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.2007 Jan;18(1):153-65.
doi: 10.1091/mbc.e06-04-0338. Epub 2006 Oct 25.

The cytoplasmic Hsp70 chaperone machinery subjects misfolded and endoplasmic reticulum import-incompetent proteins to degradation via the ubiquitin-proteasome system

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The cytoplasmic Hsp70 chaperone machinery subjects misfolded and endoplasmic reticulum import-incompetent proteins to degradation via the ubiquitin-proteasome system

Sae-Hun Park et al. Mol Biol Cell.2007 Jan.

Abstract

The mechanism of protein quality control and elimination of misfolded proteins in the cytoplasm is poorly understood. We studied the involvement of cytoplasmic factors required for degradation of two endoplasmic reticulum (ER)-import-defective mutated derivatives of carboxypeptidase yscY (DeltassCPY* and DeltassCPY*-GFP) and also examined the requirements for degradation of the corresponding wild-type enzyme made ER-import incompetent by removal of its signal sequence (DeltassCPY). All these protein species are rapidly degraded via the ubiquitin-proteasome system. Degradation requires the ubiquitin-conjugating enzymes Ubc4p and Ubc5p, the cytoplasmic Hsp70 Ssa chaperone machinery, and the Hsp70 cochaperone Ydj1p. Neither the Hsp90 chaperones nor Hsp104 or the small heat-shock proteins Hsp26 and Hsp42 are involved in the degradation process. Elimination of a GFP fusion (GFP-cODC), containing the C-terminal 37 amino acids of ornithine decarboxylase (cODC) directing this enzyme to the proteasome, is independent of Ssa1p function. Fusion of DeltassCPY* to GFP-cODC to form DeltassCPY*-GFP-cODC reimposes a dependency on the Ssa1p chaperone for degradation. Evidently, the misfolded protein domain dictates the route of protein elimination. These data and our further results give evidence that the Ssa1p-Ydj1p machinery recognizes misfolded protein domains, keeps misfolded proteins soluble, solubilizes precipitated protein material, and escorts and delivers misfolded proteins in the ubiquitinated state to the proteasome for degradation.

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Figures

Figure 1.
Figure 1.
The Hsp70 chaperone machinery of Ssa1p is required for the degradation of cytoplasmically localized misfolded proteins. Pulse-chase analysis was done inSSA1 andssa1-45ts cells. Cells expressing the substrates were lysed at the indicated times, and proteins were immunoprecipitated with anti CPY (A–C) or anti GFP (A), separated by SDS-PAGE, and analyzed using a PhosphoImager and ImagerQuaNT (Amersham Bioscience). Plotted data represent the mean values of three independent experiments. Substrates: A: ΔssCG*; B: ΔssGC*; C: ΔssCG*uv. The ERQD substrate CPY* served as a control.
Figure 2.
Figure 2.
Degradation of misfolded and ER import incompetent CPY* is dependent on the proteasome and Ssa1p but not on the Cdc48-Ufd1-Npl4 complex. Cycloheximide decay experiments were performed in the proteasomal mutantcim3-1 (A) and inufd1-1 cells (B) expressing ΔssCPY*. Cycloheximide was added (t = 0 min), and samples were collected at the indicated time points and subjected to SDS-PAGE, followed by immunoblotting. Immunoblots were analyzed with anti-CPY and anti-PGK as a loading control. Pulse-chase analysis inSSA1 andssa1-45ts cells (C) was performed and analyzed as described in the legend to Figure 1. The ERQD substrate CPY* served as a control.
Figure 3.
Figure 3.
Ubiquitin-independent degradation of GFP-cODC does not require Ssa1p activity, but its fusion to ΔssCPY* makes the process Ssa1p dependent. Pulse-chase analysis was done inSSA1 andssa1-45ts cells expressing GFPuv-cODC (A), GFP-cODC (B), ΔssCG*-cODC (C), and ΔssCG*-cODC-C441A (D).
Figure 4.
Figure 4.
The fate of the cytoplasmically mislocalized wild-type CPY is similar to its mutated counterpart. Cycloheximide decay experiments (A and B) and pulse-chase analysis (C) were performed as described in the legend to Figure 2.
Figure 5.
Figure 5.
The Hsp70 cochaperone Ydj1p promotes the degradation of cytoplasmically localized misfolded proteins. Pulse-chase analysis was performed in wild-type (WT) andydj1-151ts cells expressing ΔssCG* (A), ΔssCPY* (B), and ΔssCPY (C).
Figure 6.
Figure 6.
The Hsp70 Ssb class; the Hsp90 complex, Hsp104, Hsp110; small heat shock proteins Hsp26, Hsp42; and the yeast Bag1 homologue, Snl1p, are not involved in the degradation of ΔssCG*. Pulse-chase analysis was done in Δssb1Δssb2 (A), Δhsc82hsp82G170D (B), Δhsp104 (E), Δhsp26Δhsp42 (F), and Δsnl1 (G) cells expressing ΔssCG*, and cycloheximide decay experiments were performed insti1-1 (C) and Δsse1 (D) cells expressing ΔssCG* as described in the legend to Figure 2. PGK and CPY were served as a loading control.
Figure 7.
Figure 7.
Ssa1p and its cochaperone Ydj1p are required for rescue of aggregated ΔssCG*. Cells expressing ΔssCG* were grown at 30°C and shifted to 37°C for 60 min before the solubility assay. The solubility of ΔssCG* was assessed inSSA1, ssa1-45ts (A), wild-typeW303-1C (SSA1, SSA2, SSA3, SSA4), andydj1-151ts strains (B). The same amount of total (T), supernatant (S), and pellet (P) fraction was analyzed via SDS-PAGE and immunoblot. Immunoblots were analyzed with CPY antibody and PGK antibody as a control. The fluorescence of ΔssCG* was analyzed in living cells (C) as described inMaterial and Methods. The cells harboring overexpressed ΔssCG* or an empty plasmid were grown at 30°C and shifted to 37°C for 60 min before analysis. All the cells were visualized by fluorescence microscopy using equal exposure times and conditions. Resolubilization of aggregated ΔssCG* was assessed inSSA1 andssa1-45ts cells (D). After temperature shift of cells to 37°C for 1 h, cycloheximide was added to a final concentration of 0.5 mg/ml to block further protein synthesis. Twenty OD600 of cells were taken at the indicated time points and treated as indicated for the above solubility assay. Immunoblot of Sec61p served as control. Three independent experiments gave similar results. The fluorescence of GFP-cODC and GFP-cODC-C414A were analyzed inSSA1 cells at 37°C as stated above (E).
Figure 8.
Figure 8.
The state of ubiquitinated misfolded proteins in wild-type,SSA1,ssa1-45ts, andydj1-151ts cells at the different temperature of 25 and 37°C. The cells harboring overexpressed ΔssCG* or an empty plasmid (control) were grown at 25°C (A) and shifted to 37°C (B) for 60 min before analysis. Cell extracts were immunoprecipitated with anti-GFP antibody, separated by SDS-PAGE, followed by immunoblotting, and analyzed with anti-ubiquitin or anti-CPY antibodies.
Figure 9.
Figure 9.
Degradation of ΔssCG* requires the E2 proteins Ubc4p and Ubc5p but not the E3 ligases Doa10p and Der3p. Pulse-chase analysis was done in Δubc4Δubc5 mutant cells (A) and cycloheximide decay experiments were performed inΔdoa10Δder3 cells (B) as described in the legend to Figure 2. CPY served as a loading control.
Figure 10.
Figure 10.
Model of protein quality control in the cytoplasm. See text for details.
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