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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.

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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.

References

  1. Hartl, F. U., Bracher, A. & Hayer-Hartl, M. Molecular chaperones in protein folding and proteostasis. Nature 475, 324–332 (2011).

    Article  CAS  PubMed  Google Scholar 

  2. Needham, P. G., Guerriero, C. J. & Brodsky, J. L. Chaperoning endoplasmic reticulum-associated degradation (ERAD) and protein conformational diseases. Cold Spring Harb. Perspect. Biol. 11, a033928 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Balchin, D., Hayer-Hartl, M. & Hartl, F. U. In vivo aspects of protein folding and quality control. Science 353, aac4354 (2016).

    Article  PubMed  Google Scholar 

  4. Sontag, E. M., Samant, R. S. & Frydman, J. Mechanisms and functions of spatial protein quality control. Annu. Rev. Biochem. 20, 97–122 (2017).

    Article  Google Scholar 

  5. Pohl, C. & Dikic, I. Cellular quality control by the ubiquitin–proteasome system and autophagy. Science 366, 818–822 (2019).

    Article  CAS  PubMed  Google Scholar 

  6. Finley, D. & Prado, M. A. The proteasome and its network: engineering for adaptability. Cold Spring Harb. Perspect. Biol. 12, a033985 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Alberti, S. & Hyman, A. A. Are aberrant phase transitions a driver of cellular aging? BioEssays 38, 959–968 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Ano Bom, A. P. et al. Mutant p53 aggregates into prion-like amyloid oligomers and fibrils: implications for cancer. J. Biol. Chem. 287, 28152–28162 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Douglas, P. M. & Dillin, A. Protein homeostasis and aging in neurodegeneration. J. Cell Biol. 190, 719–729 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. De Mattos, E. P. et al. Protein quality control pathways at the crossroad of synucleinopathies. J. Parkinsons Dis. 10, 369–382 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  11. Kaganovich, D., Kopito, R. & Frydman, J. Misfolded proteins partition between two distinct quality control compartments. Nature 454, 1088–1095 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Dillin, A. & Cohen, E. Ageing and protein aggregation-mediated disorders: from invertebrates to mammals. Philos. Trans. R. Soc. Lond. B Biol. Sci. 366, 94–98 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Balch, W. E., Morimoto, R. I., Dillin, A. & Kelly, J. W. Adapting proteostasis for disease intervention. Science 319, 916–919 (2008).

    Article  CAS  PubMed  Google Scholar 

  14. Reichmann, D., Voth, W. & Jakob, U. Maintaining a healthy proteome during oxidative stress. Mol. Cell 69, 203–213 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Morales-Polanco, F., Lee, J. H., Barbosa, N. M. & Frydman, J. Cotranslational mechanisms of protein biogenesis and complex assembly in eukaryotes. Annu. Rev. Biomed. Data Sci. 5, 67–94 (2022).

    Article  PubMed  Google Scholar 

  16. Specht, S., Miller, S. B., Mogk, A. & Bukau, B. Hsp42 is required for sequestration of protein aggregates into deposition sites in Saccharomyces cerevisiae. J. Cell Biol. 195, 617–629 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Escusa-Toret, S., Vonk, W. I. & Frydman, J. Spatial sequestration of misfolded proteins by a dynamic chaperone pathway enhances cellular fitness during stress. Nat. Cell Biol. https://doi.org/10.1038/ncb2838 (2013).

  18. Miller, S. B. et al. Compartment-specific aggregases direct distinct nuclear and cytoplasmic aggregate deposition. EMBO J. 34, 778–797 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Weisberg, S. J. et al. Compartmentalization of superoxide dismutase 1 (SOD1G93A) aggregates determines their toxicity. Proc. Natl Acad. Sci. USA 109, 15811–15816 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Babazadeh, R. et al. Syntaxin 5 is required for the formation and clearance of protein inclusions during proteostatic stress. Cell Rep. 28, 2096–2110 (2019).

