Chapter Two - Methods for measuring misfolded protein clearance in the budding yeast Saccharomyces cerevisiae
Introduction
In order for a cell to remain healthy, it must maintain a balanced proteome (“proteostasis”) (Balchin, Hayer-Hartl, & Hartl, 2016; Sontag, Samant, & Frydman, 2017). Proteins that do not attain their native three-dimensional structure—e.g., nascent chains from the ribosome, or correctly folded proteins that denature due to various types of stress—must either be refolded or cleared through a process known as protein quality control (PQC). Like any quality-control system, PQC involves different machineries for detection and disposal of nonnative products. For example, molecular chaperones selectively recognize misfolded proteins and target them to refolding or clearance pathways; E3 ubiquitin ligases tag misfolded proteins with one or more molecules of ubiquitin (a posttranslational modification with many roles, one of which is as a proclearance signal); and the proteasome, which recognizes and degrades ubiquitinated proteins (Fig. 1A).
The budding yeast Saccharomyces cerevisiae has been used extensively for furthering our mechanistic understanding of protein quality control and clearance pathways (Amm, Sommer, & Wolf, 2014; Schneider, Nyström, & Widlund, 2018; Sontag et al., 2017). Not only is its core proteostasis machinery conserved through to humans, but it offers numerous clear advantages over higher eukaryotes, including an unrivaled genetic toolbox (a fully annotated genome, a complete deletion mutant strain collection, widely available GFP- and TAP-tagged libraries covering the majority of its proteome, as well as high-throughput methods to generate libraries of new genetic crosses within weeks), as well as its scale, i.e., the fact that experiments are performed with entire populations, rather than a small number of organisms representative of a population (Botstein & Fink, 2011). With regard to proteostasis specifically, the reduced complexity and redundancy of PQC systems in yeast (e.g., the human genome encodes 300–350 molecular chaperones and over 600 E3 ubiquitin ligases, compared with 69 and ~ 60, respectively, in budding yeast) have enabled dissection of protein clearance systems in a way that would not have been possible directly using mammalian models.
However, standard cell biology protocols generally do not take into account the intricacies of yeast as a model organism, or of ubiquitin as a covalent yet highly dynamic posttranslational modification. This chapter is aimed at discussing some of the unique considerations that must be addressed when studying ubiquitin-mediated clearance of misfolded proteins in budding yeast. Basic yeast maintenance and growth protocols are provided elsewhere (Bergman, 2001; Curran & Bugeja, 2014). We will also present protocols we have used successfully for quantifying relative levels of ubiquitin linkages.
Section snippets
Choice of expression system
We routinely use the Gateway® system (Alberti, Gitler, & Lindquist, 2007), which provides a quick and convenient cloning strategy with its suite of plasmids containing various fluorescent proteins or affinity tags (either N- or C-terminal fusions), promoters (GAL, inducible, or GPD, constitutive), selection markers (HIS3, LEU2, TRP1, or URA3), and origins of replication (chromosomally integrating, 2μ high-copy number, or CEN low-copy number).
The GAL1 promoter is especially useful for inducible
Clearance of misfolded protein puncta
Our lab and others have shown that proteins that misfold for a variety of reasons are all cleared within 1–2 h through the ubiquitin–proteasome system (Escusa-Toret et al., 2013; Kaganovich et al., 2008; Malinovska, Kroschwald, Munder, Richter, & Alberti, 2012; Park et al., 2013). If this process is impaired (e.g., inhibiting the proteasome, or in strains with deletions of genes involved in the clearance pathway), the misfolded proteins accumulate in puncta (Escusa-Toret et al., 2013). One of
Analysis of ubiquitin linkages
Misfolded protein clearance generally involves the posttranslational attachment of ubiquitin to one or more of the misfolded protein's Lys residues. Although well known as a proteasomal targeting signal, ubiquitination can signal other, diverse fates for the target protein, which affect signal transduction, endo-lysosomal trafficking, autophagy, NFκB activation, and DNA repair, among other processes (Kwon & Ciechanover, 2017; Yau & Rape, 2016). Ubiquitin is often linked to other ubiquitin
Acknowledgments
We thank members of the Frydman Lab past and present for development of many of the described techniques, including S. Escusa-Toret and E. M. Sontag for the puncta clearance assay, and K. C. Stein for advice on optimizing bead-beating lysis. We also thank A. Ordureau from J. W. Harper's lab (Harvard University, USA) for suggesting the use of the DUB inhibitor PR-619. R.S.S. was funded by a Human Frontier Science Program long-term fellowship (LT000695/2015-L). J.F. was funded by grants from the
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