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Hsp70–Hsp110 Chaperones Deliver Ubiquitin-Dependent

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Hsp70–Hsp110 Chaperones Deliver Ubiquitin-Dependent © 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 131, jcs210948. doi:10.1242/jcs.210948 RESEARCH ARTICLE Hsp70–Hsp110 chaperones deliver ubiquitin-dependent and -independent substrates to the 26S proteasome for proteolysis in yeast Ganapathi Kandasamy and Claes Andréasson* ABSTRACT proteasome by shuttling factors that associate with both the During protein quality control, proteotoxic misfolded proteins are ubiquitin chain and the 19S regulatory particle of the proteasome recognized by molecular chaperones, ubiquitylated by dedicated (Elsasser et al., 2004; Husnjak et al., 2008; Su and Lau, 2009). quality control ligases and delivered to the 26S proteasome for Delivered proteins are unfolded, deubiquitylated and translocated degradation. Proteins belonging to the Hsp70 chaperone and Hsp110 into the 20S proteolytic chamber of the proteasome for degradation. (the Hsp70 nucleotide exchange factor) families function in the Ubiquitin tagging is dispensable for the proteasomal degradation degradation of misfolded proteins by the ubiquitin-proteasome of a subset of cellular proteins. In such ubiquitin-independent system via poorly understood mechanisms. Here, we report that the degradation, unstructured tails that interact directly with the Saccharomyces cerevisiae Hsp110 proteins (Sse1 and Sse2) function proteasome function as degrons (Ben-Nissan and Sharon, 2014; in the degradation of Hsp70-associated ubiquitin conjugates at the Takeuchi et al., 2007; Yu et al., 2016a,b). Classical examples of post-ubiquitylation step and are also required for ubiquitin-independent proteins that undergo such ubiquitin-independent degradation in Saccharomyces cerevisiae proteasomal degradation. Hsp110 associates with the 19S regulatory include ornithine decarboxylase (ODC), particle of the 26S proteasome and interacts with Hsp70 to facilitate the Rpn4 and Pih1 (Gödderz et al., 2011; Paci et al., 2016; Xie and delivery of Hsp70 substrates for proteasomal degradation. By using a Varshavsky, 2001). Thus the physical targeting of substrate proteins highly defined ubiquitin-independent proteasome substrate, we show directly to the proteasome and the susceptibility of flexible peptide that the mere introduction of a single Hsp70-binding site renders its stretches to mediate a translocation into the core proteolytic chamber degradation dependent on Hsp110. The findings define a dedicated govern rates of cellular protein turnover. and chaperone-dependent pathway for the efficient shuttling of The abundant Hsp70 family of chaperones (hereafter Hsp70) cellular proteins to the proteasome with profound implications for functions at the heart of PQC systems, and is critical for both the understanding protein quality control and cellular stress management. folding and proteasomal degradation of misfolded proteins. Hsp70 associates with misfolded proteins in a manner controlled by KEY WORDS: Protein degradation, Proteasome, Ubiquitin, its ATPase cycle and co-chaperones. The Hsp110 sub-family of Chaperone, Hsp70, Quality control proteins (hereafter Hsp110) is an abundant co-chaperone relative of Hsp70 that plays a central role in Hsp70 function. Hsp110 INTRODUCTION [Saccharomyces cerevisiae (hereafter yeast) proteins Sse1 and Proteolytic removal of misfolded proteins is an important process to Sse2], transiently associates with Hsp70 and accelerates nucleotide maintain proteostasis and to limit the damage caused by proteotoxic exchange, which results in large conformational changes of Hsp70 stress. Cellular protein quality control (PQC) systems selectively and release of substrates from the chaperone (Andréasson et al., recognize misfolded proteins, keep them associated with molecular 2008b; Dragovic et al., 2006; Raviol et al., 2006). Such controlled chaperones and target them for proteolytic degradation (McClellan association and release play critical roles in regulating the et al., 2005; Park et al., 2007). Failure to degrade misfolded proteins proteasomal degradation of misfolded proteins during PQC by PQC results in the accumulation of proteotoxic misfolded (Abrams et al., 2014; Gowda et al., 2013). Studies in yeast have proteins and has been linked to age-associated neurodegenerative shown that reducing the Hsp70 levels (yeast proteins Ssa1, Ssa2, Ssa3 diseases, including Parkinson’s and Huntington’s disease (Forloni and Ssa4) results in the accumulation of aggregated and ubiquitin- et al., 2002). modified misfolded proteins (Fang et al., 2011; Lee et al., 2016; The ubiquitin-proteasome system (UPS) is a critical component Shiber et al., 2013). Similarly decreasing the levels of Hsp110 in PQC (Ciechanover, 1994; Glickman and Ciechanover, 