due to the stringency 
 of cellular quality control ( qc ) , protein 
 production and assembly are often inefficient in the cell .
about 30% 
 of newly synthesized proteins in mammalian cells are subject to rapid 
 degradation by proteasomes
. however , experimental 
 efforts to rationalize the interplay between the physiochemical properties 
 of polypeptide chains and their interactions with qc networks have 
 thus far been limited to a handful of soluble proteins .
far less is known about integral membrane protein folding and assembly , a process that is intimately linked to the molecular 
 basis of numerous diseases .
many of the proteins that carry 
 out the qc of nascent integral membrane proteins in the endoplasmic 
 reticulum ( er ) have been identified in recent years .
however , the structural properties subjected to surveillance by 
 qc , potentially triggering disposal by the er - associated degradation 
 ( erad ) pathway , are largely unexplored . here
we test the hypothesis 
 that the conformational stability of integral membrane proteins plays 
 a central role in their interactions with cellular qc and the efficiency 
 with which they traffic to their intended cellular compartments . to 
 test this hypothesis
, we interrogate a series of pathogenic variants 
 of human peripheral myelin protein 22 ( pmp22 ) , the cellular misfolding 
 and misassembly of which causes a spectrum of peripheral neuropathies 
 associated with charcot 
pmp22 
 is expressed in myelinating schwann cells of the peripheral 
 nervous system ( pns ) and is highly abundant in the membranes of compact 
 myelin .
strikingly , only 20% of wild - type ( wt ) pmp22 
 reaches its intended destination at the plasma membrane under physiological 
 conditions ; the majority of the nascent protein 
 is retained in the er and/or targeted for degradation .
furthermore , 
 most pathogenic variants further reduce the trafficking efficiency 
 of pmp22 , which suggests that pathogenic mutations promote misfolding 
 within the secretory pathway .
we recently reported quantitative thermodynamic measurements of 
 the conformational stability of purified wt pmp22 , the first such measurement to be made for any multispan 
 eukaryotic membrane protein .
surprisingly , we found that wt pmp22 
 exhibits marginal conformational stability in vitro , which parallels its rapid degradation and inefficient trafficking 
 in the cell .
to elucidate the nature of the interplay between the 
 folding and cellular trafficking of integral membrane proteins , we 
 here quantitatively survey the conformational stability and cellular 
 trafficking of a series of misfolding - prone pathogenic pmp22 variants . 
 remarkably
, the results not only appear to confirm a mechanistic linkage 
 between conformational stability and cellular trafficking but also 
 strongly suggest that the degree of mutagenic destabilization is directly 
 related to the severity of peripheral neuropathy in humans carrying 
 the associated mutations in the pmp22 gene . structural 
 distribution of pathogenic mutations in pmp22 .
a topology 
 diagram and a structural model depict the positions of the mutated 
 residues and the position of the myc epitope tag that was inserted 
 for immunological detection of pmp22 in mammalian cells .
( a ) a cartoon 
 depicts the topology of the sequence of pmp22 with respect to the 
 membrane .
the position of the myc epitope tag inserted 
 into the constructs used for cellular trafficking studies is indicated 
 with dashed lines .
the lowest - energy conformation of a recently published 
 structural model of the apo - form of pmp22 is viewed parallel to the 
 position of the membrane ( b ) and from its extracellular face ( c ) , with the positions of the pathogenically mutated 
 side - chains characterized in this work being highlighted for reference . 

mutated side chains associated with dss ( severe neuropathy ) , which 
 include h12q , l16p , m69k , s76i , and g150d , are shown in red .
mutated 
 side chains associated with cmt disease ( cmt1 , moderate neuropathy ) , 
 which include g107v and t118 m , are shown in purple .
mutated side chains 
 associated with hnpp ( mild neuropathy ) , which include s22f and a67 t , 
 are shown in blue . finally , the position of the side chain for the 
 nonpathogenic i137v mutation site is shown in black .
several 
 of the known pathogenic mutations in the pmp22 gene 
 have been found to enhance the misfolding and mistrafficking of pmp22 
 in the cell .
therefore , characterization of 
 the effects of these mutations provides an opportunity to elucidate 
 a potential relationship between conformational stability and cellular 
 trafficking . from the 44 known pathogenic missense mutations in pmp22 associated with peripheral neuropathies ( human gene 
 mutation database , http://www.hgmd.cf.ac.uk/ac/index.php ) , we selected 10 pathogenic variants and 1 nonpathogenic isoform 
 that are dispersed throughout the 4 transmembrane ( tm ) helices of 
 pmp22 ( figure 1a ) . 

