Compartment-specific aggregases direct distinct nuclear and cytoplasmic aggregate deposition

Abstract

Disruption of the functional protein balance in living cells activates protective quality control systems to repair damaged proteins or sequester potentially cytotoxic misfolded proteins into aggregates. The established model based on Saccharomyces cerevisiae indicates that aggregating proteins in the cytosol of eukaryotic cells partition between cytosolic juxtanuclear (JUNQ) and peripheral deposits. Substrate ubiquitination acts as the sorting principle determining JUNQ deposition and subsequent degradation. Here, we show that JUNQ unexpectedly resides inside the nucleus, defining a new intranuclear quality control compartment, INQ, for the deposition of both nuclear and cytosolic misfolded proteins, irrespective of ubiquitination. Deposition of misfolded cytosolic proteins at INQ involves chaperone-assisted nuclear import via nuclear pores. The compartment-specific aggregases, Btn2 (nuclear) and Hsp42 (cytosolic), direct protein deposition to nuclear INQ and cytosolic (CytoQ) sites, respectively. Intriguingly, Btn2 is transiently induced by both protein folding stress and DNA replication stress, with DNA surveillance proteins accumulating at INQ. Our data therefore reveal a bipartite, inter-compartmental protein quality control system linked to DNA surveillance via INQ and Btn2.

Keywords: chaperones; protein aggregation; protein disaggregation; proteostasis; ubiquitin–proteasome system.

Figure 1

Cytosolic and nuclear misfolded proteins share INQ (JUNQ) as a common deposition site

1. S. cerevisiae wt or hsp42Δ cells expressing mCherry-VHL (red) and GFP-luciferase-DM-NLS (green) were grown at 30°C and shifted to 37°C for 90 min. MG132 was added prior to temperature upshift. Changes in protein localizations were recorded. DNA was stained by DAPI (blue).

2. S. cerevisiae wt cells or hsp42Δ cells expressing GFP-VHL (green) were treated as in (A). Nuclear membranes were visualized by Nsp1 immunofluorescence labeling (red).

3. S. cerevisiae wt cells or hsp42Δ cells expressing mCherry-VHL (red) and GFP-Nup49 (green) were treated as in (A). Three-dimensional reconstructions of respective cells are given.

4. Time-lapse microscopy of wt cells expressing mCherry-VHL (green) and GFP-Nup49 (red) upon temperature upshift from 30°C to 37°C. * indicates collision of peripheral foci leading to fusion, and an arrow indicates apparent encounters of peripheral foci and nuclear INQ, followed by foci separation.

Data information: Scale bars, 2 μm.

Figure 2

INQ is located inside the nucleus

1. Cryo-sections of (A) S. cerevisiae wt cells expressing GFP-luciferase-DM-NLS, (B) hsp42Δ cells expressing GFP-VHL, and (C, D) wt or hsp42Δ cells expressing Hsp104-GFP. Sections were immunogold-labeled with GFP-specific antibodies. Gold particles are marked (black arrows). Electron-dense regions represent protein aggregates (A). Locations of cytosol (C), nuclear envelope (NE, orange arrows), and nucleus (N) are given. Scale bars, 200 nm.

2. Average numbers of gold particles associated with nuclear and cytosolic protein aggregates or distributed throughout the cytosol or nucleus were determined.

Figure 3

INQ formation does not involve ubiquitination

1. S. cerevisiae wt cells expressing GFP-VHL (green) were grown at 30°C and shifted to 37°C for 90 min in the presence of MG132. Ubiquitin (red) was stained by immunofluorescence using specific antibodies. DNA was stained by DAPI (blue). Changes in protein localizations were recorded. Scale bars, 2 μm.

2. tGnd1-GFP is stabilized in ubr1Δ san1Δ cells. S. cerevisiae wt and ubr1Δ san1Δ mutant cells expressing tGnd1-GFP were grown at 30°C. Cycloheximide was added, and protein levels were determined at the indicated time points by Western blot using GFP-specific antibodies. Zwf1 levels are given as a loading control.

3. Ubiquitination status of tGnd1-GFP expressed in S. cerevisiae wt and ubr1Δ san1Δ cells was determined by GFP pull-down and Western blot analysis using ubiquitin-specific antibodies. Levels of isolated tGnd1-GFP after GFP pull-down were determined by Western blot using GFP-specific antibodies and are given as a control.

4. S. cerevisisae wt, ubr1Δ san1Δ, and ubr1Δ san1Δ btn2Δ cells expressing tGnd1-GFP (green) were grown at 30°C. DNA and nuclear envelopes were visualized by coexpressing Htb1-mCherry or Nic96-mCherry, respectively (red). The fraction of cells showing specific tGnd1-GFP foci numbers per cell was determined. INQ formation in cells containing tGnd1-GFP foci was determined (% cells with INQ) based on close vicinity of tGnd1-GFP foci to Htb1-mCherry or localization inside Nic96-mCherry signals, yielding comparable results. Scale bars, 2 μm.

Figure 4

Defect in nuclear import delays INQ formation

1. S. cerevisiae hsp42Δ and hsp42Δ nup42Δ cells expressing mCherry-VHL (red) were grown at 30°C and shifted to 37°C in the presence of MG132. Changes in protein localization were monitored. Scale bars, 2 μm. The total number of mCherry-VHL foci per cell was determined.