    Article  CAS  PubMed  Google Scholar 

  21. Nollen, E. A. et al. Dynamic changes in the localization of thermally unfolded nuclear proteins associated with chaperone-dependent protection. Proc. Natl Acad. Sci. USA 98, 12038–12043 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Yasuda, S. et al. Stress- and ubiquitylation-dependent phase separation of the proteasome. Nature 578, 296–300 (2020).

    Article  CAS  PubMed  Google Scholar 

  23. Polling, S. et al. Misfolded polyglutamine, polyalanine, and superoxide dismutase 1 aggregate via distinct pathways in the cell. J. Biol. Chem. 289, 6669–6680 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Park, J. H. et al. Amyotrophic lateral sclerosis-related mutant superoxide dismutase 1 aggregates inhibit 14-3-3-mediated cell survival by sequestration into the JUNQ compartment. Hum. Mol. Genet. 26, 3615–3629 (2017).

    Article  CAS  PubMed  Google Scholar 

  25. Samant, R. S., Livingston, C. M., Sontag, E. M. & Frydman, J. Distinct proteostasis circuits cooperate in nuclear and cytoplasmic protein quality control. Nature 563, 407–411 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. McClellan, A. J., Scott, M. D. & Frydman, J. Folding and quality control of the VHL tumor suppressor proceed through distinct chaperone pathways. Cell 121, 739–748 (2005).

    Article  CAS  PubMed  Google Scholar 

  27. Gupta, R. et al. Firefly luciferase mutants as sensors of proteome stress. Nat. Methods 8, 879–884 (2011).

    Article  CAS  PubMed  Google Scholar 

  28. Popken, P., Ghavami, A., Onck, P. R., Poolman, B. & Veenhoff, L. M. Size-dependent leak of soluble and membrane proteins through the yeast nuclear pore complex. Mol. Biol. Cell 26, 1386–1394 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Amm, I. & Wolf, D. H. Molecular mass as a determinant for nuclear San1-dependent targeting of misfolded cytosolic proteins to proteasomal degradation. FEBS Lett. 590, 1765–1775 (2016).

    Article  CAS  PubMed  Google Scholar 

  30. Timney, B. L. et al. Simple rules for passive diffusion through the nuclear pore complex. J. Cell Biol. 215, 57–76 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Woerner, A. C. et al. Cytoplasmic protein aggregates interfere with nucleocytoplasmic transport of protein and RNA. Science 351, 173–176 (2016).

    Article  CAS  PubMed  Google Scholar 

  32. Park, S. H. et al. PolyQ proteins interfere with nuclear degradation of cytosolic proteins by sequestering the Sis1p Chaperone. Cell 154, 134–145 (2013).

    Article  CAS  PubMed  Google Scholar 

  33. Neuber, A. et al. Nuclear export receptor Xpo1/Crm1 is physically and functionally linked to the spindle pole body in budding yeast. Mol. Cell. Biol. 28, 5348–5358 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Grima, J. C. et al. Mutant huntingtin disrupts the nuclear pore complex. Neuron 94, 93–107 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Gasset-Rosa, F. et al. Polyglutamine-expanded huntingtin exacerbates age-related disruption of nuclear integrity and nucleocytoplasmic transport. Neuron 94, 48–57 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Wente, S. R. & Blobel, G. A temperature-sensitive NUP116 null mutant forms a nuclear envelope seal over the yeast nuclear pore complex thereby blocking nucleocytoplasmic traffic. J. Cell Biol. 123, 275–284 (1993).

    Article  CAS  PubMed  Google Scholar 

  37. Chen, L. et al. Sts1 plays a key role in targeting proteasomes to the nucleus. J. Biol. Chem. 286, 3104–3118 (2011).

    Article  CAS  PubMed  Google Scholar 

  38. Takeda, K. & Yanagida, M. Regulation of nuclear proteasome by Rhp6/Ubc2 through ubiquitination and destruction of the sensor and anchor Cut8. Cell 122, 393–405 (2005).