these mutations cause a spectrum of clinical phenotypes ranging from 
 hereditary neuropathy with liability to pressure palsies ( hnpp , mild 
 neuropathy ) , cmt type 1 ( cmt1 , moderate neuropathy ) , and dejerine  sottas 
 syndrome ( dss , severe neuropathy ) ( table 1 ) .
previously reported motor nerve conduction 
 velocities of patients harboring these mutations confirm that the 
 degree of dysmyelination caused by these mutations is highly variable 
 ( supplementary table 1 ) , which suggests 
 that this set of mutations is likely to manifest a range of effects 
 on the conformational equilibrium of pmp22 .
additionally , we recently 
 found that none of these mutations are predicted to cause topogenic 
 defects , which suggests that conformational 
 defects are manifested after pmp22 is cotranslationally inserted into 
 the membrane .
we recently characterized the conformational 
 equilibrium of purified wt pmp22 in n - dodecylphosphocholine 
 ( dpc ) micelles and found that only 50% of the protein is folded 
 at equilibrium . because the free energy 
 difference between the folded and unfolded ensembles is minimal under 
 this condition ( gf  u  
 0 kcal / mol ) , even small perturbations of the conformational equilibrium 
 caused by mutations should cause measurable differences in the fraction 
 of folded protein . therefore , to determine the effects of pathogenic 
 mutations on the conformational stability , we assessed the tertiary 
 folding of pathogenic pmp22 variants in dpc micelles at equilibrium 
 using near - uv circular dichroism ( cd ) spectroscopy , which is sensitive 
 to the tertiary ordering of aromatic side chains .
the shapes of the 
 near - uv cd spectra of these variants are similar to that of the wt 
 protein ( figure 2 and supplementary figure 1 ) , suggesting that the 
 mutations do not significantly alter the tertiary structure of the 
 folded protein . however , significant differences are apparent in the 
 magnitude of the tryptophan peak at 299 nm ( [ ]299 ) , which reflects the fraction of folded pmp22 ( ffold ) . with the exception of g107v and 
 t118 m pmp22 , [ ]299 is diminished in the spectra 
 of the mutant proteins ( figure 2 and supplementary figure 1 ) , which 
 confirms that these mutations reduce ffold . based on the signal of the folded protein and the signal of the 
 unfolded protein
( see experimental section ) , we determined ffold for each mutant 
 variant in dpc micelles directly from the near - uv cd spectra ( table 1 ) . moreover , because 
 pmp22 achieves conformational equilibrium under these conditions , ffold values can 
 be used to directly determine the free energy of folding ( gf  u ) and to calculate the effect of a 
 mutation on the free energy of folding ( gf  u ) .
the energetic effects of four of the pathogenic 
 mutations ( h12q , s22f , g107v , and t118 m ) appear to be modest ( |gf  u | < 1.0 kcal / mol , table 1 ) . in contrast , four other pathogenic 
 mutations ( l16p , m69k , s76i , and g93r ) significantly shift the conformational 
 equilibrium toward the unfolded state ( gf  u > 2.0 kcal / mol , table 1 ) .
furthermore , three of the four highly destabilizing 
 mutations ( l16p , m69k , and s76i ) are associated with severe pathogenic 
 phenotypes ( dss ) .
together , these data demonstrate that the majority 
 of the pathogenic mutations studied herein decrease the conformational 
 stability of pmp22 .
nevertheless , the fact that the nonpathogenic 
 i137v variant decreases the conformational stability while the severely 
 pathogenic h12q variant has a negligible effect on the conformational 
 equilibrium of the apoprotein is puzzling .
these observations suggest 
 that additional factors , such as the presence of an endogenous ligand , 
 may influence the conformational equilibrium of pmp22 in vivo .
the fraction of folded 
 protein ( ffold ) was assessed for wt and 
 mutant pmp22 using near - uv cd spectroscopy .
the average cd signal 
 from three replicate near - uv cd measurements of wt ( black ) and four 
 representative mutant variants including s22f ( cyan ) , m69k ( red ) , 
 g107v ( green ) , and i137v ( purple ) pmp22 under nondenaturing conditions 
 is plotted against the wavelength .
the magnitude of the tryptophan 
 peak , which serves as a reliable probe for the fraction of folded 
 pmp22 , is indicated for each variant with dashed 
 lines .
the magnitude of the signal of the fully folded proteins determined 
 from equilibrium unfolding of wt protein in the presence of stabilizing 
 osmolytes ( see experimental section ) is indicated 
 with a dotted line for reference .
the energetics of protein folding and misfolding 
 in the cell depend on the physiochemical properties of the cellular 
 compartments in which qc occurs .
