ß 2015. Published by The Company of Biologists Ltd | Journal of Cell Science (2015) 128, 1180–1192 doi:10.1242/jcs.165159 RESEARCH ARTICLE Assembly and quality control of the protein phosphatase 1 holoenzyme involves the Cdc48–Shp1 chaperone You-Liang Cheng1 and Rey-Huei Chen1,2,* ABSTRACT et al., 1999). Nuclear localization of Glc7 requires Sds22 and Ypi1 to form a ternary complex with Glc7 (Bharucha et al., 2008; Protein phosphatase 1 (PP1) controls many aspects of cell Hisamoto et al., 1995; MacKelvie et al., 1995; Peggie et al., physiology, which depends on its correct targeting in the cell. 2002). Sds22 is an evolutionarily conserved protein containing Nuclear localization of Glc7, the catalytic subunit of PP1 in budding leucine-rich repeats. Similar to Glc7, Sds22 counteracts Aurora B yeast, requires the AAA-ATPase Cdc48 and its adaptor Shp1 through kinase activity and is important for mitotic progression in fission an unknown mechanism. Herein, we show that mutations in SHP1 and budding yeasts (Ohkura and Yanagida, 1991; Peggie et al., cause misfolding of Glc7 that co-aggregates with Hsp104 and Hsp42 2002). In human cells, Sds22 targets PP1 to kinetochores and chaperones and requires the proteasome for clearance. Mutation or regulates kinetochore–microtubule interactions (Posch et al., 2010; depletion of the PP1 regulatory subunits Sds22 and Ypi1, which are Wurzenberger et al., 2012). Ypi1 is also an essential component of involved in nuclear targeting of Glc7, also produce Glc7 aggregates, the nuclear PP1 holoenzyme in budding yeast. Progressive indicating that association with regulatory subunits stabilizes Glc7 depletion of Ypi1 leads to a reduction of nuclear Glc7 and Sds22 conformation. Use of a substrate-trap Cdc48QQ mutant reveals that proteins and mitotic arrest (Bharucha et al., 2008; Pedelini et al., Glc7–Sds22–Ypi1 transiently associates with and is the major target 2007). A potential homolog of Ypi1 in mammals is known as of Cdc48–Shp1. Furthermore, Cdc48–Shp1 binds and prevents Inhibitor-3 (also known as PPP1R11), which forms a heterotrimeric misfolding of PP1-like phosphatases Ppz2 and Ppq1, but not other complex with Sds22 and the catalytic subunit of PP1 (Lesage et al., types of phosphatases. Our data suggest that Cdc48–Shp1 functions 2007). However, unlike yeast Sds22 and Ypi1, mammalian Sds22 as a molecular chaperone for the structural integrity of PP1 complex and Inhibitor-3 have been shown to be substrate-dependent in general and that it specifically promotes the assembly of Glc7– inhibitors of PP1 in vitro (Lesage et al., 2007). Sds22–Ypi1 for nuclear import. Genetic and biochemical analysis in budding yeast have shown that Cdc48, an abundant and evolutionarily conserved AAA- KEY WORDS: Chaperone, Protein folding, Protein phosphatase 1, ATPase family member, positively regulates nuclear PP1 activity Cdc48, Shp1 (Cheng and Chen, 2010). Cdc48 (known as VCP or p97 in metazoans) participates in numerous cellular processes such as endoplasmic reticulum (ER)-associated protein degradation INTRODUCTION (ERAD), the ubiquitin-proteasome system, autophagy, the DNA Type 1 protein serine/threonine phosphatase (PP1) functions in damage response, chromatin remodeling and cell cycle control many aspects of cell physiology in all eukaryotes (Ceulemans and (Meyer et al., 2012). These diverse functions of Cdc48/VCP/p97 Bollen, 2004). PP1 consists of the catalytic subunit and various are mediated by specific adaptors. The best known is the Npl4– regulatory subunits that confer substrate specificity, regulation of Ufd1 complex that is used for Cdc48/VCP/p97 to extract activity and subcellular localization (Ceulemans and Bollen, misfolded protein substrates from the ER (Wolf and Stolz, 2004). In the budding yeast Saccharomyces cerevisiae, the 2012) and to dissociate nonfunctional 60S ribosome subunits for essential gene GLC7 encodes the catalytic subunit of PP1, which, proteasomal degradation (Fujii et al., 2012). Cdc48–Npl4–Ufd1 in turn, regulates glycogen metabolism, actin cytoskeleton, gene also remodels chromatin-binding proteins for DNA damage repair expression, translation, mitosis and meiosis (Cannon, 2010). (Acs et al., 2011) and promotes turnover of RNA polymerase II During vegetative growth, Glc7 protein is predominantly upon UV irradiation (Verma et al., 2011). In general, Cdc48/ localized in the nucleus with enrichment in the nucleolus. The VCP/p97 acts as a segregase to dislocate ubiquitylated substrates protein also localizes to the bud neck and accumulates at the for subsequent degradation. spindle pole body during anaphase (Bloecher and Tatchell, 2000). Besides Npl4–Ufd1, Cdc48/VCP/p97 binds a family of Glc7 is targeted to and acts at these locations through specific