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. 2024 Aug 21:11:1427542.
doi: 10.3389/fmolb.2024.1427542. eCollection 2024.

Clearing the JUNQ: the molecular machinery for sequestration, localization, and degradation of the JUNQ compartment

Sarah Rolli  1 , Chloe A Langridge  1 , Emily M Sontag  1
Affiliations

Clearing the JUNQ: the molecular machinery for sequestration, localization, and degradation of the JUNQ compartment

Sarah Rolli et al. Front Mol Biosci. .

Abstract

Cellular protein homeostasis (proteostasis) plays an essential role in regulating the folding, sequestration, and turnover of misfolded proteins via a network of chaperones and clearance factors. Previous work has shown that misfolded proteins are spatially sequestered into membrane-less compartments in the cell as part of the proteostasis process. Soluble misfolded proteins in the cytoplasm are trafficked into the juxtanuclear quality control compartment (JUNQ), and nuclear proteins are sequestered into the intranuclear quality control compartment (INQ). However, the mechanisms that control the formation, localization, and degradation of these compartments are unknown. Previously, we showed that the JUNQ migrates to the nuclear membrane adjacent to the INQ at nucleus-vacuole junctions (NVJ), and the INQ moves through the NVJ into the vacuole for clearance in an ESCRT-mediated process. Here we have investigated what mechanisms are involved in the formation, migration, and clearance of the JUNQ. We find Hsp70s Ssa1 and Ssa2 are required for JUNQ localization to the NVJ and degradation of cytoplasmic misfolded proteins. We also confirm that sequestrases Btn2 and Hsp42 sort misfolded proteins to the JUNQ or IPOD, respectively. Interestingly, proteins required for piecemeal microautophagy of the nucleus (PMN) (i.e., Nvj1, Vac8, Atg1, and Atg8) drive the formation and clearance of the JUNQ. This suggests that the JUNQ migrates to the NVJ to be cleared via microautophagy.

