Ubiquitin-dependent folding of the Wnt signaling coreceptor LRP6
While protein folding may be extremely efficient , the presence of multiple domains , in soluble or membrane proteins , greatly reduces the efficacy of the overall process . Thus , a set of enzymes and chaperones assist folding and ensure that a sufficient number of active molecules reach their final destination ( Brodsky and Skach , 2011; Ellgaard et al . , 2016 ) . Even with help , folding may remain inefficient and thereby sensitive to errors or mutations , leading to disease . An illustrative example of folding inefficiency , and the consequences thereof , is the Cystic Fibrosis Transmembrane Regulator ( CFTR ) , a chloride channel expressed at the surface of lung epithelial cells ( Riordan , 2008 ) . It has been estimated that less than 25% of newly synthesized CFTR molecules actually reach the plasma membrane . When the number of functional CFTR channels is further reduced , patients suffer from Cystic Fibrosis ( Riordan , 2008 ) . This is the case for mutations that affect the kinetics or thermodynamics of CFTR folding in the ER , such as the most frequent CF mutation ΔF508 , and which are recognized by ER quality control systems and targeted for degradation ( Riordan , 2008 ) . Inefficiency in folding is a fairly common characteristic of transmembrane proteins ( Abrami et al . , 2008b; Deuquet et al . , 2009; Guerriero and Brodsky , 2012 ) . Most transmembrane proteins , which jointly compose 30% of the human proteome , are synthesized by ER-bound ribosomes and co-translationally inserted into the ER membrane ( Ellgaard et al . , 2016; Xu and Ng , 2015 ) . Subsequent folding must proceed in three topological environments: the ER lumen , the membrane and the cytosol . In each of these environments , the protein may benefit from the help of chaperones and folding enzymes . On the luminal side , these are fairly well characterized and include HSP40 , HSP70 and HSP90 family members ( Brodsky and Skach , 2011 ) , the lectin chaperones calnexin and calreticulin ( Lamriben et al . , 2016 ) and protein disulfide isomerases ( Brodsky and Skach , 2011 ) . Less is known about the assisted folding in the ER membrane and on the cytosolic side . If folding or assembly in any of these environments fails , the protein is recognized by quality control machineries and targeted to the ER associated degradation ( ERAD ) pathway ( Lemus and Goder , 2014 ) . ERAD involves polyubiquitination of the cytosolic domains of transmembrane protein , extraction from the ER membrane and degradation by the proteasome ( Lemus and Goder , 2014 ) . Here we have studied the biogenesis of Low-density lipoprotein Receptor-related Protein 6 ( LRP6 ) , a key component of the canonical Wnt signaling pathway , which has been associated with many human pathologies including cancer , osteoporosis and metabolic diseases ( Joiner et al . , 2013 ) , but also involved in the formation of gap junctions in cardiomyocytes ( Li et al . , 2016 ) . LRP6 is a type I membrane protein composed of a large extracellular domain containing multiple ß-propeller and EGF-like domains ( MacDonald et al . , 2009 ) . Proper folding of the ß-propeller domains depends of the dedicated LRP6 chaperone Mesd ( mesoderm development ) ( Hsieh et al . , 2003 ) and possibly other chaperones such as calnexin . We have previously shown that exit of LRP6 from the ER also requires a cytoplasmic post-translational modification , namely S-palmitoylation , on two cysteine residues in close proximity to the transmembrane domain ( Abrami et al . , 2008b ) . LRP6 acylation influences the conformation of this domain , possibly by alleviating the hydrophobic mismatch between the thickness of the membrane and the length of the hydrophobic stretch composing the transmembrane domain . Mutation of the palmitoylation sites leads to retention of LRP6 in the ER and ubiquitination on Lys-1403 ( Abrami et al . , 2008b ) indicative of its recognition by a quality control mechanism ( Feldman and van der Goot , 2009 ) . Once properly folded , LRP6 exits the ER , it transits through the Golgi , as indicated by the acquisition of Endoglycosidase H insensitive complex sugars ( Abrami et al . , 2008b ) , before reaching the plasma membrane where its signal role takes place . Here we have further investigated LRP6 biogenesis in the ER . We show that newly synthesized wild type LRP6 also undergoes ubiquitination , on the same site ( Lys-1403 ) , and that this post-translational modification promotes its folding . For ER exit to occur , ubiquitination must however be subsequently removed . This is mediated by the deubiquitinating enzyme USP19 , one of the isoforms of which is tail-anchored to the ER ( Hassink et al . , 2009; Wing , 2016 ) . It is thought to play an