proteins containing an ubiquitin-related (UBX) domain that is binding proteins. In the nucleus Glc7 dephosphorylates the structurally similar to ubiquitin (Yuan et al., 2001). Ubx1 (also substrates of Ipl1 (the yeast Aurora B kinase) at the kinetochore known as suppressor of high copy protein phosphatase 1, Shp1) to promote proper kinetochore–microtubule attachment, which is was originally isolated as a mutant that suppresses the toxic effect essential for mitotic progression (Francisco et al., 1994; Sassoon of Glc7 overexpression (Zhang et al., 1995). Subsequent studies further support the hypothesis that Shp1 positively regulates Glc7 1Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 114, to counteract Ipl1/Aurora B activity for mitotic progression 2 Taiwan. Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan. (Bo¨hm and Buchberger, 2013; Cheng and Chen, 2010; Robinson *Author for correspondence ([email protected]) et al., 2012). Cdc48–Shp1 acts by, at least in part, promoting nuclear localization of PP1 (Cheng and Chen, 2010; Robinson Received 27 October 2014; Accepted 15 January 2015 et al., 2012). In addition, Shp1 serves as a cofactor for Cdc48 in Journal of Cell Science 1180 RESEARCH ARTICLE Journal of Cell Science (2015) 128, 1180–1192 doi:10.1242/jcs.165159 ubiquitin-dependent protein degradation (Hartmann-Petersen Glc7 aggregates contain newly synthesized protein and et al., 2004), and it interacts with the ubiquitin-fold autophagy depend on the proteasome for clearance protein Atg8 during autophagosome biogenesis (Krick et al., To generate temperature-sensitive shp1 mutants, we amplified 2010). The mammalian homolog of Shp1, p47 (also known as SHP1 by error-prone PCR, followed by transformation into PGAL- NSFL1C), is involved in post-mitotic Golgi membrane 3HA-SHP1 strain along with a plasmid that was cut between the reassembly (Rabouille et al., 1998). upstream and downstream sequences of SHP1. We screened for Cdc48/VCP/p97 is important for several aspects of protein the transformants that grew at 25˚C, but not 37˚C, on a glucose quality control. In addition to ERAD, VCP/p97 is involved in the plate. Sequencing of the repaired plasmid carrying shp1ts revealed formation and removal of abnormal protein aggregates upon four missense mutations: I237V, K241E, L313P and F396L. To proteasome inhibition (Kobayashi et al., 2007) and in stress further confirm the shp1ts phenotypes, we transformed wild-type granule clearance (Buchan et al., 2013). It also associates with cells with the shp1ts plasmid and then deleted the genomic SHP1 polyglutamine-containing proteins and with protein inclusions in gene. This shp1ts strain was unable to grow at a temperature neurodegenerative disease samples (Hirabayashi et al., 2001). above 34˚C (Fig. 2A). We next examined the effect of shp1ts on Mutations in VCP/p97 are implicated in a variety of cancers, Glc7 and found that the nuclear GFP–Glc7 level in shp1ts at 25˚C neurodegenerative diseases and inclusion-body diseases (Haines, was lower than that in the wild type (Fig. 2B), suggesting that the 2010; Nalbandian et al., 2011; Watts et al., 2004). mutant is partially defective at permissive temperature. Upon By studying how Cdc48–Shp1 might regulate nuclear targeting shifting to 34˚C in the presence of MG132, shp1ts cells of Glc7, we reveal a new function for Cdc48 in promoting accumulated more and larger foci, whereas wild-type cells the assembly of PP1 complex consisting of Glc7–Sds22–Ypi1. contained only small puncta of GFP–Glc7 under this condition Our study also uncovers a protein quality control mechanism (Fig. 2B,C). Furthermore, GFP–Glc7 foci in the shp1ts mutant that prevents accumulation of unassembled PP1 catalytic contained Hsp104 protein, just like in Shp1-depleted cells (our subunits. unpublished data). Interestingly, in cells where both the protein synthesis was inhibited with cycloheximide (CHX) in shp1ts and RESULTS the proteasome was inhibited with MG132 there were no GFP– Shp1 depletion causes aggregation of misfolded Glc7 Glc7 foci (Fig. 2B,C). Therefore, the GFP–Glc7 aggregates are The PP1 catalytic subunit Glc7 is enriched in the nucleus of the derived from protein that is newly synthesized after the shift to budding yeast Saccharomyces cerevisiae, and this enrichment is the restrictive temperature. We further asked whether Glc7 important for mitotic progression (Pinsky et al., 2006). Previous aggregates in shp1ts were reversible and could be resolved after studies have shown that nuclear accumulation of Glc7 requires return to the permissive temperature. Upon shifting to 25˚C and the molecular chaperone Cdc48 and its adaptor Shp1 (Cheng and removing MG132, the existing Glc7 aggregates gradually Chen, 2010; Robinson et