Taiwan. o-in Cheng You-Liang 1 chaperone phosphatase Cdc48–Shp1 the the involves of holoenzyme control quality and Assembly ARTICLE RESEARCH ß 1180 2015 January 15 Accepted 2014; October 27 Received 1 Bollen, budding and gene the (Ceulemans essential localization In subcellular 2004). various of and and regulation specificity, subunit activity substrate catalytic confer the that of subunits regulatory consists and (Ceulemans PP1 in eukaryotes 2004). all functions in Bollen, physiology (PP1) of phosphatase aspects serine/threonine many protein 1 Type INTRODUCTION 1, phosphatase Shp1 Protein Cdc48, folding, Protein Chaperone, WORDS: KEY Cdc48 Glc7 substrate-trap stabilizes a subunits of Use regulatory conformation. with aggregates, association Glc7 produce that also indicating are Glc7, which of Ypi1, targeting and nuclear Sds22 in involved subunits regulatory PP1 or the Mutation of clearance. depletion for the requires Hsp42 and and Hsp104 chaperones in with co-aggregates mutations that that Glc7 of show misfolding cause we cell. Herein, mechanism. the cell unknown budding through in Shp1 an in adaptor of its PP1 and targeting of Cdc48 AAA-ATPase aspects subunit the correct requires catalytic yeast, the many its Glc7, of on localization controls Nuclear depends (PP1) which 1 physiology, phosphatase Protein ABSTRACT Ato o orsodne([email protected]) correspondence for *Author ngnrladta tseiial rmtsteasml fGlc7– of assembly the promotes import. specifically nuclear for it Sds22–Ypi1 complex that PP1 and of integrity general structural in the for chaperone molecular a other functions as Cdc48–Shp1 not that suggest but data prevents Our Ppq1, phosphatases. of and and types Ppz2 phosphatases binds PP1-like Cdc48–Shp1 of misfolding Furthermore, Cdc48–Shp1. target major the of is and with associates transiently Glc7–Sds22–Ypi1 seta o ioi rgeso Facsoe l,19;Sassoon 1994; al., et (Francisco the kinetochore progression the is mitotic dephosphorylates which for at attachment, essential ) Glc7 kinetochore– B specific proper Aurora promote nucleus through yeast to (the locations the Ipl1 these of at In substrates the acts at . and accumulates to binding and targeted 2000). Tatchell, neck is and The (Bloecher Glc7 bud . anaphase during the the body pole to in spindle localizes enrichment with also nucleus protein the in localized 2010). (Cannon, meiosis gene and , mitosis actin translation, metabolism, expression, glycogen regulates turn, in rdaeIsiueo ieSine,Ntoa ees eia etr api114, Taipei Center, Medical Defense National Sciences, Life of Institute Graduate 05 ulse yTeCmayo ilgssLd|Junlo elSine(05 2,18–12doi:10.1242/jcs.165159 1180–1192 128, (2015) Science Cell of Journal | Ltd Biologists of Company The by Published 2015. uigvgttv rwh l7poeni predominantly is protein Glc7 growth, vegetative During 2 nttt fMlclrBooy cdmaSnc,Tie 12,Taiwan. 11529, Taipei Sinica, Academia Biology, Molecular of Institute GLC7 noe h aayi uui fP1 which, PP1, of subunit catalytic the encodes 1 n e-uiChen Rey-Huei and acaoye cerevisiae Saccharomyces QQ uatrvasthat reveals mutant 1,2, * SHP1 the , rtisadmttcars Bauh ta. 08 eeiie al., et Progressive Pedelini 2008; Sds22 al., yeast. and et Glc7 (Bharucha nuclear budding arrest of mitotic reduction in and a proteins to holoenzyme leads Ypi1 of PP1 depletion of component nuclear essential and an also kinetochores the is to Ypi1 2012). al., PP1 et Wurzenberger 2010; targets al., al., et Sds22 et (Posch Peggie interactions cells, kinetochore–microtubule regulates 1991; human Yanagida, In fission and in 2002). (Ohkura progression mitotic budding for al., important and B is et containing Aurora and Peggie counteracts activity protein Sds22 kinase 1995; Glc7, conserved to al., evolutionarily Similar repeats. an et leucine-rich is MacKelvie and 2008; Sds22 1995; al., Sds22 2002). et al., (Bharucha requires Glc7 et Glc7 with complex Hisamoto of ternary a localization form to Nuclear Ypi1 1999). al., et ta. 02.I diin h1sre sacfco o d4 in Cdc48 for cofactor a Robinson as promoting serves 2010; part, Shp1 Chen, in addition, and In least 2012). (Cheng at al., PP1 et by, of progression acts localization mitotic Cdc48–Shp1 nuclear 2012). for al., activity et B Ipl1/Aurora (Bo Glc7 regulates counteract studies positively Shp1 Subsequent to that 1995). hypothesis al., the support et effect further toxic (Zhang Shp1) the overexpression suppresses 1, Glc7 that phosphatase mutant of (also a protein as Ubx1 copy isolated 2001). originally high was al., of is et suppressor that (Yuan as domain known to (UBX) similar ubiquitin-related structurally an containing proteins Cdc48/ general, substrates In ubiquitylated degradation. dislocate 2011). subsequent to segregase al., for II a polymerase et as RNA acts (Verma of VCP/p97 irradiation turnover promotes UV and upon 2011) al., repair et Stolz, damage DNA (Acs Cdc48–Npl4–Ufd1 for extract and 2012). proteins chromatin-binding al., (Wolf remodels for to also et subunits (Fujii ER Cdc48/VCP/p97 60S degradation the nonfunctional proteasomal for dissociate from to used and substrates 2012) Npl4– is the protein is that known misfolded best complex The adaptors. Cdc48/VCP/p97 Ufd1 specific of by control functions diverse mediated cycle These are cell 2012). al., and et remodeling (Meyer degradation DNA chromatin in the response, protein p97 , damage system, or ubiquitin-proteasome as (ER)-associated the VCP such (ERAD), as processes reticulum cellular (known numerous endoplasmic Cdc48 in 2010). participates AAA- metazoans) Chen, conserved activity and PP1 evolutionarily nuclear (Cheng regulates and positively member, abundant family ATPase substrate-dependent an Cdc48, be that to shown been PP1 of have inhibitors Sds22 Inhibitor-3 mammalian Ypi1, and and Sds22 al., as et yeast (Lesage unlike PP1 known However, of 2007). subunit is catalytic the mammals and Sds22 in heterotrimeric with complex a forms Ypi1 which PPP1R11), of as known (also homolog Inhibitor-3 potential A 2007). eie p4Ud,Cc8VPp7bnsafml of family a binds Cdc48/VCP/p97 Npl4–Ufd1, Besides shown have yeast budding in analysis biochemical and Genetic h n uhegr 03 hn n hn 00 Robinson 2010; Chen, and Cheng 2013; Buchberger, and ¨hm nvitro in Lsg ta. 2007). al., et (Lesage

Journal of Cell Science ult oto.I diint RD C/9 sivle nthe in membrane involved is VCP/p97 Golgi ERAD, to addition In post-mitotic control. quality in as 1998). known al., involved et (also al., (Rabouille p47 reassembly et is Shp1, of (Krick autophagy homolog NSFL1C), biogenesis ubiquitin-fold mammalian the The with 2010). during interacts Atg8 it (Hartmann-Petersen and protein 2004), degradation al., et protein ubiquitin-dependent ARTICLE RESEARCH ubro F–l7fc otya ula eihr ncells in maintain periphery to nuclear the galactose at to mostly in by foci MG132 grown small degraded GFP–Glc7 a to inhibitor of led was dot treatment number MG132 proteasome protein depletion. a Shp1 nuclear upon into the GFP–Glc7 proteasome the protein whether used of the reduction determine We of a accumulation 1A,B). caused (Fig. and h, 5 signal of than GFP–Glc7 suppression more Prolonged for medium promoter. expressed ectopically own we SHP1 its localization, from galactose- the its Glc7 of a GFP–Glc7 N-terminus detect controlled the with at To we tag promoter 3HA protein. strain, a native adding and background its promoter inducible our replacing by in Because expression viability 2012). cell al., and et for (Cheng requires Shp1 Robinson adaptor Glc7 2010; its of and Chen, accumulation Cdc48 chaperone nuclear Previous molecular 2006). that the al., shown et have the (Pinsky studies of progression nucleus mitotic the for in important enriched is yeast Glc7 budding subunit Glc7 catalytic misfolded PP1 of The aggregation causes catalytic depletion Shp1 PP1 RESULTS unassembled mechanism of control promoting quality accumulation in protein