    Article  CAS  PubMed  Google Scholar 

  39. Tatebe, H. & Yanagida, M. Cut8, essential for anaphase, controls localization of 26S proteasome, facilitating destruction of cyclin and Cut2. Curr. Biol. 10, 1329–1338 (2000).

    Article  CAS  PubMed  Google Scholar 

  40. Romero-Perez, L., Chen, L., Lambertson, D. & Madura, K. Sts1 can overcome the loss of Rad23 and Rpn10 and represents a novel regulator of the ubiquitin/proteasome pathway. J. Biol. Chem. 282, 35574–35582 (2007).

    Article  CAS  PubMed  Google Scholar 

  41. Lord, C. L. & Wente, S. R. Nuclear envelope–vacuole contacts mitigate nuclear pore complex assembly stress. J. Cell Biol. 219, e202001165 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Crisp, M. et al. Coupling of the nucleus and cytoplasm: role of the LINC complex. J. Cell Biol. 172, 41–53 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Aitchison, J. D., Blobel, G. & Rout, M. P. Nup120p: a yeast nucleoporin required for NPC distribution and mRNA transport. J. Cell Biol. 131, 1659–1675 (1995).

    Article  CAS  PubMed  Google Scholar 

  44. Heath, C. V. et al. Nuclear pore complex clustering and nuclear accumulation of poly(A)+ RNA associated with mutation of the Saccharomyces cerevisiae RAT2/NUP120 gene. J. Cell Biol. 131, 1677–1697 (1995).

    Article  CAS  PubMed  Google Scholar 

  45. Ader, C. et al. Amyloid-like interactions within nucleoporin FG hydrogels. Proc. Natl Acad. Sci. USA 107, 6281–6285 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Strawn, L. A., Shen, T., Shulga, N., Goldfarb, D. S. & Wente, S. R. Minimal nuclear pore complexes define FG repeat domains essential for transport. Nat. Cell Biol. 6, 197–206 (2004).

    Article  CAS  PubMed  Google Scholar 

  47. Parkinson, D. Y. et al. Nanoimaging cells using soft X-ray tomography. Methods Mol. Biol. 950, 457–481 (2013).

    Article  CAS  PubMed  Google Scholar 

  48. Le Gros, M. A. et al. Biological soft X-ray tomography on beamline 2.1 at the Advanced Light Source. J. Synchrotron Radiat. 21, 1370–1377 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  49. Larabell, C. A. & Nugent, K. A. Imaging cellular architecture with X-rays. Curr. Opin. Struct. Biol. 20, 623–631 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Le Gros, M. A., McDermott, G., Uchida, M., Knoechel, C. G. & Larabell, C. A. High-aperture cryogenic light microscopy. J. Microsc. 235, 1–8 (2009).

    Article  PubMed  Google Scholar 

  51. Ekman, A. et al. in Synchrotron Light Sources and Free-Electron Lasers: Accelerator Physics, Instrumentation and Science Applications (eds Jaeschke, E. et al.) 1–32 (Springer International Publishing, 2019).

  52. Ekman, A. A. et al. Mesoscale imaging with cryo-light and X-rays: larger than molecular machines, smaller than a cell. Biol. Cell 109, 24–38 (2017).

    Article  PubMed  Google Scholar 

  53. Huh, W. K. et al. Global analysis of protein localization in budding yeast. Nature 425, 686–691 (2003).

    Article  CAS  PubMed  Google Scholar 

  54. Wu, G. H. et al. Multi-scale 3D cryo-correlative microscopy for vitrified cells. Structure 28, 1231–1237 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Schuler, M. H. et al. Miro1-mediated mitochondrial positioning shapes intracellular energy gradients required for cell migration. Mol. Biol. Cell 28, 2159–2169 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Frottin, F. et al. The nucleolus functions as a phase-separated protein quality control compartment. Science 365, 342–347 (2019).

    Article  CAS  PubMed  Google Scholar 

  57. Velazquez, J. M. & Lindquist, S. hsp70: nuclear concentration during environmental stress and cytoplasmic storage during recovery. Cell 36, 655–662 (1984).