essential role in skeletal muscle atrophy through unclear mechanisms ( Wing , 2016 ) . At the more mechanistic level , USP19 has been shown to control the stability of several cytoplasmic proteins such as the inhibitors of apoptosis c-IAP1 , and c-IAP2 ( Mei et al . , 2011 ) , hypoxia-inducible factor 1-alpha ( HIF-1α ) ( Altun et al . , 2012 ) and the initiator of autophagy Beclin1 ( Jin et al . , 2016 ) . Interestingly , USP19 is a target gene of the unfolded protein response ( UPR ) and was found to rescue CFTR ΔF508 from ERAD ( Hassink et al . , 2009 ) . More recently it was found that USP19 is a key player in a novel misfolded-protein associated secretion MAPS pathway ( Lee et al . , 2016 ) . We show that USP19 controls the cellular levels of LRP6 . In the absence of USP19 , LRP6 is efficiently targeted to ERAD . Upon USP19 overexpression , ER exit of LRP6 is greatly enhanced . This work reveals the existence of a novel assisted-folding system that operates on the cytosolic side of the ER and depends on site-specific cycles of ubiquitination-deubiquitination . Upon ubiquitination on Lys-1403 , LRP6 presumably interacts with an ubiquitin-binding protein that acts as a chaperone and provides the protein with time to fold . If folding is successful , LRP6 is transported out of the ER , a step that is favored by palmitoylation ( Abrami et al . , 2008b ) . If folding is impaired or delayed , polyubiquitination of other cytosolic lysine residues takes over and targets LRP6 to ERAD . The here identified ubiquitination-deubiquitination assisted-folding cycle also appears to operate on other membrane proteins such as CFTR or the anthrax toxin receptor 2 ( also know as Capillary Morphogenesis gene 2 , CMG2 ) , loss of function of which leads to Hyaline Fibromatosis Syndrome ( Deuquet et al . , 2012 ) .
We analyzed LRP6 stability by performing a cycloheximide chase , which consists in following total protein expression levels by western blot analysis of extracts from Pigmented epithelial ( RPE1 ) cells submitted for various times to a protein synthesis block . No significant decay of LRP6 was observed over a 6 hr period ( Figure 1A , B ) . We next measured LRP6 stability by performing 35S Cys/Met metabolic pulse-chase experiments . Protein decay , monitored following a 20 min metabolic pulse , indicated that the apparent half-life ( t12app of endogenous LRP6 in RPE1 cells is approximately 3 hr ( Figure 1C , D , and supplementary information in [Abrami et al . , 2008b] ) . The same experiment was repeated on transiently expressed myc-LRP6 in HeLa cells ( Figure 1—figure supplement 1 ) . Then , we show that t12app was identical for endogenous LRP6 in RPE1 cells and transiently expressed myc-LRP6 in Hela , indicating that the potential difference in expression in these two systems does not affect degradation rates and allowing us to use both systems . 10 . 7554/eLife . 19083 . 003Figure 1 . LRP6 undergoes rapid degradation following synthesis in the ER but is stable once mature . ( A ) representative western blot of a cycloheximide ( CHX ) chase in RPE1 cells . 40 µg of total cell extracts from RPE1 cells were loaded per lane , analyzed by SDS-PAGE followed by Western blotting against endogenous LRP6 , calnexin ( Calx ) , a stable protein and Myc , a short lived protein . ( B ) Experiments as in A were quantified by ImageJ software , n = 3 . CDE: RPE1 cells were submitted to metabolic 35S Cys/Met labeling for different times and subsequently chased for different times . Endogenous LRP6 was immunoprecipitated with an anti-LRP6 antibody . A representative experiment in shown in ( C ) . Autoradiograms were quantified using the Typhoon imager and means of different experiments were calculated ( D and E ) . Error bars represent standard deviation ( n = 6 for the 20 min pulse; n = 4 for the 2 hr and 16 hr pulses ) . FG: RPE1 cells were treated or not with MG132 or Bafilomycin A and subsequently submitted , in the presence or not of the drugs , to metabolic a 20 min 35S-Cys/Met pulse followed by different chase times . A representative experiment in shown in ( F ) . Errors represent standard deviation ( n = 4 for MG132; n = 3 for Bafilomycin A , BafA , the WT control curve corresponds to that shown in Figure 1D ) . ( G ) Hela cells transiently expressing myc-tagged palmitoylation deficient LRP6 ( CC–SS ) were submitted to metabolic 35S Cys/Met labeling for different times and subsequently chased for different times . LRP6 was subsequently immunoprecipitated using an anti-myc antibody . Errors represent standard deviation ( n = 3 ) . H: Cartoon depicting the two major cellular degradation pathways for membrane proteins: ERAD ( blocked by MG132 ) and lysosomal pathway ( blocked by Bafilomycin A ) . DOI: http://dx . doi . org/10 . 7554/eLife . 19083 . 00310 . 