subunits. Cdc48 prevents a Glc7–Sds22–Ypi1. for uncovers of that function consisting also complex study new PP1 Our a of reveal assembly the we Glc7, 2004). al., of et cancers, Watts of 2011; variety al., et a (Haines, Nalbandian in 2010; -body 2001). implicated and al., diseases are neurodegenerative et VCP/p97 in (Hirabayashi with Mutations stress samples associates in also in inclusions It protein neurodegenerative and with 2013). and 2007) upon al., proteins et polyglutamine-containing al., aggregates (Buchan et protein clearance (Kobayashi abnormal inhibition of proteasome removal and formation rcino l7i ifle n ed oageaei not if aggregate to tends and a Shp1, misfolded proteasome. tagging, the without is by that GFP degraded Glc7 suggest of of results (our result fraction These cells Shp1-depleted a data). in foci unlikely unpublished al., formed et is also 2myc–Glc7 Specht phenotype because 2008; al., This shock et co-aggregate 2011). often (Kaganovich heat which proteins for 1G), misfolded (Fig. enriched with contained Hsp42 were foci and Hsp104 GFP–Glc7 they proteins The because 1F). protein, (Fig. misfolded time larger imaging became over foci brighter Timelapse pre-existing shortly the and that 1E). appeared and (Fig. puncta addition MG132 GFP–Glc7 level after multiple addition the that the that showed and antibody increased further type was wild the cells anti-GFP MG132 in Shp1-depleted that of of with in half level about blotting to GFP–Glc7 reduced the Western that confirmed 1C,D). (Fig. with foci increased cells, foci under of Shp1-depleted number proteasome and the in size by the Glc7 Notably, of conditions. turnover normal is there that indicating ysuyn o d4–h1mgtrglt ula targeting nuclear regulate might Cdc48–Shp1 how studying By protein of aspects several for important is Cdc48/VCP/p97 civdb utrn h el nglucose-containing in cells the culturing by achieved , acaoye cerevisiae Saccharomyces , 0 el arigmr hn5 than more carrying cells 80% SHP1 xrsin(i.1C,D), (Fig. expression n hsercmn is enrichment this and , SHP1 sessential is Fg BC.Frhroe F–l7fc nthe cells in condition wild-type foci this under GFP–Glc7 whereas GFP–Glc7 Furthermore, of foci, 2B,C). puncta (Fig. larger small only and contained more accumulated el ihthe with cells l7fc Fg D htwr nihdfrHp0 (our cells Hsp104 noticed Ypi1-depleted of for We fraction 3F). to enriched (Fig. a aggregation MG132 that were GFP–Glc7 enhanced Adding that further al., data). GFP– 3D) et (Fig. produced (Bharucha unpublished depletion cells foci Ypi1 some that levels in Glc7 found protein focus we Glc7 a Similarly, overall forms 2008). and protein Glc7 the nuclear while the both of of of depletion reduction use prolonged Furthermore, through 3C). Ypi1 (Fig. foci GFP–Glc7 ormses uain:I3V 21,L1PadF9L To F396L. and the L313P confirm K241E, further I237V, mutations: missense four eunt h emsietmeaue pnsitn o25 to shifting Upon temperature. permissive the to return ee This gene. h rtaoewsihbtdwt G3 hr een GFP– no were there MG132 with inhibited was proteasome the o laigteageae in aggregates required is the 2D). activity clearing (Fig. proteasome aggregates that for the MG132 and in refolded protein of h be Glc7 3 simply that presence cannot indicates to result continuous by This returned 2D). the were gradually (Fig. that in invisible cells aggregates temperature in almost permissive persisted Glc7 aggregates were the existing However, and the disappeared Glc7 MG132, whether removing asked further to shift We the in after temperature. aggregates synthesized are newly restrictive aggregates is GFP–Glc7 the that the protein from Therefore, derived 2B,C). (Fig. foci Glc7 uati atal eetv tprisv eprtr.Upon temperature. 34 permissive at the to that defective suggesting 2B), shifting partially (Fig. type is wild the mutant in that than lower was htGc smsoddudrti odto.Adn G3 to MG132 Adding condition. this indicating under 3A), misfolded (Fig. sds22-6 is Hsp104 Glc7 contained that foci These 3A,B). (Fig. SHP1 temperature-sensitive generate clearance for To proteasome and the protein on synthesized depend newly contain aggregates Glc7 estv uat h neato ewe l7adSs2is Sds22 37 at and reduced Glc7 Glc7 nuclear of the between levels In the interaction and cells Glc7. proteins disrupted the these Shp1-depleted with mutant, that in co-aggregate sensitive in showing mislocalized not foci S1), were do Fig. forming Ypi1 material and without (supplementary subunits Sds22 regulatory Both PP1 the Ypi1. the requires and Glc7 Sds22 of targeting integrity Glc7 Nuclear maintain Ypi1 and Sds22 hf o37 in foci to forms shift Glc7 Interestingly, 2002). al., et ofre h omto fGPGc oiin foci GFP–Glc7 of formation the confirmed h rnfrat htge t25 at grew that transformants of the sequences downstream and upstream 3HA-SHP1 ytei a niie ihccoeiie(H)in protein (CHX) the cycloheximide both (our with where inhibited cells cells was Shp1-depleted synthesis in in Interestingly, like data). just unpublished protein, Hsp104 contained rdal iapae n hnrapae ae Fg 3D,E), (Fig. later reappeared medium then and to shifting disappeared upon gradually that foci GFP–Glc7 contained lt.Sqecn fterpie lsi carrying plasmid repaired the of Sequencing plate. bv 34 above l7adfudta h ula F–l7lvlin level GFP–Glc7 nuclear the that found and Glc7 yerrpoePR olwdb rnfraininto transformation by followed PCR, error-prone by ora fCl cec 21)18 1019 doi:10.1242/jcs.165159 1180–1192 128, (2015) Science Cell of Journal uigtesitt 35 to shift the during ˚ Fg A.W eteaie h fetof effect the examined next We 2A). (Fig. C ˚ o sltl s3 i Pgi ta. 02.W also We 2002). al., et (Peggie min 30 as little as for C tanaogwt lsi htwsctbtenthe between cut was that plasmid a with along strain shp1 shp1 shp1 ˚ ntepeec fMG132, of presence the in C ts ts ts tanwsual ogo tatemperature a at grow to unable was strain P shp1 lsi n hndltdtegenomic the deleted then and plasmid eervril n ol ersle after resolved be could and reversible were GAL ts -3HA-YPI1 hntps etasomdwild-type transformed we phenotypes, GAL ˚ nrae h ieadnme of number and size the increased C shp1 rmtri nw ocuea cause to known is promoter ˚ ,btnt37 not but C, ts shp1 cells. el nglcoemedium galactose in cells SHP1 uat,w amplified we mutants, sds22-6 sds22-6 esree for screened We . ˚ sds22-6 ,o glucose a on C, shp1 shp1 shp1 temperature- shp1 el fe a after cells ˚ shp1 ts (Peggie C ts ts shp1 revealed t35 at ts t25 at mutant ˚ and C P SHP1 ts 1181 cells ts GAL and on ˚ ˚ C C -

Journal of Cell Science EERHARTICLE RESEARCH 1182 the the with and in misfolding Taken association prevents components. that Ypi1 PP1 Glc7. and of indicate of aggregation Sds22 results subunits stoichiometry the regulatory to the these due through probably in together, Glc7, Ypi1 of change misfolding of a cause also overexpression might promoter that suggesting 1. Fig. ocnimta l7wside ifle n aggregated and misfolded indeed was Glc7 that confirm To shp1 GAL e etpg o legend. for page next See uat,w coial xrse mcGc from 2myc–Glc7 expressed ectopically we mutants, rmtri h idtp and wild-type the in promoter shp1 ts uat The mutant. GAL eut upr h yohssta l7i ifle and misfolded is Glc7 that hypothesis These 4). the (Fig. soluble remained support cells same results the in protein Sds22 xrsinwsidcdfr1hdrn h eprtr hf to shift temperature the during h 1 for 34 induced was expression noteslbepo ntewl-yecls hra h protein in the whereas pellet cells, the wild-type in the pellet mostly in fractionated fractions was pool a was soluble these 2myc–Glc7 and the synthesized of into newly (supernatant) blotting the Western that fraction showed centrifugation. soluble by a fraction into separated ˚ ntepeec fM12 h oa ellrpoen were proteins cellular total The MG132. of presence the in C ora fCl cec 21)18 1019 doi:10.1242/jcs.165159 1180–1192 128, (2015) Science Cell of Journal shp1 ts Fg ) ycnrs,3HA– contrast, By 4). (Fig.

