protein folding in the endoplasmic reticulum ( er ) is monitored by stringent quality control mechanisms that prevent release of immature or misfolded er client proteins to travel further along the secretory pathway . notable examples include the binding - and - release cycles of bip , recognizing exposed hydrophobic stretches at the surface of nonnative conformers , and of calnexin / calreticulin , which exploit glucosylation status of n - linked glycans on er client proteins to exert quality control ( ellgaard and helenius , 2003 ) . er client proteins are rich in intra- and intermolecular disulfide bonds that are generally essential for their function . numerous oxidoreductases reside in the er to catalyze oxidative protein folding ( ellgaard and ruddock , 2005 ) . also the process of disulfide bond formation is intimately linked to the quality control systems that prevent nonnative conformers to exit from the er ( anelli and sitia , 2008 ) . the same mechanisms that exert er quality control on the folding of subunits also monitor certain steps in their oligomeric assembly . for instance , in the case of antibodies , bip binds the first constant domain ( ch1 ) of ig heavy chain ( h ) and thereby retains it in the er until it is displaced by the ig light chain ( l ) to  assemble into h2l2 structures , called  monomers  in the immunological jargon ( feige et  al . , 2009 ; haas and wabl , 1983 ; hendershot and sitia , 2005 ) . the assembly process of 2l2 monomers into igm pentamers(2l2)5j  and hexamers(2l2)6is favored by ergic-53 ( anelli et  al . , 2007 ) , which resides preferentially in the er - to - golgi intermediate compartment ( ergic ) ( schindler et  al . , 1993 ) . interaction of 2l2 monomers with ergic-53 occurs upon release by bip ( anelli et  al . , 2007 ) , suggesting that these assemblies escape from the grasp of er quality control and travel to more distal compartments of the early secretory pathway ( cortini and sitia , 2010 ) . this notion is perhaps not so surprising , as the 2l2 monomers , even if not polymerized , are correctly folded and partially assembled and therefore no longer substrate to the er chaperoning machinery . the only sign that betrays their unpolymerized state is the free tail - piece cysteine , which in mature igm polymers is disulfide linked to corresponding cysteine residues in other 2l2 monomers ( sitia et  al . , members of the pdi family of oxidoreductases associate with free cysteines of orphan assembly subunits and thereby facilitate their so - called thiol - mediated retention ( fra et  al . , 1993 ; reddy et  al . , 1996 ; sitia et  al . , we previously identified erp44 as a pdi family member that covalently binds ero1 oxidases and facilitates their intracellular localization ( anelli et  al . erp44 is special for having a single cysteine ( c29 ) in its conserved active site ( crfs ) , consistent with a dedicated role in thiol - mediated retention not only of ero1 oxidases but also of orphan subunits of otherwise disulfide - linked oligomers , including igm ( anelli et  al . moreover , erp44 localizes predominantly to the ergic ( anelli et  al . , 2007 ; , 2006 ) , unlike other pdi family members , which reside in the er ( ellgaard and ruddock , 2005 ) . still , the c - terminal rdel motif of erp44 allows its capture by kdel receptors ( kdel - r ) in distal stations of the early secretory compartment , presumably for retrieval to the er ( anelli et  al . , 2003 ) . it is yet unclear how erp44 cycles between its predominant localizations in the distal early secretory pathway and the er , and how such cycling would relate to thiol - mediated retention . here we show that the ph gradient between cisgolgi and er controls association of erp44 both with its clients and with the kdel - r . our results suggest a model in which the simultaneous unmasking of the client and kdel - r binding interfaces is facilitated by dislocation of the erp44 c - terminal tail ( c - tail ) , which in turn likely involves protonation state of the active site cysteine ( c29 ) at cisgolgi - equivalent ph . as such , both erp44 activity and its shuttling between the er and cisgolgi are ph regulated to drive a quality control cycle dedicated to the surveillance of secretory protein assembly . erp44 associates with its client proteins via disulfide linkages to residue c29 in the active site and noncovalent interactions with the surrounding hydrophobic patches at the substrate - binding site ( anelli et  al . , 2003 ; wang et  al . , 2008 ) . in the erp44 crystal structure ( wang et  al . , 2008 ) , however , the substrate binding site is shielded from the solvent by the c - tail that submerges into a groove delimited by the a and b domains ( see also figure  4a ) . the ph gradient existing in the early secretory pathway ( figure  1a , top panel ) regulates ergic-53 ( appenzeller - herzog et  al . , 2004 ) , a glycoprotein transporter lectin that shares several clients with erp44 , including igm ( anelli et  al . , 2007 ) . ph also regulates the in  vitro binding of peptides to kdel - r ( wilson et  al . , 1993 ) , which prevent erp44 secretion ( anelli et  al . , 2003 we thus surmised that the erp44 structure , obtained from crystals grown at ph 7.5 , most likely corresponds to an off conformation of the protein , found in the ph - neutral er . arrival into the distal , more acidic stations of the early secretory pathway , namely ergic and cisgolgi , could entail exposure of the substrate binding site ( figure  1a ) . we therefore assessed ph - dependent erp44 c - tail rearrangements in  vitro by 1-anilinonaphthalene-8-sulfonate ( ans ) binding fluorescence spectroscopy ( serve et  al . consistent with the exposure of hydrophobic surfaces in erp44 , the ans fluorescence peak was blue - shifted and enhanced when the ph was lowered from 7.5 to 6.5 ( figure  1b , top panel ) . this hypsochromic effect was largely abolished for a mutant in which we had engineered a disulfide bond between c29 and the c - tail ( t369c , lower panel ) to restrict its movements ( wang et  al . , 2008 ) , unless the engineered disulfide bond was disrupted with the reducing agent dtt ( figure  1b ) . we concluded that accessibility of erp44 s substrate binding site involves ph - dependent dislocation of the c - tail . next , we monitored reactivity of the active site c29 as a function of ph and found that erp44 bound more polyethylene glycol 2000-modified maleimide ( malpeg ) as the ph was lowered from 8.0 to 6.0 ( figure  1c ) , with 50% malpeg binding at ph 6.6 ( figure  1c , right panel , red dots ) . malpeg binding faithfully reported on c29 reactivity , since its substitution with serine almost abolished binding . the residual binding observed in c29s involved residue c63 ( figure  1c , right panel , turquoise dots ) , as indicated by the absence of mobility shifts in the double mutant c29s - c63a ( figure  1c ) . this finding excludes the possibility that the results obtained with erp44 wt reflected ph dependence of malpeg reactivity ( makmura et  al . , 2001 ) . the t369c mutant did not bind malpeg , reflecting the constitutive engagement of c29 in a disulfide bond with c369 ( figure  1c ; wang et  al . , 2008 ) . taken together , these results indicate that both c29 and the surrounding hydrophobic substrate binding site become more exposed at ph values similar to those encountered downstream of the er . although the rdel motif was not resolved in the crystal structure ( wang et  al . , 2008 ) , we reasoned that its accessibility would be limited by the adjacent domain a when the c - tail is in a closed conformation , in turn hindering its availability to kdel - r . t369c mutant was secreted at levels comparable to a mutant lacking the rdel motif ( rdel ) ( figure  2 , upper panels ) . insertion of a spacer peptide ( flag ) immediately upstream of the rdel tetrapeptide  to let it protrude from the protein  increased retention of the t369c mutant to levels similar to those for erp44 wt with the flag insert ( figure  2 lower panels ) . the finding that flag - tagged erp44 ( whether wt or t369c mutant ) was less well retained than  flag - less  erp44 wt may reflect that rdel recognition by kdel - r depends in part on its context . indeed , in erp44 the sequence upstream of rdel is remarkably conserved in metazoans ( data not shown ) . in all , these data support the notion that an  open and flexible conformation  of the c - tail increases accessibility of the rdel retrieval motif to its cognate receptors . having demonstrated in  vitro that accessibility of the erp44 active site is ph regulated , we set out to analyze ph dependency of erp44 activity in  vivo . to this end , we silenced expression of the golgi ph regulator ( gphr ) ( maeda et  al . , 2008 ) , which specifically raised the ph in the cisgolgi by 0.4 units ( figure  3a ) . in line with our in  vitro findings , neutralizing the er - cisgolgi ph gradient inhibited erp44 reactivity with its partners / substrates , as is evident from the gphr silencing - induced decrease of erp44 being engaged in disulfide - linked complexes ( figure  3b ) . consistently , thiol - mediated retention of erp44 client proteins was inhibited upon gphr silencing , as indicated by increased secretion of overexpressed ero1 ( figures 3c and 3f and see figure  s2 online ) , adiponectin ( apn , figures 3c and 3f ) , and igm assembly intermediates ( figures 3d and 3f ) . intracellular retention of ero1 is a task that erp44 shares with pdi , and overexpression of either erp44 or pdi enhances ero1 retention ( anelli et  al . , 2003 ; otsu et  al . , 2006 gphr silencing strongly subdued this enhanced ero1 retention when erp44 was overexpressed , but much less so in the case of pdi overexpression ( figure  3c ) . secretion of proteins that are not erp44 clients , like 1-antitrypsin ( 1at ) or a soluble gfp engineered for entry into the secretory pathway ( sgfp ) , was also unaffected by raising the cisgolgi ph ( figures 3e and 3f ) . we concluded that erp44-mediated retention in particular relies on the er - cisgolgi ph gradient . accordingly , retention of unassembled monomeric ig- chains , which is mediated not by erp44 but by the chaperone bip , was ph insensitive ( figures 3d and 3f ) . only when the ch1 domain , which is important for bip binding ( haas and wabl , 1983 ; lee et  al . , 1999 ) , is deleted ( ch1 ) or engaged in interactions with ig - l chains ( as in 22 complexes ) does erp44 become the primary retainer of the ig- chains , and hence their retention becomes ph dependent ( figures 3d and 3f ) . retention of sgfp - rdel was ph insensitive ( figure  3e ) , as expected for a protein that lacks free cysteines and hence is not an erp44 client . this finding implies at the same time that kdel - r function , which is crucial for sgfp - rdel retention , is not corrupted despite the rise in cisgolgi ph . likewise , erp44 was retrieved by kdel - r despite gphr silencing ( figure  s2b ) , whereas this treatment allowed secretion of erp44 client proteins ( figure  3f ) . perhaps a partial c - tail opening in distal stations of the secretory pathway , which are less affected by gphr silencing ( maeda et  al . , 2008 ) , is sufficient for kdel - r recognition but not for client binding . considering that the conformational changes that facilitate c - tail dislocation are induced by lowering the ph as erp44 travels to the cisgolgi , we surmised that a protonation event may lie at the basis of the switch between the on and off conformations of erp44 . to explore this notion , we analyzed the crystal structure of erp44 with the c - tail in the closed conformation using the program propka3 , a state - of - the - art semiempirical method that evaluates pka values of ionizable residues starting from protein structures ( li et  al . , 2005 ; olsson , 2011 ; olsson et  al . , 2011 ) . the most striking feature that emerged from the propka3 analysis was the downward shift of the pka of c29 . its predicted value of 7.7 units ( table 1 ) is significantly lower than the reference value for free cysteine in solution ( 9.0 ) . this shift is mainly ascribed to the propensity of the neighboring side - chain hydroxyls of s32 and t369 and the main - chain amides of residues f31 , s32 , and t369 ( table 1 and figure  4b ) to form hydrogen bonds with c29 when in the thiolate form . likewise , electrostatic interactions with the r98 side chain stabilize the thiolate form of c29 ( table 1 ) . based on this pka value , a significant percentage of c29 would be in the thiolate form ( 41% ) at ph 7.5 ( figure  4b and table 1 ) . conversely , only 6.5% of c29 was predicted to be in the thiolate form at ph 6.5 ( table 1 ) . when we removed the 16 strand from the crystal structure in  silico , simulating an c - tail conformation of erp44 , the calculated pka of c29 raised to 7.8 ( table 1 ) . the effect was even more pronounced in sample structures at the end of molecular dynamics simulations of this 16 model . as a consequence of a greater solvent exposure , loss of hydrogen bonding to t369 , and an increased distance to the side chains of both s32 and r98 , c29 had a calculated pka of 8.7 , implying that only a small percentage ( 6% ) of c29 is in the thiolate form at ph 7.5 ( table 1 and figure  4c ) . based on our in  silico analyses , we mutated the residues predicted to affect the pka of c29 in erp44 . altering the side - chain groups such that they could no longer contribute to the