7554/eLife . 19083 . 004Figure 1—source data 1 . Numeric data for graphs of Figure 1B , D , E and F and 1 hr . DOI: http://dx . doi . org/10 . 7554/eLife . 19083 . 00410 . 7554/eLife . 19083 . 005Figure 1—figure supplement 1 . Variation in USP19 cellular amount influences LRP6 degradation rates . ( A ) Metabolic 35SCys/Met pulse chase experiment ( 20 min pulse ) in the following conditions: immunoprecipitation of myc-LPR6 in HeLa cells transiently expressing myc-LRP6 wild type ( WT ) upon control silencing ( HeLa si Ctrl , n = 7 ) vs . usp19 silencing ( HeLa + siRNA USP19 , n = 6 ) or upon co-overexpression of myc-LRP6 wild type and wild type GFP-tagged USP19 ( HeLa + OE USP19 WT , n = 6 ) vs . catalytically inactive GFP-tagged USP19 ( HeLa + O . E . USP19 CS , n = 6 ) ; immunoprecipitation of endogenous LRP6 in RPE1 cells wild type ( RPE1 , n = 4 ) vs . RPE1 knout-out cells for usp19 gene ( RPE1 delta USP19 , n = 3 ) . ( B ) Total cell extract of wild type RPE1 and RPE1 knout-out cells for usp19 gene revealed with USP19 , LRP6 and Actin antibodies . DOI: http://dx . doi . org/10 . 7554/eLife . 19083 . 00510 . 7554/eLife . 19083 . 006Figure 1—figure supplement 1—source data 1 . Numeric data for graphs of Figure 1—figure supplement 1A . DOI: http://dx . doi . org/10 . 7554/eLife . 19083 . 006 The above apparent discrepancy between the cycloheximide chase and the metabolic labeling approach is due to the fact that stability of mature LRP6 is monitored through the first approach , while newly synthesized LRP6 is monitored by the second . Considering the events that occur following synthesis of a membrane protein –more or less efficient folding , ER exit , transport to destination– , the t12app of a protein determined using metabolic pulse-chase experiments may greatly dependent on the duration of the pulse . Indeed , the t12app of LRP6 increased to ≈ 5 hr for a 2 hr pulse and >15 hr for a 16 hr pulse ( Figure 1C , D , E ) . The long apparent half-lives estimated both by long metabolic labeling and cycloheximide chase suggest that mature LRP6 has a half-life that exceeds 20 hr . The rapid decay times observed for short metabolic pulses on the other hand indicate that close to 80% of newly synthesized LRP6 molecules are degraded in our cells within the first 6 hr ( Figure 1D ) , and thus do not contribute to the population of active Wnt signaling co-receptors . Cellular degradation of membrane proteins can occur by two main pathways: ERAD , where proteolysis is mediated by the proteasome , or the lysosomal pathway , which is responsible for degradation of most membrane proteins but is also the endpoint of autophagy ( Figure 1I ) . To determine the relative contribution of these pathways to the degradation of LRP6 , we repeated the 20 min metabolic pulse-chase experiments while either inhibiting the proteasome with MG132 or preventing lysosomal degradation by inhibiting the vacuolar ATPase with Bafilomycin A . MG132 protected LRP6 from degradation early after synthesis ( Figure 1F , G ) , consistent with degradation of a subpopulation of LRP6 molecules by ERAD during biogenesis . Bafilomycin A protected LRP6 at later times ( Figure 1F , G ) , consistent with transport of a portion of newly synthesized LRP6 molecules to the plasma membrane and their subsequent endocytosis and targeting to lysosomes ( Figure 1I ) . That LRP6 undergoes degradation by two distinct pathways is further support by the observation that palmitoylation deficient LRP6 , in which the two cysteines are mutated to serines ( LRP6CC–SS ) , and which does not exit the ER ( Abrami et al . , 2008b ) , is partially rescued by MG132 ( Figure 1H ) but is insensitive to inhibitors of lysosomal enzymes such as Bafilomycin A ( Figure 1H ) or leupeptin ( supplementary information in [Abrami et al . , 2008b] ) . The involvement of lysosomal enzymes in LRP6 degradation thus requires export out of the ER . We have previously shown that LRP6 undergoes palmitoylation in the ER on Cys-1394 and Cys-1399 and that palmitoylation is required for ER exit ( Abrami et al . , 2008b ) . This was based on the observation that palmitoylation deficient LRP6 did not exit the ER and underwent ubiquitination on Lys-1403 ( Abrami et al . , 2008b ) . To further investigate the importance of palmitoylation and ubiquitination on LRP6 biogenesis , we performed 35S Cys/Met metabolic pulse-chase experiments on the palmitoylation deficient LRP6CC–SS mutant , on the K1403R mutant ( LRP6CC–SS ) and on the triple mutant ( LRP6K1403RCC−SS ) . Following a 20 min pulse , no significant difference in degradation was observed between wild type LRP6 and LRP6CC–SS ( Figure 2A ) in agreement with our previous observations ( Abrami et al . , 2008b ) . In contrast , degradation was accelerated by the K1403R mutation ( Figure 2A ) . 