    Article  CAS  PubMed  Google Scholar 

  58. Falahati, H., Pelham-Webb, B., Blythe, S. & Wieschaus, E. Nucleation by rRNA dictates the precision of nucleolus assembly. Curr. Biol. 26, 277–285 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Azkanaz, M. et al. Protein quality control in the nucleolus safeguards recovery of epigenetic regulators after heat shock. eLife 8, e45205 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  60. Pan, X. et al. Nucleus–vacuole junctions in Saccharomyces cerevisiae are formed through the direct interaction of Vac8p with Nvj1p. Mol. Biol. Cell 11, 2445–2457 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Kohlwein, S. D. et al. Tsc13p is required for fatty acid elongation and localizes to a novel structure at the nuclear–vacuolar interface in Saccharomyces cerevisiae. Mol. Cell. Biol. 21, 109–125 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Levine, T. P. & Munro, S. Dual targeting of Osh1p, a yeast homologue of oxysterol-binding protein, to both the Golgi and the nucleus–vacuole junction. Mol. Biol. Cell 12, 1633–1644 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Kvam, E. & Goldfarb, D. S. Structure and function of nucleus–vacuole junctions: outer-nuclear-membrane targeting of Nvj1p and a role in tryptophan uptake. J. Cell Sci. 119, 3622–3633 (2006).

    Article  CAS  PubMed  Google Scholar 

  64. Kvam, E. & Goldfarb, D. S. Nucleus–vacuole junctions and piecemeal microautophagy of the nucleus in S. cerevisiae. Autophagy 3, 85–92 (2007).

    Article  CAS  PubMed  Google Scholar 

  65. Roberts, P. et al. Piecemeal microautophagy of nucleus in Saccharomyces cerevisiae. Mol. Biol. Cell 14, 129–141 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Krick, R. et al. Piecemeal microautophagy of the nucleus requires the core macroautophagy genes. Mol. Biol. Cell 19, 4492–4505 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. von Knebel Doeberitz, M. & Wentzensen, N. in Comprehensive Cytopathology 3rd edn, (eds Bibbo, M. & Wilbur D.) Ch. 1 (Saunders, 2008).

  68. Ponsford, A. H. et al. Live imaging of intra-lysosome pH in cell lines and primary neuronal culture using a novel genetically encoded biosensor. Autophagy 17, 1500–1518 (2021).

    Article  CAS  PubMed  Google Scholar 

  69. Campbell, T. N. & Choy, F. Y. M. The effect of pH on green fluorescent protein: a brief review. Mol. Biol. Today 2, 1–4 (2001).

    CAS  Google Scholar 

  70. Tsien, R. Y. The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544 (1998).

    Article  CAS  PubMed  Google Scholar 

  71. Haupts, U., Maiti, S., Schwille, P. & Webb, W. W. Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy. Proc. Natl Acad. Sci. USA 95, 13573–13578 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Ho, C. Y., Choy, C. H., Wattson, C. A., Johnson, D. E. & Botelho, R. J. The Fab1/PIKfyve phosphoinositide phosphate kinase is not necessary to maintain the pH of lysosomes and of the yeast vacuole. J. Biol. Chem. 290, 9919–9928 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Cormack, B. P., Valdivia, R. H. & Falkow, S. FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173, 33–38 (1996).

    Article  CAS  PubMed  Google Scholar 

  74. von Appen, A. et al. LEM2 phase separation promotes ESCRT-mediated nuclear envelope reformation. Nature 582, 115–118 (2020).

    Article  Google Scholar 

  75. Thaller, D. J. & Patrick Lusk, C. Fantastic nuclear envelope herniations and where to find them. Biochem. Soc. Trans. 46, 877–889 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Webster, B. M. et al. Chm7 and Heh1 collaborate to link nuclear pore complex quality control with nuclear envelope sealing. EMBO J. 35, 2447–2467 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Thaller, D. J. et al. An ESCRT-LEM protein surveillance system is poised to directly monitor the nuclear envelope and nuclear transport system. eLife 8, e45284 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  78. Babst, M., Odorizzi, G., Estepa, E. J. & Emr, S. D. Mammalian tumor susceptibility gene 101 (TSG101) and the yeast homologue, Vps23p, both function in late endosomal trafficking. Traffic 1, 248–258 (2000).