one 
 decisive factor is the concentration of ligands or cofactors , which can bind to and stabilize native conformations 
 relative to misfolded conformations .
pmp22 is known 
 to have an avid affinity for zn(ii ) and cu(ii ) and features two divalent 
 cation binding sites , which likely employ 
 the five histidine residues in its extracellular loops ( figure 1a ) . given that myelin is rich 
 in both zn(ii ) and cu(ii ) , it is highly probable 
 that the binding of metal ion cofactors influences pmp22 folding and 
 assembly within the cell .
we therefore sought to compare the influence 
 of metal binding on the conformational equilibrium of wt and mutant 
 pmp22 variants .
we first compared the propensity of the destabilized 
 g150d and l16p variants to bind zn(ii ) relative to wt using fluorescence 
 spectroscopy , as previously detailed .
a gain in the intrinsic tryptophan fluorescence intensity is coincident 
 with consecutive zn(ii ) binding events for each of these variants 
 ( figure 3a ) , which 
 suggests that binding is coupled to the formation of tertiary structure . 

importantly , this aspect of the titration data highlights the fact 
 that these proteins are not fully folded in the absence of metal ions 
 ( table 1 ) .
nevertheless , comparison of the apparent 
 binding affinities provides insight into whether mutations influence 
 zn(ii ) binding .
fitting of the titration data with a two - site binding 
 model reveals that these mutations significantly increase both of 
 the apparent equilibrium dissociation constants ( kd ,  app ) relative to those of wt ( figure 3a , table 1 ) .
to assess the nature of the structural changes that occur upon binding , 
 we used near - uv cd to probe the tertiary structure of the bound forms 
 of g150d pmp22 , which is mostly unfolded in dpc micelles at equilibrium 
 in the absence of zn(ii ) ( figures 3b and supplementary figure 1 ) .
the addition of a two - fold excess of zn(ii ) relative to the first 
 apparent equilibrium dissociation constant ( kd1,app ) of g150d pmp22 is coincident with an increase in the 
 near - uv cd signal at 299 nm ( figure 3b ) .
however , despite the fact that 67% of the protein 
 is saturated with a single zn(ii ) ion under this condition , only about 
 37% of the native cd signal is recovered .
this result demonstrates 
 that the saturation of the first binding site is coupled to a partial folding reaction .
a second folding reaction occurs 
 upon saturation of the second binding site . in the presence of a two - fold 
 excess of zn(ii ) relative to the second apparent equilibrium dissociation 
 constant ( kd2,app ) ( a condition under 
 which 67% of the protein is bound to two zn(ii ) ions ) ,
the similarity between the fraction of 
 doubly bound protein and the apparent fraction of folded protein under 
 this condition suggests that saturation of both binding sites is thermodynamically 
 coupled to global folding .
though we previously found 
 no evidence for the accumulation of partially folded equilibrium intermediates 
 in the absence of metal ions , these results 
 clearly show that at least three conformational states accumulate 
 during coupled zn(ii ) binding and folding ( figure 3c ) .
together , these findings confirm that 
 metal ion binding is thermodynamically coupled to pmp22 folding .
the fraction of folded protein ( ffold ) was determined from near - uv cd spectra 
 as detailed in the experimental section .
u values were determined as described 
 in the experimental section , with the associated 
 error values reflecting propagated standard deviations .
kd , app values represent the globally fit values from three 
 replicate titrations of the protein with zncl2 at ph 5.5 , 
 with the errors reflecting the standard error of the fitted value . 

gapp values were determined 
 as described in the experimental section , 
 with the associated error values reflecting propagated standard errors
elucidation of the effects of pathogenic mutations on the energetics 
 of coupled binding and folding reactions may provide insight into 
 the mechanism by which mutations in pmp22 prompt cellular misfolding . 
 unambiguous delineation of the effects of mutations on binding and 
 folding would require equilibrium unfolding measurements in the presence 
 of metal ions
. however , such measurements can not be achieved because 
 the negatively charged detergents utilized as denaturing agents for 
 -helical membrane proteins form insoluble complexes with divalent 
 metal ions . nevertheless , because binding 
 is thermodynamically coupled to refolding
, changes in the apparent 
 equilibrium dissociation constants ( kd , app ) quantitatively reflect the effects of mutations on the energetics 
 of the overall binding and folding reaction .