2. Dynamic exchange of a misfolded model protein between cytosol and nucleus. FLIP measurements of mCherry-VHL were performed in S. cerevisiae wild-type cells at 30°C. Nuclear or cytosolic areas were bleached, and loss of fluorescence intensities in non-bleached compartments was determined. Bleaching and acquisition controls are given. Error bars: SEM.

Figure 5

Hsp70 co-chaperones Sis1 and Sti1 modulate protein aggregation

1. S. cerevisiae hsp42Δ and hsp42Δ tet-off sis1 cells expressing GFP-VHL were grown for 20 h in the absence (−Dox) or presence (+Dox) of doxycycline at 30°C and shifted for 90 min to 37°C in the presence of MG132. Changes in protein localization were monitored. DNA was stained by DAPI (blue). The total number of GFP-VHL foci per cell and frequencies (%) of INQ formation were determined. Scale bars, 2 μm.

2. S. cerevisiae wt, hsp42Δ, sti1Δ, and hsp42Δ sti1Δ cells expressing GFP-VHL were grown at 30°C and shifted to 37°C in the presence of MG132. Changes in protein localization were monitored. The total number of GFP-VHL foci per cell and frequencies (%) of INQ formation were determined. Scale bars, 2 μm.

Figure 6

Btn2 triggers protein aggregation inside the nucleus

1. S. cerevisiae wt, hsp42Δ, btn2Δ, and hsp42Δ btn2Δ cells expressing GFP-VHL (green, A) or GFP-luciferase-DM-NLS (green, B) were grown at 30°C and shifted to 37°C for 90 min in the presence of MG132, and protein localizations were recorded. DNA was stained by DAPI (blue), and the nuclear envelope was stained by Nsp1 immunofluorescence (red). Solubilities of GFP-VHL and GFP-luciferase-DM-NLS were determined after stress application by Western blot using GFP-specific antibodies. Solubility of actin was determined as a control. T, total fraction; S, soluble fraction; P, pellet fraction. Scale bars, 2 μm.

2. S. cerevisiae wt, hsp42Δ, btn2Δ, and hsp42Δ btn2Δ cells expressing mCherry-VHL were heat-shocked to 38°C. Upon return to 30°C, protein synthesis was stopped by cycloheximide (CHX) addition and degradation of mCherry-VHL was monitored by Western blot analysis. The stability of GFP-luciferase-DM-NLS expressed in S. cerevisiae wt and btn2Δ cells was determined accordingly. Ccs1 or Zwf1 levels are given as loading controls.

3. S. cerevisiae hsp42Δ btn2Δ cells expressing either GFP or LuciDM-GFP and Htb1-mCherry were grown at 30°C and heat-shocked to 37°C for 30 min. Line intensity plots of luciferase-DM-GFP and Htb1-mCherry before and after heat shock are given. The ratio of nuclear and cytosolic luciferase-DM-GFP fluorescence intensity was determined at 30°C and 37°C (n > 25). Scale bars, 2 μm.

Figure 7

Hsp42 and Btn2 are compartment-specific aggregases

1. Cellular localizations of Hsp42 and Btn2 were determined at the indicated temperature by immunofluorescence. DNA was stained by either Htb1-mCherry or DAPI. Scale bars, 2 μm. In case of Hsp42 immunofluorescence, a line intensity plot of a deconvoluted widefield image is given.

2. S. cerevisiae btn2Δ hsp42Δ Hsp42-NLS cells expressing GFP-VHL (green) were grown at 30°C and heat-shocked to 37°C for 30 min in the presence of MG132. Changes in protein localizations were recorded. DNA was stained by DAPI (blue). The total number of GFP-VHL foci per cell and frequencies (%) of INQ formation were determined. Scale bars, 2 μm.

3. S. cerevisiae btn2Δ hsp42Δ Hsp42-NLS cells expressing mCherry-VHL (red) were treated as described in (C). The nuclear membrane was visualized by Nsp1 immunofluorescence (green) and colocalization of mCherry-VHL foci with Hsp42 was probed by Hsp42 immunofluorescence (green). DNA was stained with DAPI (blue). Scale bars, 2 μm.

4. Hsp42 and Btn2 levels were determined prior and post-heat shock at the indicated time points by Western blot. The numbers of molecules/cell were calculated using purified proteins as standard.

Figure 8

MMS treatment leads to formation of Btn2-dependent non-canonical DNA stress foci at the INQ

1. S. cerevisiae wt cells were treated with MMS and Btn2 levels were determined at the indicated time points. Zwf1 levels are given as a loading control.

2. S. cerevisiae wt and btn2Δ cells expressing Hos2-GFP (green, B) or GFP-VHL (green, C) were grown at 30°C and treated with MMS. Changes in protein localizations were recorded at the indicated time points. DNA was stained by DAPI (blue). The total number of GFP-VHL foci per cell and frequencies (%) of INQ formation were determined.

Figure 9

Compartment-specific aggregases control protein aggregation in S. cerevisiae Misfolded proteins are deposited at cytosolic CytoQ and nuclear INQ compartments, while amyloidogenic proteins are sequestered at IPOD next to the vacuole. CytoQ formation depends on Hsp42. Misfolded, cytosolic proteins are imported through nuclear pore complexes by Sis1 and other, so far unknown sorting factors. Protein aggregation inside the nucleus is triggered by Btn2, which transiently accumulates upon stress. INQ is located adjacent to the nucleolus and harbors cytosolic and nuclear misfolded proteins. DNA replication stress causes formation of Btn2-dependent non-canonical DNA stress foci at the INQ and might signal to checkpoint controls.