stabilization of the thiolate form of c29 through hydrogen bonds or electrostatic interactions ( s32a , r98q , or t369a ) indeed rendered erp44 constitutively accessible to malpeg and abolished the ph sensitivity of the reaction ( figure  4d ) . deletion of strand 16 gave similar results ( figure  4d ) , supporting the notion that the s32a , r98q , and t369a substitutions similarly led erp44 to adopt the we then analyzed whether the enhanced accessibility of the erp44 active site in the various mutants correlated with its affinity toward substrates . in surface plasmon resonance ( spr ) assays ( masui et  al . , 2011 ) , erp44 wt showed a ph - dependent reactivity toward ero1 , having a higher affinity for its partner at ph  6.5 than at ph 7.5 ( figure  4e ) . all the mutations that resulted in constitutive and ph - independent malpeg binding of erp44 made it also less ph dependent in spr assays and increased its affinity for ero1 ( figure  4e ) . the effect was weakest for the r98q mutant , in agreement with the modest contribution of this arginine in stabilizing the thiolate form of c29 ( figure  4c ) . in summary , our data indicate that c29 , s32 , r98 , and t369 are components of the erp44 ph - sensing mechanisms . the predicted pka of c29 approaches neutral ph due to the interaction of its side chain with the surrounding residues , suggesting that its protonation , when erp44 encounters the lower ph in the cisgolgi , is a key event for c - tail opening , and , hence , exposure of the substrate binding site . we then further substantiated our findings on the mechanisms mediating erp44 ph sensitivity by analyzing the phenotype of the above mutants in  vivo . in line with the in  vitro results , mutation of the residues expected to affect the pka of c29 ( s32 , r98 , and t369 ) dramatically increased the tendency of erp44 to form covalent complexes with client and partner proteins , reaching levels comparable to a mutant lacking the whole 16 strand ( see figure  5a and figure  s3a ) . this was particularly evident when anti - ha antibodies were used to selectively analyze the behavior of the overexpressed transgenes ( figure  s3a ) . we concluded that the s32a , r98q , and t369a mutations increased the accessibility of c29 also in  vivo , likely reflecting a more  open  conformation of the c - tail . in further support of our model , ph sensitivity of erp44 activity was lost in all these mutants , since the fraction of molecules covalently bound to client and partner proteins did not decrease upon gphr silencing in these mutants , as it did instead in erp44 wt ( figure  5a ) . in addition , the activity of these mutants in retaining adiponectin was no longer sensitive to basification of the cisgolgi ( figure  5b and figure  s3b ) . therefore , ph sensitivity is severely reduced when the critical residues s32 , r98 , and t369 are mutated and , consequently , the predicted c29 pka is raised over neutral values . the ph gradient between er and golgi may serve multiple roles  in the cell , such as calibrating activity of lectins and golgi glycan - modifying enzymes ( paroutis et  al . , 2004 ) . we have shown here that the ph gradient is exploited also for regulating quality control and secretion of a selection of key proteins . ph - regulated opening of erp44 s tail determines both client capture and kdel - r - mediated retrieval . thus , erp44 governs a ph - regulated protein quality control cycle shuttling between cisgolgi , where erp44 engages its clients , and the er , where they must be released and where erp44 s c - tail presumably closes to occlude the client binding active site . our in  vitro malpeg binding studies reveal a midpoint transition for erp44 at a ph of approximately 6.6 , a value similar to that found in the cisgolgi ( figure  3a ) . in all likelihood , however , additional mechanisms contribute to the regulation of erp44 activity in living cells . for instance , interactions with kdel - r may prevent closing of the c - tail and perhaps provide interaction with additional proteins . owing to the fact that the opening of the c - tail is ph dependent , the substrate binding site of erp44 becomes more accessible progressively from er to cisgolgi . client proteins may thus engage erp44 more proximally or distally along the early secretory pathway , according to their affinity toward the substrate binding site at a given ph . differences in the ph optimum for erp44 association may thus account for differences in the intracellular