RESEARCH ARTICLE Hsp70–Hsp110 chaperones deliver ubiquitin-dependent and -independent substrates to the 26S proteasome for proteolysis in yeast Ganapathi Kandasamy and Claes Andre ́ asson* ABSTRACT During protein quality control, proteotoxic misfolded proteins are recognized by molecular chaperones, ubiquitylated by dedicated quality control ligases and delivered to the 26S proteasome for degradation. Proteins belonging to the Hsp70 chaperone and Hsp110 (the Hsp70 nucleotide exchange factor) families function in the degradation of misfolded proteins by the ubiquitin-proteasome system via poorly understood mechanisms. Here, we report that the Saccharomyces cerevisiae Hsp110 proteins (Sse1 and Sse2) function in the degradation of Hsp70-associated ubiquitin conjugates at the post-ubiquitylation step and are also required for ubiquitin-independent proteasomal degradation. Hsp110 associates with the 19S regulatory particle of the 26S proteasome and interacts with Hsp70 to facilitate the delivery of Hsp70 substrates for proteasomal degradation. By using a highly defined ubiquitin-independent proteasome substrate, we show that the mere introduction of a single Hsp70-binding site renders its degradation dependent on Hsp110. The findings define a dedicated and chaperone-dependent pathway for the efficient shuttling of cellular proteins to the proteasome with profound implications for understanding protein quality control and cellular stress management. KEY WORDS: Protein degradation, Proteasome, Ubiquitin, Chaperone, Hsp70, Quality control INTRODUCTION Proteolytic removal of misfolded proteins is an important process to maintain proteostasis and to limit the damage caused by proteotoxic stress. Cellular protein quality control (PQC) systems selectively recognize misfolded proteins, keep them associated with molecular chaperones and target them for proteolytic degradation (McClellan et al., 2005; Park et al., 2007). Failure to degrade misfolded proteins by PQC results in the accumulation of proteotoxic misfolded proteins and has been linked to age-associated neurodegenerative diseases, including Parkinson’s and Huntington’s disease (Forloni et al., 2002). The ubiquitin-proteasome system (UPS) is a critical component in PQC (Ciechanover, 1994; Glickman and Ciechanover, 2002). Misfolded proteins are recognized by specialized ubiquitin ligases and covalently tagged with ubiquitin chains that serve as degradation signals (Eisele and Wolf, 2008; Theodoraki et al., 2012). The polyubiquitylated proteins are then delivered to 26S proteasome by shuttling factors that associate with both the ubiquitin chain and the 19S regulatory particle of the proteasome (Elsasser et al., 2004; Husnjak et al., 2008; Su and Lau, 2009). Delivered proteins are unfolded, deubiquitylated and translocated into the 20S proteolytic chamber of the proteasome for degradation. Ubiquitin tagging is dispensable for the proteasomal degradation of a subset of cellular proteins. In such ubiquitin-independent degradation, unstructured tails that interact directly with the proteasome function as degrons (Ben-Nissan and Sharon, 2014; Takeuchi et al., 2007; Yu et al., 2016a,b). Classical examples of proteins that undergo such ubiquitin-independent degradation in Saccharomyces cerevisiae include ornithine decarboxylase (ODC), Rpn4 and Pih1 (Gödderz et al., 2011; Paci et al., 2016; Xie and Varshavsky, 2001). Thus the physical targeting of substrate proteins directly to the proteasome and the susceptibility of flexible peptide stretches to mediate a translocation into the core proteolytic chamber govern rates of cellular protein turnover. The abundant Hsp70 family of chaperones (hereafter Hsp70) functions at the heart of PQC systems, and is critical for both the folding and proteasomal degradation of misfolded proteins. Hsp70 associates with misfolded proteins in a manner controlled by its ATPase cycle and co-chaperones. The Hsp110 sub-family of proteins (hereafter Hsp110) is an abundant co-chaperone relative of Hsp70 that plays a central role in Hsp70 function. Hsp110 [Saccharomyces cerevisiae (hereafter yeast) proteins Sse1 and Sse2], transiently associates with Hsp70 and accelerates nucleotide exchange, which results in large conformational changes of Hsp70 and release of substrates from the chaperone (Andréasson et al., 2008b; Dragovic et al., 2006; Raviol et al., 2006). Such controlled association and release play critical roles in regulating the proteasomal degradation of misfolded proteins during PQC (Abrams et al., 2014; Gowda et al., 2013). Studies in yeast have shown that reducing the Hsp70 levels (yeast proteins Ssa1, Ssa2, Ssa3 and Ssa4) results in the accumulation of aggregated and ubiquitin- modified misfolded proteins (Fang et al., 2011; Lee et al., 2016; Shiber et al., 2013). Similarly decreasing the levels of Hsp110 (sse1Δ) results in the accumulation of misfolded model proteins (Escusa-Toret et al., 2013; Guerriero et al., 2013; Heck et al., 2010; Mandal et al., 2010; McClellan et al., 2005) and of ubiquitin conjugates following a heat shock (Gowda et al., 2013). These findings support a role for Hsp70 and Hsp110 in misfolded protein degradation, perhaps to maintain misfolded proteins in a soluble form during transit to the proteasome, but experiments are plagued by indirect phenotypes stemming from the role of Hsp70 and Hsp110 in protein biogenesis (Gowda et al., 2013). Thus, the mechanistic function of Hsp70 and Hsp110 in proteolysis is not well understood. The transfer of misfolded proteins from chaperone systems to the UPS may rely on simple kinetic competition for substrate binding as well as on more intricate mechanisms that physically link chaperones Received 13 September 2017; Accepted 14 February 2018 Department of Molecular Biosciences, The Wenner-Gren Institute Stockholm University, SE-10691, Stockholm, Sweden. *Author for correspondence (claes.andreasson@su.se) G.K., 0000-0001-8110-2567; C.A., 0000-0001-8948-0685 1 © 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 131, jcs210948. doi:10.1242/jcs.210948 Journal of Cell Science