Keywords: microautophagy; piecemeal microautophagy of the nucleus; protein misfolding; proteostasis; spatial sequestration.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Hsp40s and 70s are important for sorting of cytoplasmic misfolded proteins to compartments. (A) Schematic showing a temperature sensitive mutant of Luciferase with a nuclear export signal tag (NES-LuciTs) misfolds when temperature is shifted to 37°C. We are investigating what pathways dictate the coalescence, sequestration, and clearance of NES-LuciTs. (B) Area under the curve (AUC) analysis of growth curves of wildtype BY4741 and chaperone deletion strains expressing NES-LuciTs at 37°C in 2% galactose and 2% raffinose selective media normalized to 2% glucose controls. Paired t-tests: *P-value<0.02. **P-value<0.001 (C) Time-resolved live-cell microcopy of wildtype BY4741 and chaperone deletion strains expressing NES-LuciTs at 37°C (magenta). Representative still frames at the times shown. Three replicates were performed. (D) Percentage of cells with puncta over time. (E) Percentage of cells with puncta at 60 min. Paired t-tests: *p-value<0.05. All error bars represent standard deviation.
FIGURE 2
FIGURE 2
Hsp70s are required for JUNQ formation. (A–C) 3D reconstructions of cells expressing NES-LuciTs (magenta) created in Volocity. (A) Representative images of nuclear pore staining with anti-Nsp1 (gold) on fixed cells in DAPI staining (blue) after 2 h heat shock with MG132 treatment. (B) FM4-64 vacuole staining of live cells (gray) with Hoechst 33342 staining (blue) after 20 min heat shock with MG132 treatment. (C) Representative images of MitoTracker Red CMXRos staining of live cells (cyan) with Hoechst 33342 staining (blue) after 20 min heat shock with MG132 treatment. All scale bars are 1 μm. (D, E) Photobleaching experiments on ssa1Δssa2Δ cells expressing NES-LuciTs or BY4741 wildtype cells expressing Flag-97QP-GFP as an IPOD control. Cells were imaged pre-bleach, bleached for 5 s, and imaged every 2 s for 90 s post-bleach. (E) Intensity of the inclusion over time normalized to the pre-bleach intensity. (F) Representative Western blot of NES-LuciTs in ssa1Δssa2Δ. Samples were collected at 0, 2, and 4 h after shifting to 37°C. Cells were treated with or without 50 μM bortezomib to inhibit the proteasome. Anti-GFP antibody was used to detect NES-LuciTs (1 s exposure). Anti-GAPDH antibody was used to detect GAPDH (1 s exposure). (G, H) Quantification of NES-LuciTs bands. Band intensities were measured using ImageJ and normalized to GAPDH control. Degradation ratios between 2 h (G) or 4 h (H) to time 0 were calculated and normalized to wildtype to allow for comparison between blots. Unpaired t-test: **P-value = 0.0061. *P-value = 0.0392. Error bars represent standard error of the mean.
FIGURE 3
FIGURE 3
Overexpression of chaperones does not affect NES-LuciTs localization. (A) Time-resolved live-cell microscopy of wildtype BY4741 expressing NES-LuciTs (magenta) and overexpressing GFP-tagged chaperones or GFP alone (green). Representative still frames at the times shown. Three replicates were performed. (B) Percentage of cells with puncta from the time-resolved imaging at 60 min. One-way ANOVA F = 0.5495, p-value = 0.6625. (C) Representative images of nuclear pore staining with anti-Nsp1 on fixed cells in DAPI staining (blue) after 2 h heat shock with MG132 treatment. (D) Representative images of MitoTracker Deep Red FM staining of live cells (cyan) with Hoechst 33342 staining (blue) after 20 min heat shock with MG132 treatment. All scale bars are 1 μm. (E) Area under the curve (AUC) analysis of growth curves of wildtype BY4741 overexpressing EGFP, Sis1, Ssa1, or Hsc82 and expressing NES-LuciTs at 37°C in 2% galactose and 2% raffinose selective media normalized to 2% glucose controls. One-way ANOVA F = 0.7747, p-value = 0.5401.
FIGURE 4
FIGURE 4
Hsp42 and Btn2 are involved in the sorting of NES-LuciTs. (A) Time-resolved live-cell microcopy of wildtype BY4741, btn2Δ, and hsp42Δ expressing NES-LuciTs at 37°C (magenta). Representative still frames at the times shown. Three replicates were performed. (B) Percentage of cells with puncta over time. (C) Percentage of cells with puncta at 0 min. One-way ANOVA F = 5.920, p-value = 0.0380. Dunnett’s multiple comparison test: *p-value = 0.0357. (D) Percentage of cells with puncta at 60 min. One-way ANOVA F = 6.504, p-value = 0.0038. Dunnett’s multiple comparison test: *p-value = 0.0323. Error bars represent standard error of the mean. (E–G) 3D reconstructions of cells expressing NES-LuciTs (magenta) created in Volocity. (E) Representative images of nuclear pore staining with anti-Nsp1 (gold) on fixed cells in DAPI staining (blue) after 2 h heat shock with MG132 treatment. (F) Representative images of FM4-64 vacuole staining of live cells (gray) with Hoechst 33342 staining (blue) after 20 min heat shock with MG132 treatment. (G) Representative images of MitoTracker Red CMXRos staining of live cells (cyan) with Hoechst 33342 staining (blue) after 20 min heat shock with MG132 treatment. All scale bars are 1 μm.
FIGURE 5
FIGURE 5
Autophagy proteins Atg1 and Atg8 are involved in the clearance of NES-Luci. (A) Time-resolved live-cell microcopy of wildtype BY4741, atg1Δ, and atg8Δ expressing NES-LuciTs at 37°C (magenta). Three replicates were performed. (B) Percentage of cells with puncta over time. (C) Percentage of cells with puncta at 30 min. One-way ANOVA F = 14.85, p-value<0.0001. Dunnett’s multiple comparison test: ***p-value = 0.0007. ****p-value<0.0001. (D) Percentage of cells with puncta at 60 min. One-way ANOVA F = 7.635, p-value = 0.0019. Dunnett’s multiple comparison test: **p-value = 0.004. Error bars represent standard error of the mean. (E) 3D reconstructions of cells expressing NES-LuciTs (magenta) with FM4-64 (gray) and Hoechst 33342 (blue) staining after 20 min heat shock with MG132 treatment. Created using Volocity. All scale bars are 1 μm. (F, G) Representative Western blots of NES-LuciTs in atg1Δ (F) and atg8Δ (G). Samples were collected at 0, 2, and 4 h after shifting to 37°C. Cells were treated with or without 50 μM bortezomib to inhibit the proteasome. Anti-GFP antibody was used to detect NES-LuciTs (1 s exposure). Anti-GAPDH antibody was used to detect GAPDH (1 s exposure).
FIGURE 6
FIGURE 6
Model showing chaperones involved in the sequestration of misfolded cytoplasmic proteins resulting in the formation of Q-bodies, JUNQ, and IPOD. The JUNQ resides at the nucleus-vacuole junction (NVJ) where autophagy proteins such as Atg1 and Atg8 and NVJ proteins Nvj1 and Vac8 are likely involved in the vacuolar degradation of the JUNQ.
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References

    1. Alberti S., Gitler A. D., Lindquist S. (2007). A suite of Gateway cloning vectors for high-throughput genetic analysis in Saccharomyces cerevisiae . Yeast 24, 913–919. 10.1002/yea.1502 - DOI - PMC - PubMed
    1. Backe S. J., Sager R. A., Heritz J. A., Wengert L. A., Meluni K. A., Aran-Guiu X., et al. (2023). Activation of autophagy depends on Atg1/Ulk1-mediated phosphorylation and inhibition of the Hsp90 chaperone machinery. Cell Rep. 42, 112807. 10.1016/j.celrep.2023.112807 - DOI - PMC - PubMed
    1. Bar-Lavan Y., Kosolapov L., Frumkin A., Ben-Zvi A. (2012). Regulation of cellular protein quality control networks in a multicellular organism. FEBS J. 279, 526–531. 10.1111/j.1742-4658.2011.08455.x - DOI - PubMed
    1. Ben-Zvi A., Miller E., Morimoto R. I. (2009). Collapse of proteostasis represents an early molecular event in Caenorhabditis elegans aging. Proc. Natl. Acad. Sci. U. S. A. 106, 14914–14919. 10.1073/pnas.0902882106 - DOI - PMC - PubMed
    1. Bhattacharya K., Picard D. (2021). The Hsp70-Hsp90 go-between Hop/Stip1/Sti1 is a proteostatic switch and may be a drug target in cancer and neurodegeneration. Cell Mol. Life Sci. 78, 7257–7273. 10.1007/s00018-021-03962-z - DOI - PMC - PubMed

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The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. Marquette University Way-Klingler Startup funds.

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