    Article  CAS  PubMed  Google Scholar 

  79. Katzmann, D. J., Babst, M. & Emr, S. D. Ubiquitin-dependent sorting into the multivesicular body pathway requires the function of a conserved endosomal protein sorting complex, ESCRT-I. Cell 106, 145–155 (2001).

    Article  CAS  PubMed  Google Scholar 

  80. Herrador, A., Léon, S., Haguenauer-Tsapis, R. & Vincent, O. A mechanism for protein monoubiquitination dependent on a trans-acting ubiquitin-binding domain. J. Biol. Chem. 288, 16206–16211 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Stack, J. H., Herman, P. K., Schu, P. V. & Emr, S. D. A membrane-associated complex containing the Vps15 protein kinase and the Vps34 PI 3-kinase is essential for protein sorting to the yeast lysosome-like vacuole. EMBO J. 12, 2195–2204 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  82. Herman, P. K. & Emr, S. D. Characterization of VPS34, a gene required for vacuolar protein sorting and vacuole segregation in Saccharomyces cerevisiae. Mol. Cell. Biol. 10, 6742–6754 (1990).

    CAS  PubMed  PubMed Central  Google Scholar 

  83. Burda, P., Padilla, S. M., Sarkar, S. & Emr, S. D. Retromer function in endosome-to-Golgi retrograde transport is regulated by the yeast Vps34 PtdIns 3-kinase. J. Cell Sci. 115, 3889–3900 (2002).

    Article  CAS  PubMed  Google Scholar 

  84. Slessareva, J. E., Routt, S. M., Temple, B., Bankaitis, V. A. & Dohlman, H. G. Activation of the phosphatidylinositol 3-kinase Vps34 by a G protein alpha subunit at the endosome. Cell 126, 191–203 (2006).

    Article  CAS  PubMed  Google Scholar 

  85. Henne, W. M., Stenmark, H. & Emr, S. D. Molecular mechanisms of the membrane sculpting ESCRT pathway. Cold Spring Harb. Perspect. Biol. 5, a016766 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  86. Hurley, J. H. & Hanson, P. I. Membrane budding and scission by the ESCRT machinery: it’s all in the neck. Nat. Rev. Mol. Cell Biol. 11, 556–566 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  87. Katayama, H., Kogure, T., Mizushima, N., Yoshimori, T. & Miyawaki, A. A sensitive and quantitative technique for detecting autophagic events based on lysosomal delivery. Chem. Biol. 18, 1042–1052 (2011).

    Article  CAS  PubMed  Google Scholar 

  88. Bialecka-Fornal, M., Makushok, T. & Rafelski, S. M. A review of fluorescent proteins for use in yeast. Methods Mol. Biol. 1369, 309–346 (2016).

    Article  CAS  PubMed  Google Scholar 

  89. Sitron, C. S., Park, J. H., Giafaglione, J. M. & Brandman, O. Aggregation of CAT tails blocks their degradation and causes proteotoxicity in S. cerevisiae. PLoS ONE 15, e0227841 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  90. Schuck, S., Gallagher, C. M. & Walter, P. ER-phagy mediates selective degradation of endoplasmic reticulum independently of the core autophagy machinery. J. Cell Sci. 127, 4078–4088 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  91. Klionsky, D. J. et al. Guidelines for the use and interpretation of assays for monitoring autophagy (4th edn). Autophagy 17, 1–382 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  92. Eising, S. et al. A lysosomal biogenesis map reveals the cargo spectrum of yeast vacuolar protein targeting pathways. J. Cell Biol. 221, e202107148 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  93. Waite, K. A., De-La Mota-Peynado, A., Vontz, G. & Roelofs, J. Starvation induces proteasome autophagy with different pathways for core and regulatory particles. J. Biol. Chem. 291, 3239–3253 (2016).