10 . 7554/eLife . 19083 . 007Figure 2 . Mutation of the palmitoylation sites and the Lys-1403 ubiquitination site accelerate LRP6 targeting to ERAD . ( A ) Metabolic 35SCys/Met pulse chase experiment ( 20 min pulse ) on transiently expressed myc-LRP6 wild type ( WT , curve corresponding to the one in Figure 1D ) , palmitoylation deficient ( CC-SS , n = 3 ) or K1403R ( KR , n = 6 ) mutants in HeLa cells . ( B ) Metabolic 35SCys/Met pulse chase experiment ( 20 min pulse ) on transiently expressed myc-LRP6 wild type ( WT , n = 7 ) , or K1403R ( K1403R , n = 7 ) mutant in HeLa cells silenced or not for mesd gene ( siRNA mesd , n = 3 ) . Errors represent standard deviation , *<p=0 . 05 calculated between LRP6 WT and K1403R . ( C ) Metabolic 35SCys/Met pulse chase experiment ( 20 min pulse ) on transiently expressed myc-LRP6K1403R in HeLa cells supplemented or not ( n = 6 , curve corresponding to the one in Figure 2A ) with MG132 ( K1403 + MG123 , n = 3 ) or Bafilomycin A ( K1403 + BafA , n = 3 ) . ( D ) Immunoprecipitation of myc-tagged LRP6 Wild Type ( WT ) and K1403 mutant ( KR ) revealed with anti-Ubiquitin antibody , with or without MG132 treatment . ( E ) Metabolic 35SCys/Met pulse chase experiment ( 2 hr pulse ) on transiently expressed myc-LRP6 wild type ( WT , n = 4 , curve corresponding to the one in Figure 1D ) or palmitoylation deficient ( CC-SS , n = 3 ) in HeLa cells . ( F ) Metabolic 35SCys/Met pulse chase experiment ( 16 hr pulse ) on transiently expressed myc-LRP6 wild type ( WT , curve corresponding to the one in Figure 1D ) , palmitoylation deficient ( CC-SS , n = 3 ) , K1403R ( K1403R , n = 3 ) or K1403R in the palmitoyl deficient background ( CC-SS + KR , n = 3 ) mutants in HeLa cells . DOI: http://dx . doi . org/10 . 7554/eLife . 19083 . 00710 . 7554/eLife . 19083 . 008Figure 2—source data 1 . Numeric data for graphs of Figure 2A , B , C and E , F . DOI: http://dx . doi . org/10 . 7554/eLife . 19083 . 008 To estimate the importance of the contribution of Lys-1403 to LRP6 folding , we compared the effect of this mutation to that of silencing the dedicated LRP6 chaperone Mesd . Remarkably , mutation of Lys-1403 had a more pronounced effect on LRP6 degradation than mesd silencing ( Figure 2B ) . Accelerated LRP6K1403R degradation did not involve lysosomes since Bafilomycin A had no effect , also suggesting that newly synthesized LRP6K1403R does not significantly exit the ER during the 6 hr that follow its synthesis . Degradation of LRP6K1403R could however be partially rescued by MG132 ( Figure 2C ) . Consistent with its targeting to the proteasome , LRP6K1403R underwent polyubiquitination as revealed when immunoprecipitating LRP6 from MG132 treated cells and blotting against ubiquitin ( Figure 2D ) . This observation also shows that ERAD targeting of LRP6 does not involve , or at least does not require , Lys-1403 polyubiquitination . Extending the pulse time to 2 or 16 hr revealed that the stability of LRP6 does depend on palmitoylation ( Figure 2E , F ) . The tripple LRP6K1403RCC−SS mutant was therefore the least stable ( Figure 2F ) . Altogether these observations indicate that spontaneous folding of LRP6 is very inefficient , and that both ubiquitination and palmitoylation promote LRP6 biogenesis and ER exit . The process is however not all-or-none , i . e . even in the absence of Lys-1403 and/or palmitoylation , a small population of molecules folds properly and exits the ER . Pulse-chase experiments with long 35S pulses indeed reveal biphasic decay curves for all mutants studied , and show the existence , irrespective of the mutations , of a minor population of extremely long-lived molecules , which presumably reside at the plasma membrane . Importantly these constitute the steady state population that is revealed by western blotting . Western blot analysis of LRP6 mutants may therefore be misleading as to the importance of specific residues for biogenesis and membrane targeting ( Abrami et al . , 2008b ) . The above findings , combined with our previous observation that LRP6CC–SSundergoes ubiquitination on Lys-1403 and fails to exit the ER ( Abrami et al . , 2008b ) , led us to propose the following working hypothesis: following synthesis and insertion into the ER membrane , LRP6 first undergoes ubiquitination , probably of a specific type , on Lys-1403 , allowing it to interact with an ER or cytosolic ubiquitin-binding protein . This interaction provides LRP6 with time to fold –and as such this ubiquitin-binding protein would act as a chaperone– protecting it from ERAD targeting . Lys-1403 is then deubiquitinated . At this stage , LRP6 can either ( 1 ) undergo palmitoylation of its two juxtamembranous cysteine residues , followed by ER exit , ( 2 ) be re-ubiquitinated on Lys-1403 , or ( 3 ) be polyubiquitinated