Journal of Cell Science ihGlc7. with xeiet.Saebr:5 bars: Scale experiments. hw.()Qatfcto ftepretg fclssoni hthave that C mean in as (E) shown presented experiments. cells are of Data percentage foci. the GFP–Glc7 of for Quantification MG132 of (D) inhibitor projections shown. proteasome by Maximum or followed h. DMSO h, 2.5 solvent 7 another control for the (Glu) of glucose addition or the (Gal) galactose containing medium (F) i.1 l7frsanra oii h1dpee cells. Shp1-depleted in foci and abnormal (RHC3048) forms Glc7 1. Fig. ARTICLE RESEARCH r4AYi.Hwvr eddntdtc n ftePP1 the dominant-negative of a To any generated 5A). we Cdc48 (Fig. detect interaction, fractions transient not pulldown any trap Shp1–TAP did the we in components However, the at 4HA–Ypi1. 2myc–Sds22, epitope 2myc–Glc7, or expressing (TAP) cells purification in affinity locus tagged chromosomal tandem Glc7– We the the interaction. with on physical Shp1 a directly through acts complex Cdc48–Shp1 Sds22–Ypi1 whether asked next Glc7–Sds22–Ypi1 We with associates transiently Cdc48–Shp1 in aggregated elcmn fguaaersde tpstos35ad58in 588 overexpression Upon and residues. Cdc48 315 glutamine of positions with domain at B residues Walker glutamate of replacement B uniiaino h ecnaeo el fec yei hthv foci. have that A in type each of cells of (C) percentage the of Quantification Cells (B) indicated. as maximum h Representative of 3–9 microscopy. projections for fluorescence medium by glucose analyzed containing to were medium shifted complete then synthetic and in galactose grown first were GFP–Glc7 eeldta l7 d2 n p1wr h anproteins main the were Ypi1 Cdc48 and with Sds22 associated Glc7, that revealed mcSs2silascae ihSp–A from Shp1–TAP with Shp1–TAP associated down still pulled 2myc–Sds22 we Cdc48–Shp1, from with interaction the 5B), PP1 (Fig. the with pulldown associates Shp1–TAP transiently complex. the Cdc48–Shp1 in that indicating components PP1 three iha nevlo 0mnaeson G F–l7fc oti s14and Hsp104 contain foci GFP–Glc7 (G) projection shown. maximum are a min Hsp42. of 10 of Images interval taken MG132. an were of with images addition time-lapse and the h, after 7 immediately for medium glucose to galactose from n I mgso h el r hw.Tepretg fGPGc foci GFP–Glc7 mean of as percentage shown The is shown. Hsp42 are or cells Hsp104 the containing of before images h of DIC 2.5 projections and for Maximum added analysis. was microscopy MG132 fluorescence from medium. shifted glucose were to (RHC3228) galactose Hsp42–mCherry or (RHC3227) mCherry evsa odn oto.Terltv eeso F–l7 omlzdto normalized GFP–Glc7, of mean levels as relative presented Pgk1 The are antibodies. were control. Pgk1, anti-Pgk1 proteins loading and cell a Total anti-GFP as with indicated. with serves h, as blotting 7 h western for 2.5 by (Glu) another analyzed YPD for or MG132 (Gal) without YPGal or in grown were GFP–Glc7 expressing oyetdswr soitdwt h1TPi el expressing cells in Shp1–TAP Cdc48 with associated were polypeptides the (Peggie mediates Glc7. Glc7 probably Cdc48–Shp1and Sds22 with between that the interaction indicates interaction result the Because our 2002), disrupt 5C). al., to (Fig. et known 2myc–Glc7 is down Shp1– mutation pull contrast, not the By did that function. Glc7 indicating from require 5C), TAP (Fig. not does temperature interaction restrictive at grown odtriewihcmoeto h P ope mediates complex PP1 the of component which determine To ylresaeSp–A uiiain efudta several that found we purification, Shp1–TAP large-scale By P P GAL GAL glc7-12 QQ QQ P -3HA-SHP1 -3HA-SHP1 GAL QQ uat hc sdfciei T yrlssoigto owing hydrolysis ATP in defective is which mutant, u o d4 Fg D.Ms pcrmtyanalysis spectrometry Mass 5D). (Fig. Cdc48 not but -3HA-SHP1 sds22-6 Z rmthe from sakiae n I mgso h el r shown. are cells the of images DIC and images -stack shp1 P and SHP1 GAL el xrsigGPGc RC15 eeshifted were (RHC3175) GFP–Glc7 expressing cells el xrsigGPGc RC15 eegonin grown were (RHC3175) GFP–Glc7 expressing cells -3HA-SHP1 ts sds22-6 uat rw ttersrcietemperature restrictive the at grown mutants RC00 and (RHC3070) uat n htSs2i o co-aggregated not is Sds22 that and mutants el xrsigetpcGPGc n Hsp104– and GFP–Glc7 ectopic expressing cells GAL m QQ m. 6 Sp,btntwt Cdc48–Shp1 with not but –Shp1, ..frtoidpnetexperiments. independent two for s.d. rmtr ecudraiydtc all detect readily could we promoter, uat.Wsenbossoe that showed blots Western mutants. RC91 el coial expressing ectopically cells (RHC2951) Z P sakiae n I mgsare images DIC and images -stack GAL 6 -3HA-SHP1 ..frtreindependent three for s.d. 6 ..frtoindependent two for s.d. RC15 cells (RHC3175) Z glc7-12 (A) sakimages -stack SHP1 sds22-6 cells hra h te he uain i o Fg A.The severe 6A). (Fig. less was not mutant did L313P original mutations the the than of three sensitivity sensitivity, other temperature temperature conferred the mutation whereas L313P the only that rnin soito fCc8Sp ihP1cmoet is Glc7. components the of PP1 integrity that structural with indicating the Cdc48–Shp1 for aggregation, important of Glc7 association with transient correlate PP1 with tcst h ed sdi h ulon h etr ltstill blot western the pulldown, the Cdc48 in Although used beads mutant. the L313P to with of consistent sticks sensitivity 6B), (Fig. temperature mutants the single-site other and type wild eeldta h muto Cdc48 of amount the that revealed niiulsnl-iemtn a eeae n expressed and generated was the in mutant ectopically single-site individual htteaonso Cdc48 of amounts the that ewe h1adP1 eaaye h ormttosi the in mutations four the analyzed we PP1, Shp1 their and Shp1 in between identity phosphatases. degree these that regulate high suggesting also 2000), a could al., et Cdc48–Shp1 (Venturi share Glc7 with which domains catalytic 5E), and (Fig. Ppz2 Ppz1, Ppq1 phosphatases, PP1-like additional with associated hra te igest uat eae ietewl type wild the like behaved Therefore, mutants 6D). (Fig. single-site other whereas ewe n n he oii el rae ihMG132 degradation cells. with Shp1-expressing the in blocking even treated that proteins these and cells of proteasome aggregation in the causes normally are by Pph3 foci and over Pph21 protein turned that three suggest results The of 7A,B). and (Fig. formed one subunits they but Pph3–GFP between cells, catalytic Shp1-expressing and in Pph21–GFP localized diffusely respectively. Pph3, were 4, also and We and 2A Shp1. phosphatase without aggregate Pph21 to tend Ppq1 examined and Ppz2 similar Glc7, Thus, in 7A,B). to By foci (Fig. 7A,B). multiple MG132 with (Fig. formed treated Shp1 cells MG132 Ppq1–GFP Shp1-depleted by of and affected Ppz2–GFP presence both not or preferentially contrast, absence was the which Ppz1–GFP in periphery, depletion cell integrity. Cdc48–Shp1 the whether to also structural localized asked Ppq1 their we and 5E), Ppz2 (Fig. promoted Shp1 Ppz1, with phosphatases associated PP1-like the Because