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Distinct proteostasis circuits cooperate in nuclear and cytoplasmic protein quality control

Abstract

Protein misfolding is linked to a wide array of human disorders, including Alzheimer’s disease, Parkinson’s disease and type II diabetes1,2. Protective cellular protein quality control (PQC) mechanisms have evolved to selectively recognize misfolded proteins and limit their toxic effects3,4,5,6,7,8,9, thus contributing to the maintenance of the proteome (proteostasis). Here we examine how molecular chaperones and the ubiquitin–proteasome system cooperate to recognize and promote the clearance of soluble misfolded proteins. Using a panel of PQC substrates with distinct characteristics and localizations, we define distinct chaperone and ubiquitination circuitries that execute quality control in the cytoplasm and nucleus. In the cytoplasm, proteasomal degradation of misfolded proteins requires tagging with mixed lysine 48 (K48)- and lysine 11 (K11)-linked ubiquitin chains. A distinct combination of E3 ubiquitin ligases and specific chaperones is required to achieve each type of linkage-specific ubiquitination. In the nucleus, however, proteasomal degradation of misfolded proteins requires only K48-linked ubiquitin chains, and is thus independent of K11-specific ligases and chaperones. The distinct ubiquitin codes for nuclear and cytoplasmic PQC appear to be linked to the function of the ubiquilin protein Dsk2, which is specifically required to clear nuclear misfolded proteins. Our work defines the principles of cytoplasmic and nuclear PQC as distinct, involving combinatorial recognition by defined sets of cooperating chaperones and E3 ligases. A better understanding of how these organelle-specific PQC requirements implement proteome integrity has implications for our understanding of diseases linked to impaired protein clearance and proteostasis dysfunction.

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Fig. 1: Combined deletion of certain E3 ligases impairs the clearance of misfolded proteins.
Fig. 2: Cytoplasmic misfolded proteins are modified with both K11- and K48- linked ubiquitin chains.
Fig. 3: K11- and K48- linked ubiquitination of misfolded proteins involves different chaperones.
Fig. 4: Confining misfolded proteins to the nucleus or cytoplasm alters their PQC requirements.

Data availability

The data sets generated and/or analysed during this study are available from the corresponding author on reasonable request. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the data identifier PXD010660. Uncropped images of all immunoblots shown in this study are in Supplementary Fig. 1.

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Acknowledgements

We thank K. Li, M. Burlingame and A. L. Burlingame for help with mass spectrometry; R. Andino and F. U. Hartl for critical reading of the manuscript; D. R. Gestaut for sharing the NLS- and NES-tagged plasmids; and all members of the Frydman laboratory for advice. R.S.S. was supported by a Human Frontier Science Program long-term fellowship (LT000695/2015-L). C.M.L. was supported by a National Institutes of Health (NIH) postdoctoral fellowship (1F32CA162919-01A1). This work was supported by an NIH grant (R37GM056433) to J.F. E.M.S. was supported by a postdoctoral fellowship from NIH (F32NS086253).

Reviewer information

Nature thanks I. Dikic, A. Dillin and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Authors

Contributions

R.S.S. and J.F. designed the study. C.M.L. performed the initial puncta screens with E3 single- and double-deletion mutants. R.S.S. performed all other experiments and analysis. E.M.S. provided insight into the contribution of Dsk2 to nuclear quality control. R.S.S. and J.F. interpreted the data and wrote the manuscript.

Corresponding authors

Correspondence to Rahul S. Samant, Christine M. Livingston or Judith Frydman.

