Monitoring Protein Misfolding by Site-Specific Labeling of Proteins <i>In Vivo</i>

<div><p>Incorporating fluorescent amino acids by suppression of the TAG amber codon is a useful tool for site-specific labeling of proteins and visualizing their localization in living cells. Here we use a plasmid encoded orthogonal tRNA/aminoacyl-tRNA synthetase pair to site-specifically label firefly luciferase with the environmentally sensitive fluorescent amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2- aminopropanoic acid (ANAP) and explore the detectability of conformational changes in labeled luciferase in the yeast cytoplasm. We find that ANAP labeling efficiency is greatly increased in [<i>PSI</i>⁺] cells and show that analysis of the ANAP fluorescence emission by confocal imaging allows for tracking the thermal unfolding and aggregation of luciferase <i>in vivo</i>. Furthermore we demonstrate that flow cytometry can be used to study conformational changes in luciferase and chaperone-mediated refolding in quantitative terms and at the level of single cells. This experimental setup for the first time allows for the direct analysis of the folding state of a protein in living cells and may serve as valuable new tool for examining mechanisms of protein folding, misfolding and aggregation.</p></div

Changes in the folding state of Luc F161ANAP detected by flow cytometry.

<p>Normalized luciferase activity of cells expressing Luc F161ANAP before heat-shock (30°C) and during pre-shock (37°C), heat shock (45°C) and recovery (30°C). The activity value measured before the temperature was elevated from 30°C to 37°C was set to 100%. CHX, addition of cycloheximide for blocking protein synthesis. (A) Histogram of the ratio of green (510 nm) to blue (450 nm) fluorescence of yeast cells expressing Luc F161ANAP at different temperatures and time-points as described in (A). The red dash line aligns to the peak position before heat-induced unfolding of Luc F161ANAP. (B) Mean of the ratio of green (510 nm) to blue (450 nm) fluorescence light emission of about 5000 yeast cells each analyzed at indicated temperatures and time-points as described in (A).</p

Luciferase mutant F161ANAP retains wild-type like activity.

<p>Crystal structure of the <i>Photinus pyralis</i> firefly luciferase (PDB ID = 1LCI). Phenylalanine 161 mutated in Luc F161ANAP for the incorporation of 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (ANAP) is highlighted in blue. (A) Gel analysis of wild-type and ANAP-incorporated luciferase variants. Expression level of luciferase and mutant derivatives was analyzed by immunoblotting with antisera specific for firefly luciferase. Actin specific antiserum (α-Actin) was used as a loading control. Luciferase (1-464aa) and Luciferase (1-160aa) represent N-terminal fragments derived from translation termination at the TAG amber codon introduced at the respective codon of the mutated luciferase genes. (B) Specific activity of Luc F161ANAP normalized to the specific activity of wild-type luciferase. The specific activity of wild-type luciferase was set to 100%. Specific activities were calculated as the ratio of the measured luciferase activity and the steady-state luciferase level (as determined by quantitative western blotting).</p

Specific incorporation of ANAP in Luc F161ANAP.

<p>Specific incorporation of ANAP in Luc F161ANAP expressed in OT56 [<i>PSI</i>⁺] cells analyzed by SDS-PAGE of total protein extracts and scanning of the gel for ANAP fluorescence (excitation wavelength 365 nm, emission wavelength 510 nm). (A) Immunoblotting analysis of Luc F161ANAP expression levels using luciferase specific antiserum. Immunodetection of α-Actin served as a loading control.</p

ANAP fluorescence emission changes report on heat-induced misfolding and aggregation of Luc F161ANAP <i>in vivo</i>.

<p>Schematic overview of the heat shock regime for analyzing Luc F161ANAP misfolding and aggregation. Cells were grown at 30°C to logarithmic phase and exposed to mild heat-shock (37°C) for 45 min. Cycloheximide (CHX) was added to arrest protein synthesis and cells were shifted to 45°C for 20 min. Samples were taken for microscopic imaging of ANAP fluorescence as indicated. (A) Microscopic analysis of yeast cells expressing Luc F161ANAP after incubation at different temperatures as indicated in (A). Heat shock changes the localization and the fluorescence emission color of Luc F161ANAP. Shown are overlays of all 32 channels. Bar 1 µm. (B) Fluorescence emission spectrum of Luc F161ANAP in cells exposed to 30°C, 37°C or 45°C. The fluorescence emission spectra of the cytosol of 50 cells (30°C and 37°C) or 50 aggregate foci (45°C) were analyzed and the average fluorescence measured at the different wavelengths was plotted. The color-coded dashed lines align to the wavelength of maximum emission. a.u.  =  arbitrary units.</p

DataSheet1_Balanced activities of Hsp70 and the ubiquitin proteasome system underlie cellular protein homeostasis.PDF