on one of the other 16 cytoplasmic lysine residues and sent to ERAD . Option 1 , re-ubiquitination on Lys-1403 , allows LRP6 to undergo a second cycle of interaction with its ubiquitin-binding chaperone , further promoting folding . Palmitoylation , which favors ER exit ( Abrami et al . , 2008b ) , could prevent LRP6 from Lys-1403 ubiquitination , possibly through a conformational change of steric hindrance given the juxtamembrane localization of both modifications . A major prediction of this model is the existence of an ER localized deubiquitinating ( DUB ) enzyme that can remove ubiquitin specifically from Lys-1403 . This DUB would control LRP6 biogenesis . The human genome encodes some 100 DUBs ( Clague et al . , 2013 ) . Of these , the ubiquitin-specific proteases ( USPs ) represent the largest family with 56 members ( Ye , 2006 ) . Because LRP6 folding takes place in the ER , we searched for ER-associated DUBs and to the best of our knowledge , only USP19 has an ER localized isoform ( Clague et al . , 2013; Hassink et al . , 2009; Wing , 2016 ) . Endogenous LRP6 and USP19 could be co-immunoprecipitated from RPE1 cells ( Figure 3A ) . While ubiquitination of LRP6 is undetectable under control condition ( Figure 3A; see also Abrami et al . , 2008b ) , silencing of usp19 ( Figure 3—figure supplement 1A ) revealed a clear LRP6 ubiquitination signal ( Figure 3A ) , often , but not always , appearing as a well identifiable band and a smear . Strikingly , LRP6 levels dropped drastically upon usp19 silencing whether in HeLa ( Figure 3—figure supplement 1B ) , RPE1 cells or primary human fibroblasts ( Figure 3B and Figure 3—figure supplement 1C ) . The same was observed upon CRISPR/Cas9-mediated usp19 knockout in RPE1 cells ( Figure 1—figure supplement 1B ) . Silencing of usp19 also led to an increase in ubiquitination of palmitoylation-deficient LRP6CC–SS and a decrease of its expression ( Figure 3—figure supplement 1D ) . In reverse , over expression of wild type USP19 , but not of its catalytically inactive mutant ( USP19C506S [Hassink et al . , 2009] ) reduced ubiquitination of LRP6CC–SS ( Figure 3—figure supplement 1E ) , indicating that USP19 influences LRP6 ubiquitination in a manner that depends on its DUB activity . Thus consistent with our working hypothesis , an ER-localized DUB , USP19 , controls the expression of LRP6 . 10 . 7554/eLife . 19083 . 009Figure 3 . De-ubiquitination of LRP6 by USP19 on Lys-1403 promotes LRP6 biogenesis . ( A ) Immunoprecipitation of endogenous LRP6 and ( B ) cellular level in RPE1 cells upon usp19 silencing . C/D/E/F: Metabolic 35SCys/Met pulse chase experiment ( 20 min pulse ) on transiently expressed myc-LRP6 wild type ( WT ) , palmitoylation deficient mutant ( CC–SS ) , K1403R mutant ( K1403R ) or K1403R mutation in the palmitoyl deficient background ( CC-SS + KR ) mutants in HeLa cells upon over expression of GFP-tagged USP19 ( O . E . USP19 ) or GFP-tagged USP19 catalytically inactive ( O . E . USP19 C-S ) or upon usp19 gene silencing ( siRNA USP19 ) . ( C ) WT n = 6 , same as in Figure 1D , other conditions n = 3 , ( D ) ( CC-SS curve corresponding to the one in Figure 2A ) and ( F ) all conditions n = 3 , ( E ) K1403R n = 6 , other conditions n = 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 19083 . 00910 . 7554/eLife . 19083 . 010Figure 3—source data 1 . Numeric data for graphs of Figure 3C , D and E , F . DOI: http://dx . doi . org/10 . 7554/eLife . 19083 . 01010 . 7554/eLife . 19083 . 011Figure 3—figure supplement 1 . Variation in USP19 cellular amount influences LRP6 ubiquitination state . ( A ) RT-PCR detecting usp19 , usp13 and LRP6 mRNA expression level upon silencing of usp19 and usp13 genes with specific RNAi . Errors represent standard deviation . B Total cell extracts of Hela transiently expressing Myc-LRP6 and silenced with 3 different RNAi targeting usp19 gene . C ) Total cell extract of primary fibroblasts silenced for usp19 gene or overexpressing GFP-tagged USP19 revealed with GFP , LRP6 and Actin antibodies . Quantification of LRP6 cellular amount is shown on the right . Errors represent standard deviation ( n = 3 ) and *<p=0 . 05 . ( C ) Immunoprecipitation of transiently expressed palmitoylation deficient myc-LRP6 ( Myc-LRP6CC-SS ) upon usp19 silencing . Quantification of Myc-LRP6CC-SS cellular amount in TCE is shown on the right . Errors represent standard deviation ( n = 3 ) and *<p=0 . 05 . ( D Immunoprecipitation of transiently expressed palmitoylation deficient myc-LRP6 ( Myc-LRP6CC-SS ) upon overexpression of GFP-tagged USP19 vs . GFP-tagged USP19 ( GFP-USP19 ) catalytically inactive mutant ( GFP-USP19C506S ) . DOI: http://dx . doi . org/10 . 7554/eLife . 19083 . 01110 . 7554/eLife . 19083 . 