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Extended data figures and tables

Extended Data Fig. 1 E3 ligases work in optimal combinations to clear misfolded proteins.

a, Assay for puncta formation distinguishes between misfolded versus natively folded proteins. WT cells expressing natively folded Ubc9WT–GFP or temperature-sensitive Ubc9ts–GFP from a galactose-inducible promoter for 4–6 h at 30 °C were shifted to glucose-containing medium for 1 h at 30 °C or 37 °C to shut off expression. Cells were fixed and imaged by fluorescence microscopy. 300 cells were counted per condition, and the percentage of cells with GFP-positive puncta is shown (means ± s.e.m. from three biologically independent experiments). Only cells expressing Ubc9ts–GFP showed a statistically significant change in the percentage of puncta-positive cells compared with WT (two-tailed Student’s t-test, ****P < 0.0001; ns, not significant). b, Deletion of individual E3 ligases does not increase puncta formation. Experiment performed as in panel a, but using strains with endogenous deletions of the genes shown on the x-axis. E3 ligases that have previously been implicated in PQC (as shown in Fig. 1d) are grouped to the right (QC, quality control). Bars represent mean ± s.e.m. from three biologically independent experiments, with the exception of rad5, hel1, etp1, irc20, hel2, apc11, hrt1, tfb3, cdc24, prp19, upf1, upf3, itt1 and rad18, where bars represent the mean from two biologically independent experiments, as well as WT, where bars represent the mean ± s.e.m. from seven biologically independent experiments. No strains showed statistically significant differences compared with WT by one-way ANOVA followed by Dunnett’s multiple comparisons test. c, Deleting certain pairs of E3 ligases increases the stability of misfolded proteins. CHX chase was followed by immunoblot to assess the stability of GFP–VHL, CPY–GFP, Ubc9ts–TAP or Ubc9WT–TAP in E3 double-deletion strains. For the WT + Bz condition, 50 μM Bz was added to the glucose-containing medium 10 min before CHX treatment. Graphs represent densitometric quantification of bands relative to t = 0 (mean ± s.e.m. from three biologically independent experiments). d, Multiple misfolded proteins are sequestered in the same subcellular location. ∆ubr1∆san1 or ∆doa10∆hrd1 strains co-expressing VHL with temperature-sensitive Ubc9ts (top) or natively folded Ubc9WT (bottom) from galactose-inducible promoters for 5–6 h at 30 °C were shifted to glucose-containing medium for 1 h at 37 °C. Fluorescence microscopy images are representative of at least 100 cells from each of three biologically independent experiments. e, Deletion of certain pairs of E3s increases puncta formation. Experiment performed as in panel a, but in strains with endogenous deletions of pairs of E3 genes. Each right-hand panel shows experiments in which cells were shifted to 37 °C for the 1 h of galactose shut-off. Bars represent means ± s.e.m. from three biologically independent experiments. Strains for which statistically significant differences were observed by one-way ANOVA followed by Dunnett’s multiple comparisons test compared with WT are indicated with the adjusted P value or with **** for P < 0.0001. f, Overexpressing a single E3 ligase does not compensate for the loss of others. Ubr1, San1 or Hrd1 were overexpressed alongside GFP–VHL in the indicated strains. The rest of the experiment was performed as in a. Bars represent means ± s.e.m. from three biologically independent experiments.

Extended Data Fig. 2 San1 forms a complex with Doa10 but not with Hrd1.

a, b, San1–V5His6 co-immunoprecipitates with Doa10–GFP but not with Hrd1–GFP. Yeast cells co-expressing Doa10–GFP (a) or Hrd1–GFP (b) from their endogenous promoters with San1–V5His6 from a galactose-inducible promoter for 16 h were shifted to 37 °C for 1 h, and immediately lysed by cryo-grinding. Native complexes were immunoprecipitated with GFP-Trap-MA nanobodies before immunoblotting with the indicated antibodies. Immunoblots are representative of three biologically independent experiments. c, d, Flag–Ubr1 does not co-immunoprecipitate with Doa10–GFP or Hrd1–GFP. The experiment was performed as in panel a and b, but with cells expressing Flag–Ubr1 (from the constitutive ADH promoter) instead of San1–V5His6. Immunoblots are representative of three biologically independent experiments.