the effect of a mutation 
 on the apparent free energy difference associated with this cooperative 
 binding and folding reaction ( gapp ) can therefore be expressed as follows:1,where r is the gas constant , t is 
 the temperature , kd , app  wt is the 
 apparent equilibrium dissociation constant of the wild - type 
 protein , and kd , app  mut is the apparent 
 equilibrium dissociation constant of the mutant protein . summing gapp values for the two sequential binding and 
 folding reactions therefore yields the effect of a mutation on the 
 free energy difference between the apoprotein ensemble and the globally 
 folded zn(ii)-bound form of pmp22 ( gapp , total ) as follows:2,which reflects 
 the energetic effects of mutations 
 on the formation of the fully folded zn(ii)-bound form of pmp22 .
the energetic effects of the g150d ( gapp , total = 1.8  0.6 kcal / mol ) and l16p ( gapp , total = 3.3  0.5 kcal / mol ) mutations 
 on the formation of zn(ii)-bound pmp22 are consistent with the magnitude 
 by which these mutations destabilize the apoprotein ( gf  u = 1.5  0.2 and > 2.0 kcal / mol 
 for the g150d and l16p , respectively ) ( table 1 ) , indicating that these mutations likely 
 disrupt similar contacts present in both the apoprotein and in the 
 zn(ii)-bound forms .
we surveyed the additional nine mutations studied 
 herein for their energetic effects on the formation of zn(ii)-bound 
 pmp22 . with the exception of s22f , which exhibits a unique decrease 
 in intrinsic tryptophan fluorescence upon saturation of its second 
 binding site ( supplementary figure 2 ) , 
 a similar increase in the intrinsic tryptophan fluorescence coincided 
 with binding and folding for each variant , which suggests similar 
 structural rearrangements
interestingly , 
 the degree to which these mutations influence the energetics of binding 
 and folding ( gapp , total ) 
 is in several cases distinct from the degree to which they destabilize 
 the apoprotein ( gf  u ) ( table 1 ) . for instance , 
 despite the fact that the apoprotein form of the nonpathogenic i137v 
 variant is destabilized relative to the wt protein ( gf  u = 1.0  0.1 kcal / mol ) , the energetics 
 of binding and folding for i137v pmp22 are indistinguishable from 
 that of wt pmp22 ( gapp , total = 0.1  0.9 kcal / mol ) . in contrast , the severely pathogenic 
 h12q mutation has minimal influence on the conformational stability 
 of the apoprotein ( gf  u = 0.37  0.07 kcal / mol ) but significantly destabilizes the 
 zn(ii)-bound form of pmp22 ( gapp , total = 2.4  0.6 kcal / mol ) .
overall nine of the ten pathogenic mutations 
 surveyed disrupt the formation of zn(ii)-bound pmp22 to a significant 
 extent ( gapp , total > 
 1.0 
 kcal / mol ) , with all four of the mutations responsible for severe disease 
 ( dss ) exhibiting the largest energetic effects on the formation of 
 zn(ii)-bound pmp22 .
these results demonstrate that pathogenic mutations 
 exhibit differential effects on the apoprotein and zn(ii)-bound forms 
 of pmp22 and suggest the effects of the mutations on the bound form 
 may be more closely related to the pathogenic misfolding mechanism .
zn(ii ) binding 
 was monitored for wt and mutant pmp22 variants in dpc micelles at 
 ph 5.5 using near - uv cd and tryptophan fluorescence .
( a ) wt ( black ) , 
 g150d ( cyan ) , and l16p ( red ) pmp22 were titrated with zncl2 , and binding was monitored by the change in the intensity of the 
 tryptophan fluorescence emission at 345 nm .
the relative fluorescence 
 intensity at 345 nm from representative replicate experiments is plotted 
 against the concentration of zncl2 , and the global fit 
 of each data set to a two - site binding model is shown .
( b ) g150d pmp22 
 was equilibrated in the presence of 0 mm ( dotted line ) , 720 m 
 ( 2  kd1,app , dashed line ) , and 14 
 mm ( 2  kd2,app , solid line ) zncl2 prior to measurement of the near - uv cd spectra , and the mean 
 residue ellipticity ( [  ] ) is plotted against the wavelength . 