To counteract proteotoxic stress and cellular aging, protein quality control (PQC) systems rely on the refolding, degradation and sequestration of misfolded proteins. In Saccharomyces cerevisiae the Hsp70 chaperone system plays a central role in protein refolding, while degradation is predominantly executed by the ubiquitin proteasome system (UPS). The sequestrases Hsp42 and Btn2 deposit misfolded proteins in cytosolic and nuclear inclusions, thereby restricting the accessibility of misfolded proteins to Hsp70 and preventing the exhaustion of limited Hsp70 resources. Therefore, in yeast, sequestrase mutants show negative genetic interactions with double mutants lacking the Hsp70 co-chaperone Fes1 and the Hsp104 disaggregase (fes1Δ hsp104Δ, ΔΔ) and suffering from low Hsp70 capacity. Growth of ΔΔbtn2Δ mutants is highly temperature-sensitive and results in proteostasis breakdown at non-permissive temperatures. Here, we probed for the role of the ubiquitin proteasome system in maintaining protein homeostasis in ΔΔbtn2Δ cells, which are affected in two major protein quality control branches. We show that ΔΔbtn2Δ cells induce expression of diverse stress-related pathways including the ubiquitin proteasome system to counteract the proteostasis defects. Ubiquitin proteasome system dependent degradation of the stringent Hsp70 substrate firefly Luciferase in the mutant cells mirrors such compensatory activities of the protein quality control system. Surprisingly however, the enhanced ubiquitin proteasome system activity does not improve but aggravates the growth defects of ΔΔbtn2Δ cells. Reducing ubiquitin proteasome system activity in the mutant by lowering the levels of functional 26S proteasomes improved growth, increased refolding yield of the Luciferase reporter and attenuated global stress responses. Our findings indicate that an imbalance between Hsp70-dependent refolding, sequestration and ubiquitin proteasome system-mediated degradation activities strongly affects protein homeostasis of Hsp70 capacity mutants and contributes to their severe growth phenotypes.</p

Table1_Balanced activities of Hsp70 and the ubiquitin proteasome system underlie cellular protein homeostasis.XLSX

To counteract proteotoxic stress and cellular aging, protein quality control (PQC) systems rely on the refolding, degradation and sequestration of misfolded proteins. In Saccharomyces cerevisiae the Hsp70 chaperone system plays a central role in protein refolding, while degradation is predominantly executed by the ubiquitin proteasome system (UPS). The sequestrases Hsp42 and Btn2 deposit misfolded proteins in cytosolic and nuclear inclusions, thereby restricting the accessibility of misfolded proteins to Hsp70 and preventing the exhaustion of limited Hsp70 resources. Therefore, in yeast, sequestrase mutants show negative genetic interactions with double mutants lacking the Hsp70 co-chaperone Fes1 and the Hsp104 disaggregase (fes1Δ hsp104Δ, ΔΔ) and suffering from low Hsp70 capacity. Growth of ΔΔbtn2Δ mutants is highly temperature-sensitive and results in proteostasis breakdown at non-permissive temperatures. Here, we probed for the role of the ubiquitin proteasome system in maintaining protein homeostasis in ΔΔbtn2Δ cells, which are affected in two major protein quality control branches. We show that ΔΔbtn2Δ cells induce expression of diverse stress-related pathways including the ubiquitin proteasome system to counteract the proteostasis defects. Ubiquitin proteasome system dependent degradation of the stringent Hsp70 substrate firefly Luciferase in the mutant cells mirrors such compensatory activities of the protein quality control system. Surprisingly however, the enhanced ubiquitin proteasome system activity does not improve but aggravates the growth defects of ΔΔbtn2Δ cells. Reducing ubiquitin proteasome system activity in the mutant by lowering the levels of functional 26S proteasomes improved growth, increased refolding yield of the Luciferase reporter and attenuated global stress responses. Our findings indicate that an imbalance between Hsp70-dependent refolding, sequestration and ubiquitin proteasome system-mediated degradation activities strongly affects protein homeostasis of Hsp70 capacity mutants and contributes to their severe growth phenotypes.</p

Table3_Balanced activities of Hsp70 and the ubiquitin proteasome system underlie cellular protein homeostasis.XLSX

