NLS-EGFP-LuciTs were treated with 8μM of FM4-64 and incubated for 2hr at 37 °C with 100μM MG132. Cells were imaged every 30 sec for 90 mins. Scale bar is 1μm. Same data shown in Fig. 7h, but only WT and with more timepoints during the entry into the vacuole. Source numerical data and unprocessed blots are available in source data.

Source data

Supplementary information

Reporting Summary

Supplementary Video 1

Live-cell time-lapse fluorescence microscopy of JUNQ and INQ formation. WT cells expressing NLS-LuciTs (left) or NES-LuciTs (right) at 37 °C, treated with 100 μM MG132. Same data shown in stills in Fig. 1c(left) and Fig. 1d (right).

Supplementary Video 2

Dynamic representation of the 3D reconstructions of SR data: shown in Fig. 1e (left) and Fig. 1g (right). Videos were created in Volocity.

Supplementary Video 3

Dynamic representation of the 3D reconstructions of SR data: shown in Fig. 2a (left) and Fig. 2b (right). Videos were created in Volocity.

Supplementary Video 4

Live-cell time-lapse fluorescence microscopy data shown in Fig. 3a.

Supplementary Video 5

Dynamic representation of data shown in Fig. 3b.

Supplementary Video 6

Dynamic representation of the 3D reconstruction of data shown in Fig. 3c. Video was created in Volocity.

Supplementary Video 7

Dynamic representation of the 3D reconstruction shown in Fig. 4c. Video was created in Amira.

Supplementary Video 8

Dynamic representation of the 3D reconstruction shown in Fig. 4d,f. Video was created in Amira.

Supplementary Video 9

Live-cell time-lapse fluorescence microscopy of the data shown in Fig. 5a.

Supplementary Video 10

Dynamic representation of the 3D reconstruction of the data shown in Fig. 5c. Video was created in Volocity.

Supplementary Video 11

Dynamic representation of the 3D reconstruction of the data shown in Fig. 5b. Video was created in Volocity.

Supplementary Video 12

Live-cell time-lapse fluorescence microscopy of the data shown in Fig. 5f shown at 5 frames s−1.

Supplementary Video 13

Live-cell time-lapse fluorescence microscopy of the data shown in Fig. 5f shown at 2 frames s−1.

Supplementary Video 14

Live-cell time-lapse fluorescence microscopy of the data shown in Fig. 5f shown at 2 frames s−1. Only the 488 nm channel is shown in greyscale.

Supplementary Video 15

Dynamic representation of the 3D reconstruction of the data shown in Fig. 5e. Video was created in Volocity.

Supplementary Video 16

Live-cell time-lapse fluorescence microscopy of WT cell data shown in Fig. 5h.

Supplementary Video 17

Live-cell time-lapse fluorescence microscopy of nvj1Δ cell data shown in Fig. 5h.

Supplementary Video 18

Live-cell time-lapse fluorescence microscopy of vps4Δ cell data shown in Fig. 5h.

Supplementary Table 1

Supplementary tables of yeast strains and plasmids.

Source data

Source Data Fig. 2

Statistical source data.

Source Data Fig. 2

Unprocessed western blots.

Source Data Fig. 3

Numerical source data.

Source Data Fig. 5

Statistical source data.

Source Data Fig. 6

Statistical source data.

Source Data Fig. 7

Statistical source data.

Source Data Fig. 7

Unprocessed western blots.

Source Data Extended Data Fig./Table 1

Unprocessed western blots.

Source Data Extended Data Fig./Table 2

Statistical source data.

Source Data Extended Data Fig./Table 5

Statistical source data.

Source Data Extended Data Fig./Table 7

Statistical source data.

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Sontag, E.M., Morales-Polanco, F., Chen, JH. et al. Nuclear and cytoplasmic spatial protein quality control is coordinated by nuclear–vacuolar junctions and perinuclear ESCRT. Nat Cell Biol 25, 699–713 (2023). https://doi.org/10.1038/s41556-023-01128-6

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