Name: coupled assays for monitoring protein refolding in saccharomyces cerevisiae
Uploaded: 2013-07-09T12:00:00
Description: Watch this Scientific Journal Video about Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae at JoVE.com

This work was supported by a grant from the National Institutes of Health (NIGMS-074696) to K.A.M. and an American Society of Microbiology Robert D. Watkins Graduate Student Research Fellowship to J.L.A. We also thank John Glover at the University of Toronto for providing the FFL-GFP source plasmid.

1. Construction of Strains Containing FFL-GFP Plasmid
For this study, Saccharomyces cerevisiae strain BY4741 (MATa, his3&Delta;1, leu2&Delta;0, met15&Delta;0, ura3&Delta;0) was used along with an HSP104 deletion strain from the yeast knockout collection (Open Biosystems/Thermo Scientific). The deletion was confirmed using an Hsp104-specific antibody for Western blot analysis.
FFL-GFP was expressed ...

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In this article the model protein FFL-GFP was used to show that the yeast disaggregase, Hsp104 contributes to protein re-solubilization and repair. The enzymatic assays and microscopy differentially interrogated the status of the same substrate protein to determine refolding efficiency and yield. Results of the enzymatic recovery assays suggest that not only is the maximal recovery in the hsp104D mutant strain inefficient, but the initial magnitude of the unfolding stress was greater in the mutant strain (<stron...

In humans neurodegenerative disorders including Alzheimer's, Parkinson's, and Huntington's diseases have been linked to protein misfolding and aggregation 10. Cells employ molecular chaperones to prevent kinetic trapping of cellular proteins into misfolded inactive structures 6. Chaperones participate in intricate interaction networks within the cell, but it is not completely understood how the sum of these interactions contributes to organismal proteostasis. One of the main chaperones responsible for the majority of cytosolic protein folding is the 70 kD heat shock protein (Hsp70) family 19. It has been shown that in yeast loss of Hsp70 decreases the ability to fold nascent heterologously expressed FFL and to refold the endogenous protein, ornithine transcarbamoylase, in vivo 18, 7. The ability to analyze folding with near-real time resolution will facilitate understanding how additional cellular factors contribute to this Hsp70-dependent process. In addition, folding/refolding reactions may not be completely dependent on these contributing proteins, so assays must be sensitive enough to detect both large and small changes in kinetics and efficiency.
The yeast cell disaggregase, Hsp104, plays a vital role in repairing aggregated misfolded proteins. Although Hsp104 homologs have been identified in fungi and plants, this family appears to be absent in metazoans. It has been proposed that other chaperones such as those of the Hsp110 family perform some of the known Hsp104 activities in mammals 16. Hsp104 is an AAA+, hexameric protein complex that functions in yeast to remodel protein aggregates, contributing to refolding and repair 13. Hsp104, along with the yeast Hsp70, Ssa1, and the yeast Hsp40, Ydj1, is required for recovery of denatured FFL in yeast cells 5, 8. The small heat shock protein, Hsp26, has also been shown to be required for Hsp104-mediated disaggregation of FFL 2.
FFL is a two-domain protein that binds the substrate luciferin in the active site and following a conformational change that requires ATP and oxygen, decarboxylates the substrate releasing oxyluciferin, carbon dioxide (CO2), adenosine monophosphate (AMP), and light 11, 9, 3. The commercially available FFL substrate, D-luciferin, results in light emission between 550-570 nm that can be detected using a luminometer 15. FFL is exquisitely sensitive to denaturation from chemical or heat treatments and aggregates rapidly upon unfolding. When exposed to temperatures between 39-45 &deg;C FFL is reversibly unfolded and inactivated 12. In contrast, GFP and its derivatives are highly resistant to protein unfolding stresses 14. Therefore fusion of these two proteins allows FFL to function as an experimentally labile moiety capable of targeting functional GFP to intracellular deposits that can be visualized using fluorescence microscopy at both the population and single cell levels. Application of the enzymatic assay in a semi-automated multimode plate reader coupled with automated microscopy allows unprecedented simultaneous assessment of kinetics and yield of refolding reactions. In addition, the facile molecular genetics of the model eukaryote Saccharomyces cerevisiae allows both precise manipulation of the protein quality control network and the opportunity for discovery-based approaches to identify novel players contributing to cellular stress response and proteostasis.
In this study, wild-type (WT) and HSP104 deletion strains expressing FFL-GFP are subject to protein denaturing heat shock. FFL-GFP refolding is monitored through both an enzymatic assay and microscopy as a proxy readout for repair of the expressed proteome over a recovery time course. When compared to WT cells, we show the Hsp104 deletion strain is ~60% less efficient at refolding FFL-GFP, supporting previous findings establishing a role for Hsp104 in reactivation of denatured FFL 2.

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 <td>Synergy MX Microplate Reader</td>
 <td>BioTek</td>
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 <td>Olympus IX81-ZDC Confocal Inverted Microscope</td>
 <td>Olympus, Tokyo Japan</td>
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 <td>Lumitrac 200 white 96-well plates</td>
 <td>USA Scientific</td>
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 <td>SlideBook 5.0 digital microscopy software</td>
 <td>Intelligent Imaging Innovations, Inc. Denver, CO, USA</td>
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 <td>Tris Base</td>
 <td>Fisher</td>
 <td>BP152-1</td>
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 <td>(LiAc) Lithium Acetate</td>
 <td>Sigma</td>
 <td>L6883</td>
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 <td>PEG (polyethelene glycol)</td>
 <td>Fisher</td>
 <td>BP233-1</td>
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 <td>EDTA</td>
 <td>Fisher</td>
 <td>BP120-1</td>
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 <td>DMSO</td>
 <td>Malinckrodt</td>
 <td>4948</td>
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 <td>Sunrise</td>
 <td>1300-030</td>
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 <td>SC-URA</td>
 <td>Sunrise</td>
 <td>1306-030</td>
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 <td>cycloheximide</td>
 <td>Acros Organics</td>
 <td>357420050</td>
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 <td>D-luciferin</td>
 <td>Sigma</td>
 <td>L9504</td>
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 <td>low melt agarose</td>
 <td>NuSieve</td>
 <td>50082</td>
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 <td>immersion oil</td>
 <td>Cargille</td>
 <td>16484</td>
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</table>

coupled assays for monitoring protein refolding in saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Yeast is dependent on the disaggregase, Hsp104 to efficiently refold heat-denatured proteins. The activity of FFL-GFP was monitored after a 25 min heat shock, using a luminescence flash assay Figure 1. The results of this automated assay shown in Figure 2 revealed a stepwise increase in activity over 90 min that ultimately led to a &gt;80% recovery in WT cells. The hsp104&Delta; strain recovered 19% of the original activity over the same time frame. Moreover, the extent of initi...

Watch this Scientific Journal Video about Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae at JoVE.com

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

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