Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Summary

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.

Abstract

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.

Introduction

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 Hsp....

Protocol

1. Construction of Strains Containing FFL-GFP Plasmid

For this study, Saccharomyces cerevisiae strain BY4741 (MATa, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0) 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 from p426MET25-FFL-GFP, constructed from a LEU2-based source plasmid obtained from the Glover laboratory at the University of Toronto ¹⁷ an....

Results

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 >80% recovery in WT cells. The hsp104Δ strain recovered 19% of the original activity over the same time frame. Moreover, the extent of initial ina.......

Discussion

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 (

Materials

List of materials used in this article

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