Extended Data Fig. 3 K48–Ub and K11–Ub linkages are reduced in ∆ubr1∆san1 and ∆doa10∆hrd1 strains, respectively.

a, Diagram showing the Ub-linkage ELISA used to quantify Ub linkages. Flag–VHL from a yeast lysate was immunoprecipitated in an anti-Flag-conjugated 96-well plate (using four wells per sample), and incubated with antibodies against GFP (negative control), Flag, K11–Ub, or K48–Ub. Following incubation with a secondary antibody (anti-rabbit-HRP), the strength of each signal was detected by electrochemiluminescence at 450 nm. To quantify the K11–Ub or K48–Ub linkages on Flag–VHL, we subtracted the anti-K11 or anti-K48 signal from the negative control (anti-GFP) and normalized to the total Flag–VHL signal for each sample. b, Ub-linkage ELISA confirms that K48–Ub and K11–Ub linkages are reduced on Flag–VHL in ∆ubr1∆san1 and ∆doa10∆hrd1 strains, respectively. WT or E3 double-deletion strains expressing Flag-VHL at 30 °C for 4–6 h were lysed after 1 h Bz treatment, also at 30 °C. Ub-linkage ELISA was then performed as described in a. Bars represent Flag-normalized values from each strain (mean ± s.e.m. from three biologically independent experiments), expressed as a proportion of the Flag-normalized WT values. Strains with statistically significant differences compared with WT by one-way ANOVA followed by Dunnett’s multiple comparisons test are indicated (****P < 0.001). c, GFP–VHL denaturing immunoprecipitation (1% SDS + 8 M urea) followed by immunoblot for K48–Ub or K11–Ub in WT or E3 double-deletion strains. Immunoblots are representative of three independent experiments. d, Relative amounts of K11–Ub and K48–Ub linkages present on GFP–VHL in ∆ubr1∆san1 or ∆doa10∆hrd1 strains compared with WT. WT or E3 double-deletion strains expressing GFP–VHL at 30 °C for 5–6 h were lysed in denaturing conditions (1% SDS + 8 M urea) after 1 h Bz treatment, also at 30 °C. Ub-linkage ELISA was then performed using GFP-multiTrap plates. Bars represent GFP-normalized values from each strain (means ± s.e.m. from three biologically independent experiments) expressed as a proportion of the GFP-normalized WT values. Strains for which statistically significant differences were observed by one-way ANOVA followed by Dunnett’s multiple comparisons test compared with WT are indicated with the adjusted P value, or with **** for P < 0.0001.

Extended Data Fig. 4 K11–Ub linkages are not necessary for proteasomal degradation of all cytoplasmic substrates.

a–d, WT or UbK11R cells expressing stable Ub-M-GFP (a), the N-end-rule substrate Ub-R-GFP (b), the ubiquitin fusion degradation (UFD) substrate UbG76V–GFP (c) or GFP fused to the artificial degron CL1 (d) from galactose-inducible promoters for 4–6 h at 30 °C were shifted to glucose-containing medium for 1 h at 30 °C or 37 °C to shut off expression. Cells were fixed and imaged by fluorescence microscopy. 300 cells were counted per condition, and the percentage of cells with GFP-positive puncta is shown (mean ± s.e.m. from three biologically independent experiments). There was a statistically significant increase in puncta compared with WT when GFP-CL1 (which contains a short amphipathic CL1 helix that could mimic a partially unfolded protein) was expressed in UbK11R cells, as judged by two-tailed Student’s t-test (P = 0.0127). The differences for all other substrates were not significant (ns, P > 0.05). DUB, deubiquitinating enzyme, which cleaves Ub from Ub-M-GFP or Ub-R-GFP.