Ppz2 of Ppq1 aggregation and and misfolding prevents Cdc48–Shp1 with treated cells in those mutant to L313P similar temperature, GFP– Shp1 two restrictive Furthermore, the in data). at other foci MG132 unpublished (our formed the Cdc48 Glc7 and addition, binding reduced PP1 to foci, GFP–Glc7 to In together of formation act show Glc7. also Shp1 produced and of Cdc48 temperature-sensitive misfolding that Fig. notion prevent material the (supplementary supports temperature S2) restrictive in The at Cdc48. foci MG132 with formed associate GFP–Glc7 to that inability finding of consequence a be might r loipratfrtesrcueo ucino h1 h TAP- The Shp1. of function Shp1 or structure tagged the for important also are antre fCc8Sp.Itrsigy Cdc48 the Interestingly, is Cdc48–Shp1. complex of Glc7–Sds22–Ypi1 the target that main indicating 5E), (Fig. o le h rti tblt.Pldw nlsssoe htthe Shp1 associated that 8myc–Ypi1 with showed and analysis 2myc–Sds22, 2myc–Glc7, Pulldown of did stability. amounts mutations protein the the that indicating alter 6B), not (Fig. wild-type the as levels ihShp1 with onwsas eue Fg B.Tose A uiiainof purification TAP Two-step Shp1 6B). and (Fig. Shp1 reduced also was down ofrhrivsiaeteiprac fteinteraction the of importance the investigate further To ts ora fCl cec 21)18 1019 doi:10.1242/jcs.165159 1180–1192 128, (2015) Science Cell of Journal rti:I3V 21,L1PadF9L Each F396L. and L313P K241E, I237V, protein: ts ts ts Fg C,sgetn httepeoye of phenotypes the that suggesting 6C), (Fig. n h1L1Pwr eue oprdwt the with compared reduced were L313P Shp1 and n igest uat eeepesdt similar to expressed were mutants single-site and ts rmCdc48 from shp1 shp1 shp1 ts shp1 ugsigta h te he residues three other the that suggesting , D QQ tan eildlto ltsshowed plates dilution Serial strain. uain htdsutteinteraction the disrupt that mutations lee htw aeioae also isolated have we that alleles QQ d2 n l7wr l reduced all were Glc7 and Sds22 , epesn el ute confirms further cells -expressing QQ cdc48-3 nteShp1 the in el rae with treated cells QQ ts TPpull- –TAP Sp also –Shp1 shp1 shp1 1183 ts ts ,

Journal of Cell Science nofc nSp-eltdcls(i.7,) niePq and Ppq1 Unlike 7C,D). concentrated (Fig. ( protein cells the cells Shp1-depleted Ppq1 whereas in Shp1-expressing puncta, foci few in into a GFP–Ppq1 only of produced induction the copper-inducible the EERHARTICLE RESEARCH 1184 full- expressed we (Ppq1 is domain depletion, non-catalytic domain its Shp1 which and upon Ppq1, determine length To aggregation Glc7. to in prone absent is that extension Pph3. and Pph21 the of in integrity Shp1 role 7A), significant structural (Fig. no cells, foci has the probably Cdc48–Shp1 Shp1-expressing of that size suggesting or than intensity the enhance Shp1-depleted be not did to depletion in seemed Shp1-depleted there foci Although in 7A). more (Fig. foci MG132 formed with treated also cells Pph3–GFP and Pph21–GFP clearance. for proteasome the require and protein synthesized newly of consist SHP1 foci GFP–Glc7 2. Fig. 3)adctltcdmi (Ppq1 domain catalytic and 237) fe – as (B) days. 2–3 after o.Saebr:5 bars: Scale top. fM12o MOa niae n rw t25 at grown and indicated as DMSO or MG132 of el otiigvrosnmeso F–l7fc nteeprmn hw n()i lte smean as plotted is (B) in shown of experiment projections in the Maximum produced in microscopy. first foci fluorescence GFP–Glc7 by were of analyzed numbers and various h containing 1 cells for (CHX) cycloheximide and MG132 p1 p2adPq aea -emnlnon-catalytic N-terminal an have Ppq1 and Ppz2 Ppz1, C RC96 and (RHC1946) Ppq1 , N a xrse oahge ee n a enriched was and level higher a to expressed was m SHP1 m. shp1 shp1 CUP1 RC78 and (RHC3738) ts ts el RC79 rae ihM12fr1ha 34 at h 1 for MG132 with treated (RHC3739) cells RC97 ee1-odsra iue nS-R ltsadgona h niae eprtr.Tepae eephotographed were plates The temperature. indicated the at grown and plates SC-URA on diluted serial 10-fold were (RHC1947) rmtr ntepeec fMG132 of presence the In promoter. C mn cd 3–4)through 238–549) acids amino , shp1 ts ˚ o – .Mxmmpoetosof projections Maximum h. 1–3 for C RC79 el xrsigGPGc eesitdfo 25 from shifted were GFP–Glc7 expressing cells (RHC3739) N mn cd 1– acids amino , GAL ) ˚ .AtrwsigotM12 h utr a pi notowt h addition the with two into split was culture the MG132, out washing After C. eurdfrtesrcua nert fteP1cmlxin complex PP1 the of integrity structural is chaperone the Cdc48–Shp1 the for that demonstrate required we report, this In DISCUSSION d4–h1i motn o rprfligo P catalytic PP1 that of suggest folding results proper our for the together, subunits. important in Taken is cells not. full- Cdc48–Shp1 Shp1-depleted the did though in even Ppz1 data), foci length unpublished (our formed MG132 also of presence region alone domain treatment non-catalytic catalytic Ppz1 the of Similarly, MG132 of structure. extension Ppq1 N-terminal upon the stabilize the Ppq1 might number that that shows and and result Glc7 size This 7C,D). in (Fig. the and increased treatment MG132 foci without even cells Shp1-depleted some rae ihM12(i.7,) oee,Ppq1 However, cells 7C,D). Shp1-depleted (Fig. in MG132 aggregates with forming treated without nucleus the in Z sakiae r hw.Teshm fteeprmn ssona the at shown is experiment the of scheme The shown. are images -stack ora fCl cec 21)18 1019 doi:10.1242/jcs.165159 1180–1192 128, (2015) Science Cell of Journal 6 ..frtoidpneteprmns D F–l7foci GFP–Glc7 (D) experiments. independent two for s.d. Z A A (A) sakiae r hw.()Tepretg of percentage The (C) shown. are images -stack ˚ Cto34 shp1 ˚ D ntepeec rasneof absence or presence the in C eeinsri arigplasmid-born carrying strain deletion C eae iial to similarly behaves C omdfc in foci formed

Journal of Cell Science EERHARTICLE RESEARCH GAL repressing By yeast. budding erddtruhtepoesm nteasneo functional of and misfolded absence is the Glc7 in subunit proteasome catalytic the PP1 through the degraded that show we rmtro yuigatemperature-sensitive a using by or promoter SHP1 xrsintruhueo the of use through expression shp1 ts mutant, n p1 h nsebe l7i rn omsodn and misfolding to prone is Glc7 Sds22 unassembled subunits regulatory the to Ypi1, binding and is without function proteasome Similarly, cannot the unless intact. that Shp1 aggregates restoring upon irreversible resolved be forms Glc7 Misfolded Shp1. ora fCl cec 21)18 1019 doi:10.1242/jcs.165159 1180–1192 128, (2015) Science Cell of Journal r hw.Saebr:5 bars: Scale shown. are Maximum or h. of DMSO 2.5 projections of last presence the the for glucose in MG132 to h shifted 7 were for D medium The in (F) as time. cells over same plotted containing is D foci in GFP–Glc7 shown cells of percentage of projections Z Maximum time. medium indicated glucose the to for shifted then galactose and in medium grown first were (RHC3397) Glc7 i.3 F–l7frsageae in aggregates sds22-6 forms GFP–Glc7 3. Fig. niae.Mxmmpoetosof projections Maximum indicated. eesitdt 35 to shifted were Hsp104–mCherry and GFP–Glc7 expressing and (RHC3368) (D) of or projections Z DMSO Maximum of indicated. presence as the MG132 in min 120 for F–l7(H36)wr hfe o35 to shifted were (RHC3369) GFP–Glc7 is foci (C) time. GFP–Glc7 over containing plotted A of in percentage shown The cells (B) shown. are images sakiae r hw.()The (E) shown. are images -stack sakiae n I mgsaeshown. are images DIC and images -stack P GAL -3HA-YPI1 n p1dpee cells. Ypi1-depleted and Z sakiae n I images DIC and images -stack sds22-6 ˚ o –2 i as min 0–120 for C el xrsigGFP– expressing cells sds22-6 RC39 cells (RHC3369) m m. el expressing cells (A) Z -stack SDS22 1185 ˚ C