distribution of ero1 , which is enriched at mitochondrial - associated er membranes ( anelli et  al . , 2012 ; simmen et  al . , 2010 ) and resides overall more distally in the early secretory pathway , as compared to another erp44 client , sumf1 , which localizes to the more proximal stations ( fraldi et  al . , , the active site c29 is buried at the interface between domain a and the c - tail ( wang et  al . , 2008 ) . the environment in which c29 resides induces a downward pka shift of this residue toward neutral values , de facto stabilizing the thiolate form of the amino acid . the negative charge of the deprotonated c29 restrains the c - tail movements via hydrogen bonds to s32 and t369 and electrostatic interactions with r98 , which also interacts with the tail backbone . upon entry of erp44 into the cisgolgi compartment this event , in turn , weakens c29 interactions with surrounding residues , as a cysteine with a side - chain thiol is less effective in forming the same hydrogen - bonding and long - range coulombic interactions that the thiolate can maintain . how c29 of erp44 reacts with clients to form mixed disulfides and whether erp44 can act alone or requires assistance for this process deserves further investigation . in line with our model , silencing of erp44 causes more abundant secretion of many proteins with one or more exposed reactive cysteine , including ig- ( anelli et  al . , 2007 ; ronzoni et  al . , 2010 ) , adiponectin ( wang et  al . , 2007 ; qiang et  al . , 2007 ) , sumf1 ( fraldi et  al . , 2008 ; mariappan et  al . , 2008 ) , and peroxiredoxin 4 ( t. kakihana , k. araki , s.v . , i. shun - ichiro , m.c . , similarly , upon neutralization of the golgi ph , erp44 substrates like ero1 , adiponectin , and igm subunits are secreted more abundantly , presumably because the active site of erp44 remains largely inaccessible . this observation suggests that erp44 could partially open the c - tail , exposing its rdel , and interact with kdel receptors without binding client proteins through an as - yet - unknown mechanism . erp44 receives substrates from , and associates with , ergic-53 ( anelli et  al . , 2007 ) , a lectin that also embraces glycoproteins released by the calnexin / calreticulin quality control ( ellgaard and helenius , 2003 ) . the erp44-ergic-53 tandem may thus integrate thiol - mediated and glycan - trimming - dependent er quality control cycles . similarly , we have shown that upon displacement by ig - l , bip no longer warrants retention of orphan ig - h chains but leaves this role to erp44 instead . erp44 indeed is strongly upregulated in the course of b cell differentiation ( van anken et  al . , 2003 ; anelli et  al . , 2007 ) to help avoid release of incompletely assembled igm , which would jeopardize the efficiency of the humoral immune response . the erp44 assembly control cycle thus embodies the last - resort quality control mechanism to prevent the untimely exit from the early secretory compartment of orphan subunits of otherwise disulfide - linked oligomeric secretory proteins . the cdna encoding human erp44 without the signal sequence was amplified by pcr and cloned into pet28 vector ( from novagen ) at nhei and xhoi sites . the recombinant form of erp44 , which was overproduced in e.  coli , lacks the  rdel motif at the c terminus . vectors for expression of ha - erp44 in  mammalian cells were previously described ( anelli et  al . , 2002 ) . mutants were obtained by polymerase chain reaction ( pcr ) or site - directed mutagenesis ( sdm ) ( table s1 ) . human erp44-flag - rdel was a kind gift of professor k. nagata ( kyoto - sangyo university ) . plasmids encoding wt and mutated secretory ig- chains ( s and ch1 ) were described in detail previously ( cenci et  al . , 2006 ; mattioli et  al . , 2006 ) . the purification of wild - type and mutant erp44 and ero1 was performed as described ( inaba et  al . , 2010 ) . the association and dissociation rate constants ( kon or koff ) for the direct binding of erp44 to immobilized ero1 were determined by spr measurements on a biacore2000 system ( ge , healthcare ) , in the presence of 1  mm gsh and 0.25  mm gssg as described ( inaba et  al . , 2010 ) . hyperactive ero1 ( with cysteines 104 , 131 , and 166 replaced by ala ) was used in spr assays to limit variations caused by the autoregulatory mechanisms controlling ero1 activity ( inaba et  al . , 2010 ) . ans fluorescence spectra were recorded in 1  cm cuvettes on a hitachi f-4500 