012Figure 3—figure supplement 1–source data 1 . Numeric data for graphs of Figure 3—figure supplement 1A , C . DOI: http://dx . doi . org/10 . 7554/eLife . 19083 . 012 We next investigated whether USP19 indeed affects LRP6 biogenesis . siRNA-mediated silencing of usp19 in HeLa cells and usp19 knock-out in RPE1 cells ( Figure 1—figure supplement 1B ) both led to a strong decrease in t12app of LRP6 , droping from 3 to 1 hr , as monitored by 35S Cys/Met metabolic pulse-chase experiments ( Figure 3C and Figure 1—figure supplement 1A ) . Overexpression of wild type USP19 led to a marked increase in t12app to above 6 hr , beyond the time frame of these experiments . Importantly , overexpression of catalytically inactive USP19C506S had no effect . This not only shows that the catalytic activity of USP19 is required , but the complete absence of rescue also indicates that USP19 does not act as a chaperone , as proposed for the unconventional misfolded protein-associated secretion MAPS pathway ( Lee et al . , 2016 ) . The palmitoylation deficient LRP6CC–SS was affected in a qualitatively similar manner: silencing usp19 decreased t12app to less than 1 hr , while over expression of USP19 increased t12app to 6 hr ( Figure 3D ) . Remarkably , silencing or overexpression of USP19 had no effect whatsoever on LRP6K1403R ( Figure 3E ) . Mutation of Lys-1403 similarly abolished the regulatory effect of USP19 on LRP6CC–SS , LRP6K1403RCC−SS being insensitive to the cellular USP19 levels ( Figure 3F ) . Altogether these experiments clearly point to Lys-1403 as the specific target site of USP19 , revealing the crucial role of this residue in preventing early LRP6 degradation . These experiments also show that USP19 , even when overexpressed , cannot reverse the polyubiquination undergone by LRP6K1403R and thus ERAD targeting , further highlighting the specificity of this DUB . Finally , these observations demonstrate that USP19 promotes the biogenesis of LRP6 , with only 18% of newly synthesized LRP6 molecules surviving beyond 6 hr in the absence of USP19 and 60% surviving upon USP19 overexpression . We finally tested the importance of USP19 for LRP6 function , as the co-receptor in Wnt signaling . Overexpression of USP19 in HeLa cells lead to a 50% increase in the Wnt signaling capacity ( Figure 4A ) , monitored using the TOPFLASH reporter assay ( Abrami et al . , 2008a ) . Most strikingly , silencing of usp19 , but not usp13 , in RPE1 cells stably expressing 7xTCF-FFluc directly activated by the TCF/-βcatenin complex led to a more than 80% drop in Wnt signaling ( Figure 4B ) . In agreement , surface biotinylation showed that usp19 silencing lead to a drastic drop LRP6 at the cell surface ( Figure 4—figure supplement 1 ) . 10 . 7554/eLife . 19083 . 013Figure 4 . USP19 controls the Wnt signaling capacity of the cell . ( A ) Wnt signaling measured in HEK293 cells carrying the TOPFLASH reporter assay , transiently transfected for the indicated constructs ( n = 5 ) ( B ) Wnt signaling measured in RPE1 cells stably expressing lentiviral vector possessing a 7xTCF-FFluc upon usp19 or usp13 silencing ( n = 4 ) . ( C ) Working model ( described in the text ) . ( D ) cellular level of the indicated endogenous proteins in RPE1 cells upon usp19 silencing ( siRNA ) or overexpression ( O . E . ) of GFP-tagged USP19 . ( E ) cellular level of transiently co-transfected CFTR ( constant amount ) and GFP-tagged USP19 ( increasing amount ) in RPE1 cells . DOI: http://dx . doi . org/10 . 7554/eLife . 19083 . 01310 . 7554/eLife . 19083 . 014Figure 4—source data 1 . Numeric data for graphs of Figure 4A , B . DOI: http://dx . doi . org/10 . 7554/eLife . 19083 . 01410 . 7554/eLife . 19083 . 015Figure 4—figure supplement 1 . usp19 silencing leads to decrease in LRP6 cell surface expression . Surface Biotinylation assay performed in RPE1 cells upon 24 , 48 and 72 hr of usp19 or usp13 gene silencing . Quantification of endogenous LRP6 surface expression at 48 hr of gene silencing in Streptavidin-mediated pull down is shown above the western blot . Errors represent standard deviation ( n = 3 ) and ***<p 0 . 0005 . DOI: http://dx . doi . org/10 . 7554/eLife . 19083 . 01510 . 7554/eLife . 19083 . 016Figure 4—figure supplement 1–source data 1 . Numeric data for graphs of Figure 4—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 19083 . 