To counteract proteotoxic stress and cellular aging, protein quality control (PQC) systems rely on the refolding, degradation and sequestration of misfolded proteins. In Saccharomyces cerevisiae the Hsp70 chaperone system plays a central role in protein refolding, while degradation is predominantly executed by the ubiquitin proteasome system (UPS). The sequestrases Hsp42 and Btn2 deposit misfolded proteins in cytosolic and nuclear inclusions, thereby restricting the accessibility of misfolded proteins to Hsp70 and preventing the exhaustion of limited Hsp70 resources. Therefore, in yeast, sequestrase mutants show negative genetic interactions with double mutants lacking the Hsp70 co-chaperone Fes1 and the Hsp104 disaggregase (fes1Δ hsp104Δ, ΔΔ) and suffering from low Hsp70 capacity. Growth of ΔΔbtn2Δ mutants is highly temperature-sensitive and results in proteostasis breakdown at non-permissive temperatures. Here, we probed for the role of the ubiquitin proteasome system in maintaining protein homeostasis in ΔΔbtn2Δ cells, which are affected in two major protein quality control branches. We show that ΔΔbtn2Δ cells induce expression of diverse stress-related pathways including the ubiquitin proteasome system to counteract the proteostasis defects. Ubiquitin proteasome system dependent degradation of the stringent Hsp70 substrate firefly Luciferase in the mutant cells mirrors such compensatory activities of the protein quality control system. Surprisingly however, the enhanced ubiquitin proteasome system activity does not improve but aggravates the growth defects of ΔΔbtn2Δ cells. Reducing ubiquitin proteasome system activity in the mutant by lowering the levels of functional 26S proteasomes improved growth, increased refolding yield of the Luciferase reporter and attenuated global stress responses. Our findings indicate that an imbalance between Hsp70-dependent refolding, sequestration and ubiquitin proteasome system-mediated degradation activities strongly affects protein homeostasis of Hsp70 capacity mutants and contributes to their severe growth phenotypes.</p

Hsp104-dependent refolding of Luc F161ANAP analyzed by flow cytometry.

<p>Histogram of the ratio of green (510 nm) to blue (450 nm) fluorescence of wild-type Sup35-destabilized W303 and W303<i>hsp104Δ</i> cells expressing Luc F161ANAP. After pre-shock and heat shock, cells were allowed to recover for 2 hours at 30°C. Flow cytometry was performed every 15, 5, and 15 minutes during pre-shock, heat shock and recovery, respectively. Red dash lines align to the peak position before heat shock. (A) Quantification of Luc F161ANAP refolding shown as mean of the ratio of green (510 nm) to blue (450 nm) fluorescence. Green circles represent fluorescence ratios from wild-type cells, and the red squares represent fluorescence ratios from <i>hsp104Δ</i> cells. (B) Normalized luciferase activity of wild-type and <i>hsp104Δ</i> cells expressing Luc F161ANAP before heat shock, during heat shock and during recovery. The luciferase activity measured before temperature upshift from 30°C to 37°C was set to 100%. Green circles indicate the relative luciferase activity of wild-type cells, and the red squares of <i>hsp104Δ</i> cells.</p

Table2_Balanced activities of Hsp70 and the ubiquitin proteasome system underlie cellular protein homeostasis.XLSX

To counteract proteotoxic stress and cellular aging, protein quality control (PQC) systems rely on the refolding, degradation and sequestration of misfolded proteins. In Saccharomyces cerevisiae the Hsp70 chaperone system plays a central role in protein refolding, while degradation is predominantly executed by the ubiquitin proteasome system (UPS). The sequestrases Hsp42 and Btn2 deposit misfolded proteins in cytosolic and nuclear inclusions, thereby restricting the accessibility of misfolded proteins to Hsp70 and preventing the exhaustion of limited Hsp70 resources. Therefore, in yeast, sequestrase mutants show negative genetic interactions with double mutants lacking the Hsp70 co-chaperone Fes1 and the Hsp104 disaggregase (fes1Δ hsp104Δ, ΔΔ) and suffering from low Hsp70 capacity. Growth of ΔΔbtn2Δ mutants is highly temperature-sensitive and results in proteostasis breakdown at non-permissive temperatures. Here, we probed for the role of the ubiquitin proteasome system in maintaining protein homeostasis in ΔΔbtn2Δ cells, which are affected in two major protein quality control branches. We show that ΔΔbtn2Δ cells induce expression of diverse stress-related pathways including the ubiquitin proteasome system to counteract the proteostasis defects. Ubiquitin proteasome system dependent degradation of the stringent Hsp70 substrate firefly Luciferase in the mutant cells mirrors such compensatory activities of the protein quality control system. Surprisingly however, the enhanced ubiquitin proteasome system activity does not improve but aggravates the growth defects of ΔΔbtn2Δ cells. Reducing ubiquitin proteasome system activity in the mutant by lowering the levels of functional 26S proteasomes improved growth, increased refolding yield of the Luciferase reporter and attenuated global stress responses. Our findings indicate that an imbalance between Hsp70-dependent refolding, sequestration and ubiquitin proteasome system-mediated degradation activities strongly affects protein homeostasis of Hsp70 capacity mutants and contributes to their severe growth phenotypes.</p