26 research outputs found
Guk1-7 is thermally unstable.
<p>(A) Ribbon structure of Guk1 (PDB 1EX7). Positions of the four missense mutations and predicted ∆∆G values are indicated. Loss of fluorescence measured by flow cytometry after a two hour incubation at 37°C with cycloheximide is indicated in brackets. (B) Cellular thermal shift assay of Guk1 and Guk1-7 fused to a six histidine tag in lysates derived from cells grown at 25°C. One representative anti-His Western Blot is shown. The graph represents the means and standard deviations of Guk1 levels from three independent experiments. (C) Guk1 and Guk1-7 fused to a six histidine tag were expressed in cells grown at 25°C or shifted to 37°C for 20 min. Total cell lysate (T), soluble (S), and pellet fractions (P) were immunoblotted with anti-His antibodies.</p
Guk1-7 degradation is proteasome dependent.
<p>(A) Flow cytometry profiles of wild type cells expressing Guk1-GFP or Guk1-7-GFP were incubated at 25°C or 37°C for two hours in the presence of CHX. Fluorescence in cells with the control empty vector (EV) are also shown. Lines demark median GFP fluorescence values and i denotes the difference in median intensity values used to measure protein stability. (B) Comparison of quantitation of Guk1-7 levels in a CHX chase assay by Western blot or flow cytometry. (C) Wild type and <i>rpt6-20</i> cells expressing Guk1-7-GFP were incubated with CHX at 25°C or 37°C and samples were analysed by flow cytometry at the indicated time points. The results represent the means and standard deviations of three independent experiments. (D) Guk1-7-GFP expressing wild type or <i>pep4∆prb1∆</i> cells were incubated at 25°C or 37°C in the presence of CHX and samples were analyzed by flow cytometry at the indicated time points. The results represent the means and standard deviations of three independent experiments. (E) <i>rpt6-20</i> cell expressing Guk1-GFP or Guk1-7-GFP were grown at 25°C and then shifted to 37°C for 1 hour prior to their fixation and imaging. Scale bar represents 5μm. (F) Guk1-GFP and Guk1-7-GFP expressing cells were incubated at 25°C and cell lysates were immunoprecipitated using GFP-Trap beads and then immunoblotted with anti-ubiquitin, anti-GFP, and anti-Pgk1 antibodies.</p
Thermosensitive alleles are stabilized by prefoldin subunits.
<p>(A) Individual prefoldin subunit mutants expressing Guk1-7-GFP were incubated at 37°C in the presence of CHX for 2 hours. The percentage of cells with puncta was calculated from 30–50 GFP positive cells. Scale bar represents 5μm. (B) Four thermosensitive alleles were expressed as GFP fusion proteins in the six prefoldin deletion strains. Cells were incubated with CHX at 25°C and 37°C for 2 hours and fluorescence intensity was measured by flow cytometry. (C) Guk1-7-GFP was expressed in wild type, <i>gim3∆</i>, <i>tcp1-1</i>, or <i>tcp4-1</i> cells and incubated with CHX for two hours at either 25°C or 37°C before flow cytometry analysis. (D) Wild type, <i>gim3∆</i>, <i>tcp1-1</i>, and <i>tcp4-1</i> cells expressing Guk1-7-GFP were incubated with CHX for 2 hours at 25°C or 37°C prior to fixation and imaging. Scale bar represents 5μm.</p
Prefoldin Promotes Proteasomal Degradation of Cytosolic Proteins with Missense Mutations by Maintaining Substrate Solubility
<div><p>Misfolded proteins challenge the ability of cells to maintain protein homeostasis and can accumulate into toxic protein aggregates. As a consequence, cells have adopted a number of protein quality control pathways to prevent protein aggregation, promote protein folding, and target terminally misfolded proteins for degradation. In this study, we employed a thermosensitive allele of the yeast Guk1 guanylate kinase as a model misfolded protein to investigate degradative protein quality control pathways. We performed a flow cytometry based screen to identify factors that promote proteasomal degradation of proteins misfolded as the result of missense mutations. In addition to the E3 ubiquitin ligase Ubr1, we identified the prefoldin chaperone subunit Gim3 as an important quality control factor. Whereas the absence of <i>GIM3</i> did not impair proteasomal function or the ubiquitination of the model substrate, it led to the accumulation of the poorly soluble model substrate in cellular inclusions that was accompanied by delayed degradation. We found that Gim3 interacted with the Guk1 mutant allele and propose that prefoldin promotes the degradation of the unstable model substrate by maintaining the solubility of the misfolded protein. We also demonstrated that in addition to the Guk1 mutant, prefoldin can stabilize other misfolded cytosolic proteins containing missense mutations.</p></div
Model for stabilization of temperature sensitive alleles by Gim3.