spectrofluorometer . erp44 and mutants ( 5  m ) were mixed with 100  m ans in 20  mm tris - hcl ( ph 7.5 ) or mes ( ph 6.5 ) containing 150  mm nacl with or without 10  mm dtt and incubated at 293 k for 10  min . cells were starved for 5  min in cysteine and methionine - free dmem ( gibco , invitrogen ) , pulsed for 10  min with ( s ) cysteine and methionine ( easy tag , perkin - elmer ) , washed twice , and chased for the indicated periods in complete medium . cell lysates and supernatants were immunoprecipitated with anti - ha or anti - flag antibodies and resolved by sds - page . fluorograms or western blot images were acquired with the chemidoc - it imaging system ( uvp , upland , ca ) or with fla-900 starion ( fujifilm life science , usa ) and quantified with image quant 5.2 as described ( anelli et  al . , 2007 ) . tissue culture , transfection , and silencing were performed as described previously ( anelli et  al . , 2007 ) . fetal calf serum and cell culture medium were from gibco , invitrogen . unless otherwise indicated , chemicals were from sigma . polyclonal anti - mouse  and anti - erp44 ( b68 ) antibodies were purchased from primm srl ( milano , italy ) . monoclonal anti - gfp ( clones 7.1 and 13.1 ) was purchased from roche ( usa ) and polyclonal anti-1 antitrypsin ( ncl a1ap ) from novocastra . monoclonal anti - erp44 antibody ( 36c9 ) was previously described ( anelli et  al . , 2007 ) . goat anti - mouse ig and anti - rabbit ig horse - radish peroxidase ( hrp ) were from jackson immunoresearch laboratories , inc . mouse monoclonal antibodies specific for myc ( 9e10 ) , ha ( 12ca5 ) , and flag ( m2 ) were immobilized by crosslinking to protein g and protein a beads , respectively . hrp rabbit anti - mouse igg ( hl ) was from dako cytomation ( glostrup , co ) . each erp44 derivative ( 5  m ) was incubated on ice for 30  min in various ph buffers containing 100  mm sodium phosphate and 150  mm nacl , followed by incubation with maleimidyl peg2k ( 300  m ) for 10  min at room temperature . the reaction was stopped by the addition of 5% trichloroacetic acid , and the protein pellet was washed with acetone and dissolved in buffer containing 50  mm tris - hcl ( ph 7.0 ) and 1% sds before loading onto a reducing sds gel ( 10% ) . hela cells expressing pme - zeo - gpp130-phluorin ( miesenbck et  al . , 1998 ) were cultured on glass - bottomed dishes and analyzed under a tcs sp2 laser scanning confocal ( leica confocal ) equipped with an ld laser 405 ( 405  nm ) and a multiline ar laser ( 457 , 488 , and 515  nm ) at room temperature . data acquisition and analysis of intensity ratios ( 405  nm ; 457  nm ) were performed as described ( miesenbck et  al . , 1998 ) . statistical significance was calculated , comparing between conditions indicated by brackets using the two - tailed student s t test . oligonucleotides for rna interference ( silencer select predesigned sirna products ) were purchased from ambion ( sirna i d # s56768 ) . lipofectamine rnai max was from invitrogen ( carlsbad , usa ) and used for silencing in hela cells , according to the suppliers instructions . we used the propka 3.0 software ( http://propka.ki.ku.dk ) to predict the pka value of erp44 c29 and the role of surrounding amino acids starting from the crystal structure of the entire molecule ( wang et  al . , 2008 ; pdb i d code 2r2j ) and from in  silico models of the 16 mutant .
summaryto warrant the quality of the secretory proteome , stringent control systems operate at the endoplasmic reticulum ( er)-golgi interface , preventing the release of nonnative products . 
 incompletely assembled oligomeric proteins that are deemed correctly folded must rely on additional quality control mechanisms dedicated to proper assembly . here 
 we unveil how erp44 cycles between cisgolgi and er in a ph - regulated manner , patrolling assembly of disulfide - linked oligomers such as igm and adiponectin . at neutral , er - equivalent ph , 
 the erp44 carboxy - terminal tail occludes the substrate - binding site . at the lower ph of the cisgolgi , 
 conformational rearrangements of this peptide , likely involving protonation of erp44 s active cysteine , simultaneously unmask the substrate binding site and rdel motif , allowing capture of orphan secretory protein subunits and er retrieval via kdel receptors . 
 the erp44 assembly control cycle couples secretion fidelity and efficiency downstream of the calnexin / calreticulin and bip - dependent quality control cycles .