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The Hsp70/90 cochaperone, Sti1, suppresses proteotoxicity by regulating spatial quality control of amyloid-like proteins.

Wolfe KJ, Ren HY, Trepte P, Cyr DM - Mol. Biol. Cell (2013)

Bottom Line: Under toxic conditions, Htt103Q accumulates in unassembled states and speckled cytosolic foci.Sti1 also suppresses cytotoxicity of the glutamine-rich yeast prion [RNQ+] while reorganizing speckled Rnq1-monomeric red fluorescent protein into distinct foci.Sti1 is an Hsp70 cochaperone that regulates the spatial organization of amyloid-like proteins in the cytosol and thereby buffers proteotoxicity caused by amyloid-like proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular Biology and Physiology, University of North Carolina, Chapel Hill, NC 27599 Neuroproteomics, Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany.

ABSTRACT
Conformational diseases are associated with the conversion of normal proteins into aggregation-prone toxic conformers with structures similar to that of β-amyloid. Spatial distribution of amyloid-like proteins into intracellular quality control centers can be beneficial, but cellular mechanisms for protective aggregation remain unclear. We used a high-copy suppressor screen in yeast to identify roles for the Hsp70 system in spatial organization of toxic polyglutamine-expanded Huntingtin (Huntingtin with 103Q glutamine stretch [Htt103Q]) into benign assemblies. Under toxic conditions, Htt103Q accumulates in unassembled states and speckled cytosolic foci. Subtle modulation of Sti1 activity reciprocally affects Htt toxicity and the packaging of Htt103Q into foci. Loss of Sti1 exacerbates Htt toxicity and hinders foci formation, whereas elevation of Sti1 suppresses Htt toxicity while organizing small Htt103Q foci into larger assemblies. Sti1 also suppresses cytotoxicity of the glutamine-rich yeast prion [RNQ+] while reorganizing speckled Rnq1-monomeric red fluorescent protein into distinct foci. Sti1-inducible foci are perinuclear and contain proteins that are bound by the amyloid indicator dye thioflavin-T. Sti1 is an Hsp70 cochaperone that regulates the spatial organization of amyloid-like proteins in the cytosol and thereby buffers proteotoxicity caused by amyloid-like proteins.

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Sti1-inducible foci colocalize with markers for cytosolic protein-handling depots. (A, B) Localization of endogenous (A) Hsp104 or (B) Hsp42 as monitored by immunofluorescence using Htt103Q as a StiF marker. (C, D) Effect of Hsp104 inhibition on Rnq1-mRFP localization at the StiF. Fluorescence microscopy was carried out in strains with (C) Spc42 or (D) Hsp42 tagged at the endogenous locus with GFP. Rnq1-mRFP was induced with 50 μM CuSO4, and Hsp104 was inhibited with 3 mM GndHCl for 2 h before cells were fixed. Nuclei were visualized using DAPI staining. Dotted lines indicate the outline of the cell.
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Figure 5: Sti1-inducible foci colocalize with markers for cytosolic protein-handling depots. (A, B) Localization of endogenous (A) Hsp104 or (B) Hsp42 as monitored by immunofluorescence using Htt103Q as a StiF marker. (C, D) Effect of Hsp104 inhibition on Rnq1-mRFP localization at the StiF. Fluorescence microscopy was carried out in strains with (C) Spc42 or (D) Hsp42 tagged at the endogenous locus with GFP. Rnq1-mRFP was induced with 50 μM CuSO4, and Hsp104 was inhibited with 3 mM GndHCl for 2 h before cells were fixed. Nuclei were visualized using DAPI staining. Dotted lines indicate the outline of the cell.

Mentions: Sti1-inducible foci (StiF) are perinuclear and contain amyloid-like material, yet it is unclear whether they have features of protein-handling depots such as the IPOD and JUNQ. To characterize the StiF in more detail, we determined whether these foci contain PQC machinery that would help protect cells from proteotoxic stress. Under normal conditions, Hsp104 and Hsp42 were not detected in the speckled foci that contain Htt103Q-GFP (Figure 5, A and B), but Hsp104 and Hsp42 were present in StiF. Sti1 expression caused Hsp104 to become concentrated in Stif because its signal intensity in the cytosol versus the Stif has a 1:1.5 ratio. Although Hsp42 did colocalize with Htt103Q, the signal for Hsp42 in foci and cytosol had a 1:1 ratio, so it is difficult to judge whether the colocalization observed is due to overlap in staining or concentration of Hsp42 to the StiF.