Extended Data Fig. 5 Misfolded VHL is modified with branched K11/K48 ubiquitin chains.

a, b, Both K11–Ub and K48–Ub linkages are present on the same VHL molecule. a, This experiment was designed to determine whether both K48–Ub and K11–Ub linkages are present in the same VHL population. Sequential immunoprecipitation was carried out, first with anti-Flag antibody, then with an anti-K11–Ub or anti-K48–Ub antibody. The resulting negative control (‘no Flag’, with mock Flag plus K11 or K48 immunoprecipitation with lysate from cells expressing GFP–VHL instead of Flag–VHL), bead control (‘Control’, with no K11–Ub or K48–Ub antibody), ‘Bound’ and ‘Flow-through’, in addition to samples with just the first Flag immunoprecipitation (Input), were subjected to SDS–PAGE and immunoblotted for the presence of the other Ub linkage (b).Immunoblots representative of three biologically independent experiments are shown. The arrow indicates the size of un-ubiquitinated Flag–VHL. The asterisks indicate proteins in the stacking gel that did not enter the resolving gel. c, This bispecific anti-K11/K48–Ub antibody was designed to bind ubiquitin chains with K11 and K48 linkages branching off the same ubiquitin moiety. d, Misfolded VHL co-localizes with K11/K48–Ub chains. WT cells expressing GFP–VHL from a galactose-inducible promoter for 4–6 h at 30 °C were shifted to glucose-containing medium with 50 μM bortezomib for 1 h to shut off expression. Cells were fixed, spheroplasted and detergent permeabilized before immunostaining with an antibody designed to recognize ubiquitin that had K11 and K48 linkages emanating from the same moiety (K11/K48). Confocal fluorescence microscopy images are representative of at least 100 cells from each of three biologically independent experiments. Scale bars represent 2 μm. e, VHL is modified with branched K11/K48–Ub chains. GFP–VHL denaturing immunoprecipitation was followed by immunoblot for K11/K48–Ub or GFP (VHL) in WT or E3 double-deletion strains. Immunoblots representative of three biologically independent experiments are shown.

Extended Data Fig. 6 Nuclear and cytoplasmic proteins require different PQC pathways for clearance.

a, NLS–GFP–VHL and NES–GFP–VHL form a single punctum in the nucleus or cytoplasm, respectively, upon proteasome inhibition. WT cells expressing NLS–GFP–VHL or NES–GFP–VHL from a galactose-inducible promoter for 4–6 h at 30 °C were shifted to glucose-containing medium with 50 μM Bz for 1 h at 30 °C to shut off GFP–VHL expression. Fixed and spheroplasted cells were immunostained for the nuclear pore complex protein Nsp1 (red) before imaging by fluorescence microscopy. Representative cells from three biologically independent repeats are shown. bd, Misfolded luciferasets (Lucts) confined to the nucleus can be cleared by San1-mediated K48-linked ubiquitination. b, The increase in the percentage of cells containing puncta of NLS–GFP–Lucts and NES–GFP–Lucts across the E3 single- and double-deletion strains is similar to the pattern observed with NLS–GFP–VHL and NES–GFP–VHL in Fig. 4. Shown is the percentage of cells (mean ± s.e.m. from three biologically independent experiments, each with n = 300) containing NLS–GFP–Lucts or NES–GFP–Lucts puncta in WT, single- or double-deletion strains after 4–6 h expression of the protein at 30 °C followed by 1 h shut-off at 37 °C. Strains for which statistically significant differences were observed by one-way ANOVA followed by Dunnett’s multiple comparisons test compared with WT are indicated with the adjusted P value, or with **** for P < 0.0001. c, Misfolded nuclear Lucts has severely reduced K11–Ub linkages (****P < 0.0001 by one-way ANOVA followed by Dunnett’s multiple comparisons test). Ubiquitin-linkage ELISA was performed on lysates of WT yeast expressing NLS-, NES- or unaltered GFP–Lucts at 37 °C as described in Extended Data Fig. 2c, but in GFP-multiTrap 96-well plates instead of anti-Flag-conjugated 96-well plates. Anti-Flag was used instead of anti-GFP as the ELISA negative control. Bars represent means ± s.e.m. from three biologically independent experiments. d, Misfolded luciferasets confined to the nucleus does not require K11–Ub linkages for clearance. The experiment was performed as in panel b, but with yeast strains expressing WT or mutant K11R–Ub as their sole source of ubiquitin. 300 cells were counted per condition, and the percentages of cells with GFP-positive puncta are shown in (means ± s.e.m. from three biologically independent experiments). Only NES–GFP–Lucts had a statistically significant change in puncta-positive cells in the K11R strain when compared with WT (one-way ANOVA followed by Dunnett’s multiple comparisons test; ****P < 0.0001; ns = P > 0.05). e, VHL confined to the nucleus (NLS) or cytoplasm (NES) requires different chaperones for clearance. The experiment was performed as in panel b, but with the indicated chaperone-deletion strains. Bars represent means ± s.e.m. from three biologically independent experiments. Strains for which statistically significant differences were observed compared with WT by one-way ANOVA followed by Dunnett’s multiple comparisons test are indicated with the adjusted P value, or with **** for P < 0.0001.