Journal of Cell Science ossetwt hsnto,w eosrt htGc aggregates Shp1. Glc7 depleting that in after demonstrate long we formed notion, over, this turned fraction with 2002)], been Consistent significant al., has a Glc7 et a after exiting (Nigavekar for only of Glc7 expected apparent for As become 8). phenotypes min (Fig. the 180 Ypi1 [over and and/ half-life Sds22 Glc7 long with nascent of assembly Rather, folding its complex. the the or affects of probably maintenance depletion the Cdc48– for Shp1 that required implying not depletion, promoter is Shp1 Shp1 by the the affected not of complex probably after appearance Glc7–Sds22–Ypi1 is existing slow the depleted The that 2010). indicates quickly phenotypes Chen, and is (Cheng protein shutdown Shp1 though EERHARTICLE RESEARCH 1186 conditional used we 2010), Chen, and mutants, (Cheng integrity. background a structural as its acts promote Cdc48–Shp1 to chaperone that of physically PP1 proposes absence general study also the Our in Ppq1, aggregates Shp1. form functional and and the Cdc48–Shp1 Ppz2 Glc7, with interact Besides phosphatases, Glc7-Sds22-Ypi1. PP1-like of, other might assembly and the with, promote associates transiently Cdc48–Shp1 aggregation. grgtsatrpoogdsprsinof suppression misfolded prolonged after buffering aggregates not of might Glc7 strains. (Bo capacity some of in aggregation level their apparent and be degradation in or that different so activity vary localization proteins, that Glc7 reflect strains its might reduces yeast discrepancy on The background 2013). effect strain Buchberger, an DF5 al., et the without the (Robinson that in shown domain has mutant report UBX recent C-terminal a However, the 2012). lacks and in acids reduced is Glc7–mCitrine of a level nuclear the with and Consistent background, Glc7. of localization finding, nuclear our the for important is htls fSs2cue ifligo xsigGc.The Glc7. existing of misfolding restrictive indicates causes result the Sds22 our 2002), to of temperature-sensitive al., et loss shift (Peggie that binding the Glc7 the affect after Because to 3A). min (Fig. 30 temperature Sds22 within and Glc7–Sds22 Glc7 once aggregates dispensable with contrast, be By complex nucleus. might large the it enters a 2007), in al., is et all (Pedelini protein although Ypi1 Thus, 2008). Glc7 the al., nuclear et of the (Bharucha later of much to reduction occurs and level Similar arrest gene mitotic 2B). essential whereas (Fig. protein, the protein of synthesized suppression newly from Fg B.Telvlo ula l7i hsmtn salready is mutant this in Glc7 temperature nuclear restrictive the of to level shift The the 2B). of (Fig. h 1 within aggregates, shp1-105 Because efudta l7dsper rmtencesand nucleus the from disappears Glc7 that found We P GAL shp1 uatta otisol h -emnl15amino 105 N-terminal the only contains that mutant SHP1 SHP1 -3HA-SHP1 ts el ttersrcietmeaueaederived are temperature restrictive the at cells sesnili tandrvdfo h S288C the from derived strain a in essential is sa seta eei u 33strain W303 our in gene essential an is shp1 ts and el loqikyacmlt Glc7 accumulate quickly also cells sds22-6 shp1 YPI1 ts sds22-6 odmntaeta Shp1 that demonstrate to , uat cuuaeGlc7 accumulate mutants ucl eltsYpi1 depletes quickly SHP1 uaini known is mutation Fg A,even 1A), (Fig. shp1 h and ¨hm deletion SHP1 , hsepan h l7ageainapast efse in faster be temperature, to high appears aggregation the Glc7 at why turnover explains protein this fast with combination n p2as neatwt d2 n p1(riket tal., et (Breitkreutz Ypi1 and Sds22 with Ppz1 interact 7A). in also (Fig. aggregates Ppz2 inhibition form and proteasome and upon 5E) (Fig. cells Shp1 Shp1-depleted with ubiquitin associate also of Ppq1) form new a Cdc48–Shp1. or and we to minor binding involves modification However, process manner. ubiquitin the that ubiquitin-independent transient possibility a the exclude in cannot PP1 probably on Cdc48–Shp1 (our is that acts viability imply domain cell results and These ubiquitin-binding of PP1 data). unpublished of UBA analysis localization known deletion nuclear for serial the dispensable addition, that In shows components. Shp1 form ubiquitin-modified PP1 any the detect of to unable were we substrates, the at Cdc48 with interaction domain. high the UBX a affects nearby the at it distort conformation that SEP Shp1 such might the affect temperature mutation linking further region L313P and a the backbone in peptide Thus, the (Dreveny located domains. flies is UBX confers and residue and rats This humans, alone 2004). conserved from al., is homologs mutation et Shp1 L313 p47 that L313P the shows in Shp1 alignment 6). (Fig. Sequence the aggregation is phenotypes. Glc7 particular, interaction cause also of direct In interaction the completion the because disrupt Glc7, the that that molecular of integrity after demonstrate structural a for substrate further important for its We expected releases action. is that which chaperone 5A,B), (Fig. aggregated. is Sds22–Ypi1 protein the that notion the supports centrifugation eue tteprisv eprtr Fg B,idctn that indicating 2B), (Fig. temperature Shp1 permissive the at reduced uclua oain.Sm ftergltr uuis including or subunits, substrates regulatory specific the to of enzyme Some the locations. target subcellular or activity the an control of instead of Glc7, suppression on prolonged depletion from Shp1 effect of indirect effect of direct a suppression suggests upon than temperature SHP1 restrictive to shifted cells h rtaoe(i.8.Orfnigta el yteie Glc7 synthesized newly that finding by in Our removed 8). normally is might (Fig. and proteasome the aggregation is subunits Glc7 and Glc7 misfolding of unassembled to regulatory self-aggregation Therefore, prone surface. prevent the hydrophobic and or its Wu structure through of 2002; Glc7 al., the Binding et stabilize (Ceulemans 2001). region distinct interacts Tatchell, a hydrophobic Sds22 at whereas a 2001), Glc7 to Tatchell, with binds and (Wu that Glc7 sequence of groove RV/IXF an contain Ypi1, nadto oGc,ohrP1lk hshtss(p2and (Ppz2 phosphatases PP1-like other Glc7, to addition In ubiquitylated on act to thought generally is Cdc48 Although Glc7– with transiently interacts Cdc48–Shp1 that show We l7ascae ihavreyo euaoysbnt that subunits regulatory of variety a with associates Glc7 shp1 ts xrsinb s fthe of use by expression ucini opoie n P atal fetd In affected. partially PP1 and compromised is function ora fCl cec 21)18 1019 doi:10.1242/jcs.165159 1180–1192 128, (2015) Science Cell of Journal ts uatsdmnsit h eltfato upon fraction pellet the into sediments mutant RC77 el xrsig3ASs2wr is grown 25 first at were 3HA–Sds22 expressing cells in (RHC3727) aggregated is Glc7 shp1 synthesized Newly 4. Fig. ceeo apepeaaini hw nright. on shown is preparation The sample antibodies. of anti-HA scheme and western and anti-Myc by (S) followed with soluble centrifugation, blotting into by lysate fractions cell (P) the pellet separate in to (T) performed proteins was total temperature assay the sedimentation during A galactose shift. 