    Article  CAS  PubMed  Google Scholar 

  94. Kvam, E. & Goldfarb, D. S. Nvj1p is the outer-nuclear-membrane receptor for oxysterol-binding protein homolog Osh1p in Saccharomyces cerevisiae. J. Cell Sci. 117, 4959–4968 (2004).

    Article  CAS  PubMed  Google Scholar 

  95. Shinoda, H., Shannon, M. & Nagai, T. Fluorescent proteins for investigating biological events in acidic environments. Int. J. Mol. Sci. 19, 1548 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  96. Vida, T. A. & Emr, S. D. A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast. J. Cell Biol. 128, 779–792 (1995).

    Article  CAS  PubMed  Google Scholar 

  97. Thorn, K. Genetically encoded fluorescent tags. Mol. Biol. Cell 28, 848–857 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  98. Costantini, L. M. et al. A palette of fluorescent proteins optimized for diverse cellular environments. Nat. Commun. 6, 7670 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  99. Amm, I., Sommer, T. & Wolf, D. H. Protein quality control and elimination of protein waste: the role of the ubiquitin–proteasome system. Biochim. Biophys. Acta 1843, 182–196 (2014).

    Article  CAS  PubMed  Google Scholar 

  100. Eisele, F. et al. An Hsp90 co-chaperone links protein folding and degradation and is part of a conserved protein quality control. Cell Rep. 35, 109328 (2021).

    Article  CAS  PubMed  Google Scholar 

  101. Arrasate, M., Mitra, S., Schweitzer, E. S., Segal, M. R. & Finkbeiner, S. Inclusion body formation reduces levels of mutant huntingtin and the risk of neuronal death. Nature 431, 805–810 (2004).

    Article  CAS  PubMed  Google Scholar 

  102. Schneider, K. L. et al. Using reporters of different misfolded proteins reveals differential strategies in processing protein aggregates. J. Biol. Chem. 298, 102476 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  103. Kumar, B. et al. ESCRT-I protein Tsg101 plays a role in the post-macropinocytic trafficking and infection of endothelial cells by Kaposi’s sarcoma-associated herpesvirus. PLoS Pathog. 12, e1005960 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  104. Gottschling, D. E. & Nystrom, T. The upsides and downsides of organelle interconnectivity. Cell 169, 24–34 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  105. Zhang, K. et al. The C9orf72 repeat expansion disrupts nucleocytoplasmic transport. Nature 525, 56–61 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  106. Fallini, C., Khalil, B., Smith, C. L. & Rossoll, W. Traffic jam at the nuclear pore: all roads lead to nucleocytoplasmic transport defects in ALS/FTD. Neurobiol. Dis. 140, 104835 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  107. Shang, J. et al. Aberrant distributions of nuclear pore complex proteins in ALS mice and ALS patients. Neuroscience 350, 158–168 (2017).

    Article  CAS  PubMed  Google Scholar 

  108. Chou, C. C. et al. TDP-43 pathology disrupts nuclear pore complexes and nucleocytoplasmic transport in ALS/FTD. Nat. Neurosci. 21, 228–239 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  109. Brickner, D. G., Light, W. & Brickner, J. H. Quantitative localization of chromosomal loci by immunofluorescence. Methods Enzymol. 470, 569–580 (2010).

    Article  CAS  PubMed  Google Scholar 

  110. Weiss, D. et al. Computed tomography of cryogenic biological specimens based on X-ray microscopic images. Ultramicroscopy 84, 185–197 (2000).

    Article  CAS  PubMed  Google Scholar 

  111. Tan, S. A modular polycistronic expression system for overexpressing protein complexes in Escherichia coli. Protein Expr. Purif. 21, 224–234 (2001).

    Article  CAS  PubMed  Google Scholar 

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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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  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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