Extended Data Fig. 7 Mass spectrometry of the VHL interactome identifies distinct PQC circuitries for nuclear and cytoplasmic VHL.

a, Triple SILAC-base mass spectrometry of VHL immunoprecipitates. WT yeast cells transfected with one of NLS–GFP–VHL, NES–GFP–VHL or Flag–VHL were grown overnight at 30 °C in raffinose-synthetic media supplemented with light Lys0, heavy Lys8 or medium Lys4, respectively. Growth of VHL was induced in galactose for 4–5 h before shut off in glucose for 90 min. Next, 1.5 mg of protein from each of the three lysed samples were mixed before immunoprecipitation using GFP-TRAP_MA magnetic bead on-bead restriction digestion and peptide clean-up. Peptides were identified using liquid-chromatography/mass-spectrometry analysis before analysis using MaxQuant. b, Strong correlation between the four biological repeats (R1–R4). Raw intensities for light (NLS–GFP–VHL; top), heavy (NES–GFP–VHL; middle) and medium (VHL–Flag control, bottom) were log10-transformed and plotted as scatterplot matrices. The Pearson correlation coefficient for each pairwise comparison is indicated, and the density distribution of intensities within each repeat is shown in the diagonal axis of the matrices. c, Enriched PQC proteins in NLS–GFP–VHL and NES–GFP–VHL interactomes. Normalized median light/medium (NLS–GFP–VHL) and heavy/medium (NES–GFP–VHL) SILAC ratios were log2-transformed. Proteins with log2(SILAC ratio) of greater than 0.5 were considered as enriched, yielding 49 and 56 proteins for the NLS and NES interactomes, respectively. Enriched proteins known to play a role in PQC are shown. Both nuclear and cytoplasmic VHL share enrichments in proteasomal subunits, the Hsp70 chaperones Ssa1, Ssa2, Ssa4 and Ssb2, and the thioredoxins Trx1, Trx2 and Tsa1 (previously implicated in misfolded-protein management). All enriched proteins are shown in Extended Data Table 1. d, Enriched PQC pathways in NLS–GFP–VHL and NES–GFP–VHL interactomes. The enriched proteins from each interactome (median values from four biologically independent experiments) were subjected to pathway analysis to search for enriched GO terms, KEGG pathways and PFAM protein domains in either interactome using the STRING database. Selected enriched PQC pathways are shown (P < 0.05 using Fisher’s exact test followed by Benjamini–Hochberg multiple testing correction).

Extended Data Table 1 Protein and pathways enriched in nuclear and cytoplasmic interactomes

Supplementary information

Supplementary Figure 1

This file contains the uncropped images of immunoblots shown in this study. Parts of the images showing the protein of interest are indicated with a red rectangle. Specific primary antibodies used are indicated next to the images

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Samant, R.S., Livingston, C.M., Sontag, E.M. et al. Distinct proteostasis circuits cooperate in nuclear and cytoplasmic protein quality control. Nature 563, 407–411 (2018). https://doi.org/10.1038/s41586-018-0678-x

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Keywords

  • Ubiquitin Chains
  • Distinct Chaperone
  • Proteasome
  • Stable Isotope Labeling By Amino Acids In Cell Culture (SILAC)
  • Synthetic Glucose Medium

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