0.2% of addition from the induced was 2myc–Glc7 MG132. ˚ ts n hnsitdt 34 to shifted then and C mutants. SHP1 GAL RC76 and (RHC3726) rmtr hsrsl also result This promoter. ˚ o ntepeec of presence the in h 1 for C SHP1 GAL shp1 shp1 expression. rmtrby promoter ts mutations shp1 ts

Journal of Cell Science EERHARTICLE RESEARCH H18) ellsts(nu)adteTPpldw yIGspaoewr nlzdb etr ltigfrTP y n A B Cdc48 (B) HA. and Myc TAP, for blotting (RHC1967, western 4HA–Ypi1 by or analyzed RHC3510), were (RHC3509, IgG–sepharose 2myc–Sds22 by RHC1814), pulldown (RHC1810, TAP 2myc–Glc7 the express and ectopically (input) to lysates used Cell were RHC1987). 4) and 2 (lanes Glc7-Sds22-Ypi1. locus with associates transiently Cdc48–Shp1 5. Fig. E uiidsmlsfo eesbetdt assetoer nlss w needn eut r shown. are results independent Two analysis. spectrometry mass to subjected were D from samples Purified (E) mca h hoooa ou nwl-yeclsadepesdwt nagdSp RC85,i idtp el ihcrmsmlyTPtge Shp1 TAP-tagged chromosomally with cells wild-type in (RHC2885), Shp1 untagged with expressed and cells in wild-type and in (RHC2898) locus chromosomal the (RHC29 Shp1 the and untagged TAP (RHC2977) at with and cells Shp1 2myc wild-type (input) TAP-tagged in lysates chromosomally expressed ectopically cell with was 2myc–Sds22 The cells (C) RHC2071). type blotting. (RHC2070, western 8myc–Ypi1 by analyzed or were IgG–sepharose RHC2077) by (RHC2076, pulldown 2myc–Sds22 RHC1898), (RHC1896, 2myc–Glc7 expressed eeaaye ywsenbotn.()Tose A uiiainwspromdwt el xrsigglcoeidcdCc8(T n nagdShp untagged and (WT) Cdc48 galactose-induced expressing Cdc48 cells expressing with cells performed with was and purification (RHC2965) TAP Shp1-TAP Two-step or (D) (RHC2964) blotting. western by analyzed were hog the through GAL rmtri el otiigutge h1(ae n )o A-agdSp ttecrmsmllcs(ae n )adectopically and 4) and 2 (lanes locus chromosomal the at Shp1 TAP-tagged or 3) and 1 (lanes Shp1 untagged containing cells in promoter sds22-6 el ihcrmsmlyTPtge h1(H29) fe Cdc48 After (RHC2899). Shp1 TAP-tagged chromosomally with cells A el otiigutge h1(ae n )o A-agdSp ttechromosomal the at Shp1 TAP-tagged or 3) and 1 (lanes Shp1 untagged containing Cells (A) glc7-12 QQ Q)adSp–A RC09.Tesmlswr nlzdb ivrstaining. silver by analyzed were samples The (RHC2069). Shp1–TAP and (QQ) uatwt hoooal A-agdSp RC96.Gc a agdwith tagged was Glc7 (RHC2976). Shp1 TAP-tagged chromosomally with mutant ora fCl cec 21)18 1019 doi:10.1242/jcs.165159 1180–1192 128, (2015) Science Cell of Journal QQ a xrse,cl yae n h A pulldown TAP the and lysates cell expressed, was QQ a expressed was 0,wild- 50), 1 1187

Journal of Cell Science l7adTAP-tagged and Glc7 EERHARTICLE RESEARCH Fg C.I otat h aayi oan fPq n Ppz1 and Ppq1 of 1188 cells domains Shp1-depleted catalytic in the aggregates contrast, catalytic the In forms the and 7C). rarely full-length (Fig. of Glc7 it the than N-terminus and for level is domain, the higher Ppq1 as a of at region to Ppq1, non-catalytic expressed extension N-terminal with The additional and domain. Ppz2 catalytic an interact Ppz1, have to Furthermore, S3). Ppq1 Sds22 Shp1 Fig. notion, PP1 material for this of (supplementary with assembly important Consistent the Ypi1. in is and role that Sds22 general and a containing subunits have regulatory might some Cdc48–Shp1 share phosphatases PP1-like Garcı 2010; 6. Fig. A-agdwild-type TAP-tagged mcYi RC29 93 90 95 97 99.Clswr is rw t25 at grown first were Cells 3909). 3907, 3905, 3930, 3903, (RHC2279, 8myc–Ypi1 shp1 eeaaye ywsenbotn o h niae rtis C w-tpTPprfcto a efre ihclsepesn h1TP(RHC2237) Shp1–TAP expressing cells with performed was purification TAP Two-step (C) proteins. indicated the for blotting Shp1 western by analyzed were rw t25 at grown ee1-odsra iue nYDpae n rw tidctdtmeaue.()Cdc48 (B) temperatures. indicated at grown and plates YPD on diluted serial 10-fold were ts uat n hoooa mcGc RC25 84 86 88 81 83,etpc2y–d2 RC27 83 85 88 90 91 or 3901) 3900, 3898, 3895, 3823, (RHC2237, 2myc–Sds22 ectopic 3893), 3891, 3888, 3886, 3824, (RHC2275, 2myc–Glc7 chromosomal and mutants TP(H32)a hw nB h ape eeaaye ysle tiig D el eee o endogenous for deleted Cells (D) staining. silver by analyzed were samples The B. in shown as (RHC3823) –TAP shp1 ˚ n hnsitdt 34 to shifted then and C ´ L313P -ieoe l,20) ugsigta l7adother and Glc7 that suggesting 2003), al., et a-Gimeno uainafcsitrcinwt P n assGc aggregation. Glc7 causes and PP1 with interaction affects mutation SHP1 SHP1 (RHC4072), (RHC3984), ˚ o ntepeec fDS ( DMSO of presence the in h 1 for C shp1 shp1 ts ts (RHC4074), (RHC3985), shp1 shp1 I237V I237V (RHC4076), (RHC3986), 2 rM12() aiu rjcin of projections Maximum (+). MG132 or ) lopoue F–l7pnt pnM12tetet whereas treatment, MG132 upon puncta Glc8 subunit GFP–Glc7 regulatory of produces deletion that addition also found In we degraded. Ypi1, become and Sds22 and PP1 to misfold the to components tend subunits regulatory catalytic with association Without and unstable. subunits regulatory the might of chaperone. subunits perturbation Cdc48–Shp1 to the catalytic extension sensitive the more lacking PP1 Glc7 is whereas some structure, protein the of the stabilize Thus, data). extension unpublished our N-terminal do 7C; than (Fig. aggregates proteins more full-length the form cells Shp1-depleted in synthesized shp1 shp1 ˚ u td eel htpoenpopaae r intrinsically are phosphatases protein that reveals study Our n hnsitdt 34 to shifted then and C K241E K241E QQ ora fCl cec 21)18 1019 doi:10.1242/jcs.165159 1180–1192 128, (2015) Science Cell of Journal A el eee o endogenous for deleted Cells (A) (RHC4078), a nue nclsta xrs TAP-tagged express that cells in induced was (RHC3987), shp1 shp1 ˚ o .Lsts(nu)adTPpull-downs TAP and (input) Lysates h. 1 for C L313P L313P Z sakiae r hw.Saebr 5 bar: Scale shown. are images -stack RC00 or (RHC4080) RC98,and (RHC3988), SHP1 SHP1 n coial xrsigGFP– expressing ectopically and shp1 n coial expressing ectopically and shp1 F396L F396 RC02 were (RHC4082) SHP1 (RHC3989) rvarious or and m m.