2002). (sse1Δ) results in the accumulation of misfolded model proteins Misfolded proteins are recognized by specialized ubiquitin ligases (Escusa-Toret et al., 2013; Guerriero et al., 2013; Heck et al., 2010; and covalently tagged with ubiquitin chains that serve as Mandal et al., 2010; McClellan et al., 2005) and of ubiquitin degradation signals (Eisele and Wolf, 2008; Theodoraki et al., conjugates following a heat shock (Gowda et al., 2013). These 2012). The polyubiquitylated proteins are then delivered to 26S findings support a role for Hsp70 and Hsp110 in misfolded protein degradation, perhaps to maintain misfolded proteins in a soluble form Department of Molecular Biosciences, The Wenner-Gren Institute Stockholm during transit to the proteasome, but experiments are plagued by University, SE-10691, Stockholm, Sweden. indirect phenotypes stemming from the role of Hsp70 and Hsp110 in protein biogenesis (Gowda et al., 2013). Thus, the mechanistic *Author for correspondence ([email protected]) function of Hsp70 and Hsp110 in proteolysis is not well understood. G.K., 0000-0001-8110-2567; C.A., 0000-0001-8948-0685 The transfer of misfolded proteins from chaperone systems to the UPS may rely on simple kinetic competition for substrate binding as Received 13 September 2017; Accepted 14 February 2018 well as on more intricate mechanisms that physically link chaperones Journal of Cell Science 1 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs210948. doi:10.1242/jcs.210948 to the UPS machinery. For example in the yeast cytoplasm, the analysis was expanded with the distinct UFD substrate UbV76–EGFP degradation of misfolded proteins that are associated with the (Dantuma et al., 2000). This UFD substrate was also dependent on chaperone Hsp70 depends on the nucleotide exchange factor Fes1 Hsp110 for its degradation, and again the unmodified and being released from the chaperone and then recognized by PQC monoubiquitylated forms were found to be distributed in the ubiquitin ligases (Gowda et al., 2016, 2013, 2018). In metazoan cells, soluble and insoluble fractions (Fig. 1D). To assess the functional mechanisms involve the direct docking of the ubiquitin ligase CHIP requirements of Sse1 in the protein degradation pathway, we tested to Hsp70, and the nucleotide exchange factor BAG-1 coordinates the previously described Sse1 mutants deficient in ATP binding release of the misfolded protein from Hsp70 with proteasome (G205D), ATP hydrolysis (K69M) and substrate binding (denoted interactions (Demand et al., 2001; Hohfeld and Jentsch, 1997; Lüders sbd; L433A, N434P, F439L and M441A) (Andréasson et al., 2008b; et al., 2000). A third example involves the ubiquitylation of proteins Garcia et al., 2017; Shaner et al., 2004). Sse1 depends on ATP damaged by acute heat shock, which has been proposed to depend on binding but not on hydrolysis to interact with Hsp70 (Andréasson the coordinated interactions of the PQC ubiquitin ligase Rsp5, the et al., 2008b). We found that the proteasomal degradation of UbV76– Hsp40 chaperone Ydj1 and the misfolded protein (Fang et al., 2014). Ura3–HA was strongly impeded by the lack of ATP binding, but not Nevertheless, our understanding of the basic PQC mechanisms that by ATP hydrolysis or substrate binding (Fig. 1F). Thus, Hsp110 link chaperone systems and the UPS is at the best rudimentary. functions together with Hsp70 in the proteasomal degradation Here, we investigate the function of Hsp110 in proteasomal pathway. degradation by employing a novel yeast strain with a temperature- Next, we focused on the potential post-ubiquitylation role of sensitive Hsp110 function (sse1-200 sse2Δ). We find that Hsp110 Hsp110 in proteasomal degradation following the turnover of functions together with Hsp70 in the degradation of aggregation- misfolded ubiquitylated proteins after heat shock (Fang et al., 2011; prone proteins involving both ubiquitin-dependent and -independent Gowda et al., 2013). Consistent with the previous reports, western proteasomal pathways. Hsp110 is required to keep Hsp70-associated blot analysis of ubiquitin-conjugate levels in wild-type (WT) cells proteasome substrates soluble and interacts with 19S regulatory transferred from 25°C to 37°C showed an accumulation of particle of the proteasome, suggesting coordinated recruitment of ubiquitylated proteins during the first 20 min after the temperature Hsp70–substrate complexes to the 26S proteasome for degradation. upshift, which were subsequently cleared by proteasomal The data defines a novel PQC pathway that enables the direct and degradation to reach the initial levels after 30 min (Fig. S1A). In efficient delivery of Hsp70-associated aggregation-prone proteins to sse1-200 sse2Δ cells, the accumulation of ubiquitin-conjugate the proteasome. levels was exacerbated and the accumulated ubiquitylated
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