The Hsp70/90 cochaperone, Sti1, suppresses proteotoxicity by regulating spatial quality control of amyloid-like proteins.

Wolfe KJ, Ren HY, Trepte P, Cyr DM - Mol. Biol. Cell (2013)

Sti1-inducible foci colocalize with markers for cytosolic protein-handling depots. (A, B) Localization of endogenous (A) Hsp104 or (B) Hsp42 as monitored by immunofluorescence using Htt103Q as a StiF marker. (C, D) Effect of Hsp104 inhibition on Rnq1-mRFP localization at the StiF. Fluorescence microscopy was carried out in strains with (C) Spc42 or (D) Hsp42 tagged at the endogenous locus with GFP. Rnq1-mRFP was induced with 50 μM CuSO4, and Hsp104 was inhibited with 3 mM GndHCl for 2 h before cells were fixed. Nuclei were visualized using DAPI staining. Dotted lines indicate the outline of the cell.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 5: Sti1-inducible foci colocalize with markers for cytosolic protein-handling depots. (A, B) Localization of endogenous (A) Hsp104 or (B) Hsp42 as monitored by immunofluorescence using Htt103Q as a StiF marker. (C, D) Effect of Hsp104 inhibition on Rnq1-mRFP localization at the StiF. Fluorescence microscopy was carried out in strains with (C) Spc42 or (D) Hsp42 tagged at the endogenous locus with GFP. Rnq1-mRFP was induced with 50 μM CuSO4, and Hsp104 was inhibited with 3 mM GndHCl for 2 h before cells were fixed. Nuclei were visualized using DAPI staining. Dotted lines indicate the outline of the cell.
Mentions: Sti1-inducible foci (StiF) are perinuclear and contain amyloid-like material, yet it is unclear whether they have features of protein-handling depots such as the IPOD and JUNQ. To characterize the StiF in more detail, we determined whether these foci contain PQC machinery that would help protect cells from proteotoxic stress. Under normal conditions, Hsp104 and Hsp42 were not detected in the speckled foci that contain Htt103Q-GFP (Figure 5, A and B), but Hsp104 and Hsp42 were present in StiF. Sti1 expression caused Hsp104 to become concentrated in Stif because its signal intensity in the cytosol versus the Stif has a 1:1.5 ratio. Although Hsp42 did colocalize with Htt103Q, the signal for Hsp42 in foci and cytosol had a 1:1 ratio, so it is difficult to judge whether the colocalization observed is due to overlap in staining or concentration of Hsp42 to the StiF.

Bottom Line: Under toxic conditions, Htt103Q accumulates in unassembled states and speckled cytosolic foci.Sti1 also suppresses cytotoxicity of the glutamine-rich yeast prion [RNQ+] while reorganizing speckled Rnq1-monomeric red fluorescent protein into distinct foci.Sti1 is an Hsp70 cochaperone that regulates the spatial organization of amyloid-like proteins in the cytosol and thereby buffers proteotoxicity caused by amyloid-like proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular Biology and Physiology, University of North Carolina, Chapel Hill, NC 27599 Neuroproteomics, Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany.

ABSTRACT
Conformational diseases are associated with the conversion of normal proteins into aggregation-prone toxic conformers with structures similar to that of β-amyloid. Spatial distribution of amyloid-like proteins into intracellular quality control centers can be beneficial, but cellular mechanisms for protective aggregation remain unclear. We used a high-copy suppressor screen in yeast to identify roles for the Hsp70 system in spatial organization of toxic polyglutamine-expanded Huntingtin (Huntingtin with 103Q glutamine stretch [Htt103Q]) into benign assemblies. Under toxic conditions, Htt103Q accumulates in unassembled states and speckled cytosolic foci. Subtle modulation of Sti1 activity reciprocally affects Htt toxicity and the packaging of Htt103Q into foci. Loss of Sti1 exacerbates Htt toxicity and hinders foci formation, whereas elevation of Sti1 suppresses Htt toxicity while organizing small Htt103Q foci into larger assemblies. Sti1 also suppresses cytotoxicity of the glutamine-rich yeast prion [RNQ+] while reorganizing speckled Rnq1-monomeric red fluorescent protein into distinct foci. Sti1-inducible foci are perinuclear and contain proteins that are bound by the amyloid indicator dye thioflavin-T. Sti1 is an Hsp70 cochaperone that regulates the spatial organization of amyloid-like proteins in the cytosol and thereby buffers proteotoxicity caused by amyloid-like proteins.

Show MeSH
Related in: MedlinePlus