<p>(A) Proposed model for how Gim3 promotes degradation of temperature sensitive alleles destabilized by missense mutations.</p
Guk1-7-GFP puncta colocalize with Q-body markers.
<p>(A) Cells with Hsp104 endogenously tagged with mCherry and ectopically expressing Guk1-GFP or Guk1-7-GFP were grown at 25°C and then incubated at 25°C or 37°C for 30 minutes before fixation and imaging. Scale bar represents 5μm. (B) Hsp42-mCherry cells ectopically expressing Guk1-GFP or Guk1-7-GFP were grown at 25°C prior to incubation at 25°C or 37°C for 30 minutes. Cells were then fixed before imaging. The scale bar represents 5μm. (C) Enlarged images from cells collected as in A. Scale bar represents 2.5μm. (D) Enlarged images from cells collected as in B. Scale bar represents 2.5μm.</p
Gim3 facilitates clearance of insoluble Guk1-7.
<p>(A) <i>GIM3</i> or <i>gim3∆</i> cells expressing Guk1-7-GFP were grown at 25°C and then incubated at either 25°C or 37°C for 2 hours in the presence of CHX before fixation and imaging. (B) <i>GIM3</i> and <i>gim3∆</i> cells expressing Guk1-7-GFP along with an empty vector control or <i>GIM3</i> were incubated at 25°C or 37°C for two hours in the presence of CHX before fixation and imaging. (C) <i>GIM3</i> or <i>gim3∆</i> cells expressing Guk1-7-GFP were incubated at 25°C and then shifted to 37°C. Samples were collected at the indicated time points and then fixed before imaging. For all images, the scale bar represents 5μm and dotted lines demark cell boundaries.</p
Gim3 helps maintain Guk1-7 solubility.
<p>(A) <i>GIM3</i> or <i>gim3∆</i> Guk1-7-GFP expressing cells were grown at 25°C or shifted to 37°C for 20 min. The ratio of the pellet fraction to total cell lysate is noted and represents the mean and standard deviation of three independent experiments. (B) Guk1-7-GFP was immunoprecipitated from Gim3-TAP expressing cells incubated at 25°C and then immunoblotted with anti-TAP, anti-GFP, or anti-Pgk1 antibodies.</p
Ubr1 promotes Guk1-7-GFP degradation.
<p>(A) Wild type and <i>ubr1∆</i> cells expressing Guk1-7-GFP were incubated with CHX at 25°C or 37°C and samples were analysed by flow cytometry at the indicated time points. The results represent the means and standard deviations of three independent experiments. P values were calculated with an unpaired Student’s t test, *, ** and *** denote P < 0.05, 0.005, and 0.0005, respectively. (B) <i>UBR1</i> and <i>ubr1∆</i> cells expressing Guk1-7-GFP along with an empty vector (EV) control or <i>UBR1</i> were incubated at 37°C and samples were collected at the indicated time points for flow cytometry analysis. Results represent the means and standard deviations of three independent experiments. P values were calculated with a one-way ANOVA and post-hoc Tukey HSD to assess significance, ** denotes P < 0.005. (C) <i>ubr1∆</i> cells coexpressing Guk1-7-GFP and an empty vector control or either <i>UBR1</i> or <i>UBR1</i> (C1220S) were incubated at 37°C with CHX and samples were collected at the indicated time points. (D) Wild type or <i>ubr1∆</i> cells expressing Guk1 (T290G) fused to GFP were incubated with CHX at 37°C for two hours before being analyzed by flow cytometry. The results represent the relative fluorescence intensities from three independent experiments (with standard deviations). P values were calculated with a one-way ANOVA and post-hoc Tukey HSD to assess significance, *** denotes P < 0.0005.</p