016 We here reveal the existence of an ubiquitin-dependent folding machinery , which operates on the cytosolic side of the ER membrane ( Figure 4C ) . More specifically we show that following synthesis , LRP6 undergoes ubiquitination on Lys-1403 , most likely of a specific form . In conceptual analogy to the binding of newly synthesized mono-glucosylated proteins to calnexin in the ER lumen ( Lamriben et al . , 2016 ) , we hypothesize that Lys-1403-ubiquitin can interact with a putative ER-ubiquitin binding protein that would also act as a chaperone . Upon release from this putative ubiquitin-binding chaperone , LRP6 is deubiquitinated by USP19 . Following USP19 deubiquitination , LRP6 can , if properly folded , exit the ER , an events possibly promoted by palmitoylation of cysteines in the vicinity of Lys-1403 ( Abrami et al . , 2008b ) . Or , LRP6 can undergo a new cycle of Lys-1403-specific ubiquitination and chaperone binding . Upon prolonged presence in the ER , LRP6 undergoes polyubiquitination on other cytosolic lysines , leading to ERAD targeting . In the ER , LRP6 can thus undergo two types of ubiquitination events , which operate with different kinetics: ( 1 ) specific ubiquitination on Lys-1403 which promotes folding , ( 2 ) the slower polyubiquitination on other lysines which promotes ERAD . These two types of ubiquitination events are reminiscent of de-glucosidation and de-mannosidation of glycoproteins in the ER lumen , which also occur with different kinetics . De-glucosidation promotes folding of glycosylated protein , but if folding is too lengthy or fails , de-mannosidation takes place and targets the protein to ERAD . While the here described ubiquitin-dependent folding system was identified for LRP6 , it likely also operates on other membrane proteins . We indeed found that usp19 silencing also led to a pronounced decrease in the levels of CMG2 ( Figure 4D ) , defective folding of which lead to Hyaline Fibromatosis Syndrome ( OMIM #228600 ) ( Deuquet et al . , 2009 , 2011 ) . Silencing of usp19 however did not affect the levels of the CMG2-related protein TEM8 ( Tumor endothelial marker 8 ) , nor those of the Transferrin and the EGF receptors ( Figure 4D ) . USP19 was reported to rescue the CFTR Δ508 mutant , which is an ERAD substrate ( Hassink et al . , 2009 ) . USP19 however also controls proteostasis of wild type CFTR ( Figure 4E ) . Future studies are required to establish the generality of this ubiquitin-dependent folding system , determine whether it has a built-in quality control , and identify the missing components such as the folding-promoting ubiquitin ligase , the putative ubiquitin-binding chaperone and the degradation-targeting ligase ( Figure 4C ) . All these enzymes could be exploited to control Wnt signaling in the context of disease .
Hela and RPE1 cells were used in this study . These cells are not on the list of commonly misidentified cell lines maintained by the International Cell Line Authentication Committee . They were mycoplasma negative as tested on a trimestral basis using the MycoProbe Mycoplasma Detection Kit CUL001B . RPE1 cells were maintained in DMEM high glucose supplemented with 10% Fetal Calf Serum , 2 mM penicillin and Streptomycin and HeLa cells were maintained in MEM supplemented with 10% FCS , 2 mM L-Glutamine and antibiotics . RPE1 USP19 knockout cell lines were generated using the CRISPR/Cas9 technology . Briefly , the cells were co-transfected with three plasmids: MLML3636 , JDS246 carrying the gRNA sequence TCTGGCGGGGCCAGTGCCAC and GFP encoding plasmid . Single GFP transfected cells were sorted by FACS in 96 well plates . The Knockout clones were detected by western blot ( Figure 1—figure supplement 1B ) . We used the following primary antibodies: Rabbit anti-LRP6 mAb ( Cell Signaling , #2560 RRID:AB_2139329 ) , Mouse anti-Actin mAb ( Millipore , MAB1510 ) , Mouse anti-Myc 9E10 mAb ( Covance MMS-150R RRID:AB_291327 ) , Mouse Ubiquitin ( Santa Cruz sc-8017 RRID:AB_628423 ) , Goat anti-CMG2 ( R and D systems #AF2940 ) , TEM8 ( Sigma-Aldrich , St . Louis , SAB2501028 ) , Mouse anti-Tf-R ( Zymed , #13–6800 ) , Mouse anti-EGF-R ( Sigma E3138 , RRID:AB_476925 ) , CFTR ( home-made ) , Mouse anti-Tubulin ( Sigma T5168 ) , Mouse anti-GFP ( Roche , #11814460001 ) , Rabbit anti-USP19 ( Bethyl , A301-587A , RRID:AB_1078839 ) . We used the following beads for immunoprecipitations: Protein G Sepharose 4 Fast Flow ( GE Healthcare , 17–0618-01 ) , Streptavidin Agarose ( Sigma , S1638 ) , anti-Myc Affinity Gel ( Thermo Scientific # 20169 ) . LRP6 encoding plasmids as been previously described ( Abrami et al . , 2008b ) . The catalytically inactive mutant was obtained by Quik Change Technology ( Agilent ) according to manufacturer’s instruction . Plasmids were transfected into RPE1 and HeLa cells for 24 hr in cDNA/9 . 6 cm2 plate using Fugene ( Roche Diagnostics ) . For the dual Luciferase assay , plasmids and reagents were from Promega . For immunoprecipitation , cells were PBS washed and lysed 30 min at 4°C in IP Buffer ( 0 . 5% Nonidet P-40 , 500 mM Tris pH 7 . 