Journal of Cell Science EERHARTICLE RESEARCH i.7 h1dpeincue grgto fPz n Ppq1. and Ppz2 of aggregation causes depletion Shp1 7. Fig. oguoemdu o ,floe ycpe nuto o . ntepeec fDS rM12 aiu rjcin of projections Maximum mean MG132. The or DMSO quantified. of were presence foci the GFP in containing h C 1.5 in for induction shown copper cells by of followed fractions h, The 1 for medium glucose to RC48,Ph1(H32)o p3(H32)a h hoooa oiwr anandi aats rsitdt lcs-otiigmdu o h. of 7 projections for Maximum medium microscopy. mean glucose-containing fluorescence The to by quantified. shifted were analysis or foci before GFP galactose h containing in 2.5 A maintained in last were shown the cells loci during of chromosomal added fractions the was at MG132 (RHC3422) or Pph3 DMSO or (RHC3420) Pph21 (RHC3418), F-agdPq RC63,o t o-aayi oan(Ppq1 domain non-catalytic its or (RHC3623), Ppq1 GFP-tagged N H34)o aayi oan(Ppq1 domain catalytic or RHC3640) , (A) P GAL -3HA-SHP1 6 ..o w needn xeiet sson (C) shown. is experiments independent two of s.d. 6 ..o w needn xeiet sson cl as 5 bars: Scale shown. is experiments independent two of s.d. el xrsigGPtge p1(H31) p1(H31) Ppz2 (RHC3416), Ppz1 (RHC3414), Ppq1 GFP-tagged expressing cells ora fCl cec 21)18 1019 doi:10.1242/jcs.165159 1180–1192 128, (2015) Science Cell of Journal C H32) otoldby controlled RHC3624), , Z sakiae r hw.()The (B) shown. are images -stack Z P sakiae r hw.(D) shown. are images -stack GAL CUP1 -3HA-SHP1 rmtrwr shifted were promoter el carrying cells m m. 1189

Journal of Cell Science and A,40b fteusra euneadtecdn einof region pRS404 coding the and sequence at upstream cloned the was of bp 400 TAP, eelnalzdwith linearlized were at codon stop of downstream bp (500 region of promoter codon) the start Ypi1, of upstream 8myc-tagged and 4HA- of expression with at linearlized codon stop start of of upstream bp at (500 region promoter of codon) the Sds22, 2myc-tagged and EERHARTICLE RESEARCH 1190 S1. Table material supplementary in shown is genotypes their and strains SHP1-TAP TRP1 epitope C-terminal for deletions, substitution Gene promoter W303. and of tagging derivatives are strains strains yeast All and plasmids of Construction METHODS AND MATERIALS always regulatory events. is the dephosphorylation by PP1 fortuitous substrates avoid of the to to activity subunits targeted catalytic and This control controlled the quality absent. properly that protein is a ensuring Reg1 represent might when mechanism PP1 in of that fill instability such might intrinsic 2003), subunits. al., subunits et lower regulatory regulatory (Ghaemmaghami a at other Glc8 major expressed and is the Sds22 Reg1 than that are level predicted integrity Glc8 been Glc7 has and it on Consistently, deletions Sds22 material their (supplementary that of impact effect suggest differential obvious The no S4). has Fig. Reg1 of deletion ptp a at tag epitope the at integration at codon stop or of downstream start bp of 200 upstream at bp sequence (500 epitope region GFP- promoter of of the codon) expression Glc7, ectopic For 2myc-tagged loci. and chromosomal the at recombination YPI1 Xba pRS405 Sal eegnrtdb C-eitditgainadhomologous and integration PCR-mediated by generated were ou.The locus. and I ie,ad50b ontemrgo at region downstream bp 500 and sites, I h lsi a ierie with linearlized was plasmid The . SDS22 GLC7 h lsiswr ierie with linearlized were plasmids The . Bam yst-ietdmtgnss h opeels fyeast of list complete The mutagenesis. site-directed by Eco Xho Ists n h oigrgo ls20b downstream bp 200 plus region coding the and sites, HI Eco a lndat cloned was shp1 a lndat cloned was URA3 Bam Iand RI and I Xba Vt nert tthe at integrate to RV Sna Iand HI igest uat eegnrtdfrom generated were mutants single-site or and I Ifritgaina the at integration for BI Sal Xho YPI1 LEU2 ie,adtecdn einpu 0 bp 200 plus region coding the and sites, I Sal ie,teTPeioesqec at sequence epitope TAP the sites, I Sal Bam Sac and I a lndat cloned was Sac ie in sites I ou.Fretpcepeso f3HA- of expression ectopic For locus. and I Ists n h oigrgo plus region coding the and sites, HI and I Bam Not P GAL Xba ie in sites I Iand HI Xba pRS405 -3HA-SHP1 Sna ie,teeioesequence epitope the sites, I LEU2 Bam ie,teGPo 2myc or GFP the sites, I Ifritgaina the at integration for BI Hind TRP1 pRS404 Sal Iand HI h lsiswere plasmids The . Stu ou.Frectopic For locus. and I I ie in sites III ou.FrShp1– For locus. Ior and h plasmids The . 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Ohno, and T. Sakata, M., Kitabatake, K., Fujii, S. C. Chan, and W. Wang, L., Francisco, S. P. Freemont, and X. Zhang, A., Shaw, K., Uchiyama, H., Kondo, I., Dreveny, H. R. Chen, and L. Y. Cheng, and W. Stalmans, K., Tatchell, M., Maeyer, De V., Vulsteke, M. H., Ceulemans, Bollen, and H. Ceulemans, oe o ehia sitnei mgn,adD.Ta-a e (IPMB analysis. Wen Tuan-Nan spectrometry Dr. mass and for imaging, Core) in Proteomics assistance technical for Core) uhn .R,Klii,R . alr .P n akr R. F. Parker, J. Cannon, and P. J. Taylor, M., R. Kolaitis, R., B., J. Larsen, V., Buchan, Neduva, L., Boucher, R., J. Sharom, H., Choi, A., Breitkreutz, Bo K. Tatchell, K. and Tatchell, and K. A. Bloecher, L. Daves, L., Gao, R., J. Larson, P., J. Bharucha, T) qiaetprin ftettlpoen oul n pellet and soluble mM 10 protein, and SDS total 1% the 8.0, 95 5 pH of with Tris-HCl supplemented were portions mM fractions 50 Equivalent urea, M DTT). (8 buffer HU MET,1 MMgCl mM EDTA, 10 mM 1 7.2, EGTA, pH phosphate, potassium mM mM mM 5 25 (10 buffer sorbitol, lysis in phosphate, M lysed potassium 1 mM (100 7.5, buffer pH spheroplasting in zymolyase with uhrcontributions Author interests. financial or competing no declare authors The interests Competing inputs, scientific for providing Stark and reagents Michael sharing Dr for Wang Chao-Wen Dr thank We Acknowledgements OD at Cells assay Solubility c,K,Lisebr,M . cemn,L,Slmn,F . op,T and T. Hoppe, A., F. Salomons, L., Ackermann, S., M. Luijsterburg, K., Acs, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.165159/-/DC1 online available material Supplementary material Supplementary Science Taiwan. National 101-2311-B-001-021-MY3], and NSC Sinica; Academia number from [grant grants Council by supported was study This manuscript. the wrote Funding R.-H.C 1E. Figure for except data, experiments the the analyzed performed Y.-L.C. and experiments. the designed R.-H.C. and Y.-L.C. eppi,ppttn n hmsai)fr1 i nie h lysates The ice. on min 10 700 10 at and for spun PMSF chymostatin) were mM and 1 pepstatin, TX-100, leupeptin, 0.5% vanadate, sodium rcin eete eaae noslbe()adple P rcin by fractions (P) pellet and (S) 16,000 soluble protein at into total centrifugation separated The (T). then fraction were protein fractions total as collected were supernatants m .adBcbre,A. Buchberger, and S. hm, ¨ 20) tutrlbsso h neato ewe h A Taep97/VCP ATPase AAA the p47. between protein interaction adaptor the its of and basis Structural (2004). activity. B Aurora balancing 123 by bi-orientation chromosome promote phosphatase-1. protein of 47331-47337. 6-triangle 5/alpha 4/alpha alpha M. Bollen, button. reset and economizer cellular a phosphatase-1, iscainadpoesm-eedn N erdto nnonfunctional in degradation chromosome RNA yeast decay. rRNA regulating proteasome-dependent 25S in and kinase dissociation protein IpL1 to opposition segregation. in acts cerevisiae. function. Cdc48/VCP and autophagy by cleared 1461-1474. are al. granules et stress G. yeast. Liu, in C., network Stark, interaction phosphatase J., and B. kinase Breitkreutz, Y., Z. Lin, 1 phosphatase protein of regulation positive by (Glc7). progression cycle cell cerevisiae. in promotes Saccharomyces activity of cycle 1 cell mitotic type the in phosphatase 1 protein nuclear of regulator cerevisiae. Saccharomyces positive a Ypi1, erimn yrmvn 3BL rmDAdul-tadbreaks. double-strand DNA from L3MBTL1 Biol. Mol. removing Struct. by P. recruitment N. Dantuma, ˚ o i n eovdb D-AEfrwsenblotting. western for SDS-PAGE by resolved and min 3 for C 2025-2034. , LSONE PLoS ora fCl cec 21)18 1019 doi:10.1242/jcs.165159 1180–1192 128, (2015) Science Cell of Journal 20) idn ftecnaesraeo h d2 uehlxt the to superhelix Sds22 the of surface concave the of Binding (2002). 600 d.Ap.Microbiol. Appl. Adv. 21) ucino rti hshts-,Gc,i Saccharomyces in Glc7, phosphatase-1, protein of Function (2010). o.Cl.Biol. Cell. Mol. 5 , 18 MOJ. EMBO sds22-6 .– eecletdadshrpat eeprepared were spheroplasts and collected were 0.5–1 g 8 21) h A-TaeVPp7pooe 53BP1 promotes VCP/p97 AAA-ATPase The (2011). 1345-1350. , e56486. , o 0mna 4 at min 10 for g 20) yai oaiaino rti hshts type phosphatase protein of localization Dynamic (2000). o t4 at h 1 for and 21) h A-TaeCc8adcfco Shp1 cofactor and Cdc48 AAA-ATPase The (2010). 31 o.Bo.Cell Biol. Mol. 14 MOJ. EMBO 21) h udn es d4(h1 complex Cdc48(Shp1) yeast budding The (2013). 2579-2589. , 4731-4740. , glc7-12 73 2 b ,50mM 27-59. , mratehnl.Shrpat were Spheroplasts -mercaptoethanol). 20) ucinldvriyo protein of diversity Functional (2004). 6 23 D-AEsml ufr etdat heated buffer, sample SDS-PAGE ˚ tan,SePn e IBImaging (IMB Lee Sue-Ping strains, .Tepleswr eupne in resuspended were pellets The C. ˚ 1030-1039. , ormv eldbi n the and debris cell remove to C 19) ye1poenphosphatase protein 1 Type (1994). 19 1032-1045. , b gyeohsht,1mM 1 -glycerophosphate, .Cl Biol. Cell J. Science 21) lblprotein global A (2010). hso.Rev. Physiol. 21) 0 subunit 40S (2012). .Bo.Chem. Biol. J. 21) Eukaryotic (2013). m 328 /lec of each g/ml 149 1043-1046. , .Cl Sci. Cell J. 125-140. , Cell 84 (2008). 1-39. , 1191 277 153 Nat. , ,

Journal of Cell Science hua .adYngd,M. Yanagida, F. and J. H. Cannon, Ohkura, and S. Y. Tan, S., S. Nigavekar, eeii . aqia . Arin M., Marquina, L., Pedelini, Garcı ARTICLE RESEARCH 1192 P., Rana, V., Katheria, M., Badadani, E., Dec, S., Donkervoort, A., Nalbandian, J. M. Stark, and D. P. Andrews, H., S. MacKelvie, E., Waelkens, A., M. Garcia-Gimeno, L., Pedelini, M., Beullens, B., Lesage, L. E. Eskelinen, Y., Muehe, P., Schlotterhose, E., Welter, S., Bremer, R., Krick, A. Kakizuka, and A. Manno, T., Kobayashi, J. Frydman, and R. Kopito, D., Kaganovich, ee,H,Bg .adBee,S. Bremer, and M. Bug, H., Meyer, K. Matsumoto, and K. Tatchell, K., Sugimoto, L., D. Frederick, N., Hisamoto, Y., Kamei, Y., Ohsawa, K., Nakadate, K., Tanaka, K., Inoue, M., Hirabayashi, H., A. Johnsen, G., Koch, K., Hofmann, A., M., Wallace, Belle, R., W., Hartmann-Petersen, R. S. Howson, D. K., Haines, Bower, K., W. Huh, S., Ghaemmaghami, ersil ciao fGc rti phosphatase-1. protein Glc7 404 of activator repressible imttctasto noe ecn-ihrpa rti htpositively that protein repeat leucine-rich a phosphatase-1. protein encodes modulates transition midmitotic yptywt ae’ ies fbn,fottmoa eeta and dementia, frontotemporal bone, body of inclusion disease sclerosis. diseases: lateral Paget’s amyotrophic protein-associated with al. et valosin-containing myopathy B. Martin, of V., Caiozzo, faces J., Mukherjee, C., Nguyen, inhibitor-3. and Sds22 8909-8919. between M. sandwiched Bollen, phosphatase-1 and P. Sanz, Atg8. ubiquitin-like with concert in biogenesis M. Thumm, and protein abnormal of clearance and formation aggregates. the in (VCP)/p97 protein containing compartments. control quality distinct two between 3776. az .adGri-ieo .A. M. Garcia-Gimeno, and P. Sanz, A-Taei h bqii system. ubiquitin the of in AAA-ATPase function mitotic the phosphatase. of protein regulator 1 potential type a yeast encodes SDS22 gene cerevisiae cerevisiae. Saccharomyces type of phosphatase protein control of growth regulator the positive a in be 1 may gene EGP1 The (1995). phenotypes . , cell to and al. relevant , et Y. cytoplasmic Kimura, aggregates, A., protein Iwamatsu, abnormal A., Sinohara, H., A. Popiel, processes. ubiquitin-dependent nonproteolytic Biol. and Curr. C. proteolytic Gordon, to activity and Cdc48 B. K. Hendil, association? by guilt or culprits emerging cancer: S. J. yeast. Weissman, in and expression protein K. protein E. 1 O’Shea, type N., Dephoure, cerevisiae Saccharomyces novel a inhibitor. phosphatase Ypi1, of characterization aGmn,M . 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Yuan, Hyman, I., Poser, A., M. Lampson, M., Held, C., Wurzenberger, hn,S,Gh,S n okr,F C. F. Volkert, and S. Guha, S., Zhang, oisn .C,Pilp,J,Bo,L,Bsel .P n acel K. Tatchell, and P. E. Boswell, L., Brou, J., Phillips, C., L. Robinson, G. Warren, and P. Freemont, N., Hui, R., Newman, H., Kondo, C., Rabouille, oc,M,Kodl,G . wf,S,Kn,E . eua .G n Swedlow, and G. J. Deluca, M., E. King, S., Swift, A., G. Khoudoli, M., S. Posch, Biggins, and A. C. Breed, Y., S. Tatsutani, V., C. and Kotwaliwale, A., K. B. Tatchell, Pinsky, V., E. Knatko, A., Bloecher, H., S. MacKelvie, W., M. Peggie, soitdpoendegradation. protein associated etok . ht,M .adKmns .E. V. dementia protein. frontotemporal Kimonis, valosin-containing and mutant bone and by of caused P. disease is Paget M. with associated Whyte, myopathy A., II. Pestronk, Pol RNA of turnover cerevisiae. 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