4 , 20 mM EDTA , 10 mM NaF , 2 mM benzamidin and protease inhibitor cocktail ( Roche ) , and centrifuged 3–5 min at 5000 rpm . Supernatants were incubated overnight with Sepharose beads . In case of non-coupled Sepharose G beads , supernatants were subjected to preclearing with the beads prior to the actual immunoprecipitation reaction . Cell surface protein biotinylation was performed as described ( Abrami et al . , 2008b ) . Briefly , silenced cells were treated 30 min with 0 . 17 mg/ml sulfo-NHS-Biotin ( Pierce ) at 4°C , quenched with 100 mM NH4Cl , and lysed in IP Buffer ( as described above ) . The lysate was immunoprecipitated with streptavidin-coated sepharose beads ( Sigma , S1638 ) . The dual luciferase assays were performed with plasmids and reagents from Promega . HEK293 cells were transfected with 0 . 1 µg TOP-Luciferase , 50 ng TK-Renilla , 0 . 25 µg Mesd encoding plasmid and 0 . 5 µg myc-LRP6 wild type or mutant encoding plasmids/9 . 6 cm2 plate . The cells were lysed 24 hr after transfection and the luciferase activity was determined according to the manufacturer’s instructions . The drugs are used in complete medium at the final concentration of 10 μM for MG132 and 100 nM for Bafilomycin A 2 hr before the starvation and are kept during the whole experiment . For the metabolic chases , the cells were starved in DMEM HG devoid of Cys/Met for 40 min at 37°C , pulsed with the same medium supplemented with 140 μCi of 35S Cys/Met for the indicated time , washed and incubated in DMEM complete medium for the indicated time of chase ( Abrami et al . , 2008a ) . For the cycloheximide chases , the cells were incubated in medium supplemented with 10 µg/ml of cycloheximide for 2 hr , washed and incubated in complete medium for the indicated time .
Many membrane proteins fold inefficiently and require the help of enzymes and chaperones . Here we reveal a novel folding assistance system that operates on membrane proteins from the cytosolic side of the endoplasmic reticulum ( ER ) . We show that folding of the Wnt signaling coreceptor LRP6 is promoted by ubiquitination of a specific lysine , retaining it in the ER while avoiding degradation . Subsequent ER exit requires removal of ubiquitin from this lysine by the deubiquitinating enzyme USP19 . This ubiquitination-deubiquitination is conceptually reminiscent of the glucosylation-deglucosylation occurring in the ER lumen during the calnexin/calreticulin folding cycle . To avoid infinite futile cycles , folded LRP6 molecules undergo palmitoylation and ER export , while unsuccessfully folded proteins are , with time , polyubiquitinated on other lysines and targeted to degradation . This ubiquitin-dependent folding system also controls the proteostasis of other membrane proteins as CFTR and anthrax toxin receptor 2 , two poor folders involved in severe human diseases .
Proteins carry out almost every process that happens inside a cell . Like all machines , their ability to work properly depends on their three-dimensional shape and structure . To make proteins , building blocks called amino acids are first assembled into a string that , like wool in a sweater , needs to be knitted into the final three-dimensional structure . How proteins reach their 3D structure is called “folding” , and when protein folding fails , or is not so efficient , it can cause very severe diseases . Protein folding is not as nicely progressive as knitting a sweater: it is more like putting all the wool into a big messy blob that then suddenly turns into a protein with the right three-dimensional structure . Cells have machinery that can detect messy-looking molecules and destroy them . Therefore , new proteins need to be hidden from this machinery until they have finished folding . A human protein called LRP6 is found on the surface of cells and it plays an important role in allowing cells to communicate with each other . Like many other proteins , LRP6 is produced inside the cell in a compartment called the endoplasmic reticulum and is then exported to the cell surface . In 2008 , a team of researchers found that LRP6 is modified in a particular way known as S-palmitoylation before it leaves the endoplasmic reticulum . This suggested that there is a system that helps this protein to fold correctly . Here Perrody , Abrami et al . – including some of the researchers from the previous work – used biochemical techniques to investigate how LRP6 folds . The experiments show that another type of protein modification that involves attaching a molecule called ubiquitin to LRP6 promotes this protein’s folding . Once the protein is folded , the ubiquitin is removed from LRP6 by an enzyme called USP19 . Further experiments show that this system also helps to ensure that two other important proteins fold correctly . The next steps following on from this work are to identify the other molecules involved in this protein folding system . A future challenge is to find out how this system protects new proteins from being degraded while they are still folding .