( c ) a cartoon depicts a hypothetical reaction coordinate for the coupled 
 binding and folding of the g150d pmp22 variant . to assess the linkage between the destabilization 
 imparted by these mutations and the mechanisms of pathogenic cellular 
 misfolding
, we sought to relate in vitro folding 
 measurements to the degree with which these variants are retained 
 by cellular qc .
recognition of misfolded pmp22 by cellular qc machinery 
 is a key factor leading to the retention of pathogenic variants in 
 the secretory pathway and the decrease in the concentration of mature 
 pmp22 at the plasma membrane .
we therefore 
 devised a means to quantitatively assess the efficiency with which 
 these variants escape qc and traffic to the plasma membrane .
pmp22 
 variants were transiently expressed in madin  darby canine kidney 
 ( mdck ) cells , and pmp22 at the plasma membrane was then labeled with 
 a pe - conjugated primary antibody . to detect retained intracellular 
 pmp22 , the cells
were then fixed , permeabilized , and again immunostained 
 with an alexa fluor 647-conjugated primary antibody .
flow cytometry 
 was then employed to quantify the immunostaining of pmp22 at the plasma 
 membrane ( folded ) and in the intracellular compartments ( misfolded ) 
 for each cell .
cells expressing wt pmp22 
 consistently exhibited significant trafficking of the protein to the 
 plasma membrane ( figure 4a ) , though its trafficking efficiency varied considerably across 
 the cellular population ( figure 4b ) . the population average for wt pmp22 ( 17  
 3% ) is consistent with previously reported estimates of the wt pmp22 
 trafficking efficiency in schwann cells ( 20% ) .
furthermore , the relative trafficking efficiencies 
 of pathogenic variants ( table 2 ) are generally consistent with previous qualitative observations 
 in other cell lines .
the trafficking efficiencies 
 of the nonpathogenic i137v pmp22 and those of the s22f and a67 t variants 
 associated with mild disease phenotypes are quite similar to that 
 of the wt protein ( table 2 ) . however , the other eight pathogenic variants exhibit markedly 
 decreased trafficking efficiencies ( figure 4 , table 2 ) . considering
that total cellular pmp22 expression 
 levels were comparable for all variants tested ( table 2 ) , these differences likely reflect the degree 
 of cellular misfolding and the resulting intracellular retention by 
 the cellular qc machinery .
importantly , five of the six variants with 
 the highest degree of intracellular retention are highly destabilized 
 and are associated with severe disease phenotypes .
these observations 
 quantitatively affirm the notion that a reduction in cellular trafficking 
 is a common effect of pathogenic mutations and 
 suggests that the intracellular retention of pathogenic pmp22 variants 
 is related to their reduced conformational stability .
wt and mutant 
 pmp22 variants were transiently expressed in mdck cells , and the cellular 
 trafficking of these proteins was quantitatively assessed using flow 
 cytometry .
( a ) contour plots depict the distribution of 2500 gfp - positive 
 cells from a representative flow cytometry experimental replicate 
 expressing wt ( black ) , t118 m ( cyan ) , or l16p ( red ) pmp22 according 
 to their relative immunostaining of pmp22 at the plasma membrane ( stained 
 with a pe - conjugated monoclonal antibody ) and intracellular pmp22 
 ( stained with an alexa fluor 647-conjugated monoclonal antibody ) . 

( b ) a histogram depicts the fraction of the cellular population with 
 the indicated ratio of the staining of pmp22 at the plasma membrane 
 to that of the total pmp22 staining for cells expressing wt ( black ) , 
 t118 m ( cyan ) , or l16p ( red ) pmp22 .
calculated ratios account for both 
 the effect of nonspecific binding of the antibodies as well as the 
 intrinsic difference in the intensities of the signal of the two fluorescently 
 labeled antibodies ( see experimental section ) .
the heights of the bars reflect the average fractional population 
 from three replicate experiments ( 7500 cells total per variant ) and 
 the error bars reflect the standard deviations of these values . to our knowledge
, the relationship 
 between the conformational stability of an -helical membrane 
 protein and its cellular trafficking in mammalian cells has not been 
 previously explored .
notably , most highly destabilized 
 proteins exhibit reduced trafficking ( tables 1 and 2 ) . however , 
 there are two exceptions .
the s22f mutant pmp22 exhibits a unique 
 increase in its cellular trafficking efficiency , which may be related 
 to the distinct structural properties of the zn(ii)-bound form of 
 s22f pmp22 ( supplementary figure 2 ) .
also , 
 the g107v variant appears to have comparable conformational stability 
 to that of wt pmp22 ( table 1 ) , yet exhibits impaired cellular trafficking ( table 2 ) .
however , we noticed that 
 the intrinsic tryptophan fluorescence intensity of g107v pmp22 is 
 similar to that of wt pmp22 under our experimental conditions , which 
 has been optimized for biophysical studies , but greatly diminished 
 at physiological ph ( supplementary figure 3 ) .
this finding suggests the structural defects caused by this particular 
 mutation may not be manifested under the conditions used for our folding 
 reactions . for these reasons ,
we have excluded the s22f and g107v 
 variants from our analysis of the relationship between the conformational 
 stability and cellular trafficking of pmp22 variants . in order 
 to assess the mechanism of pathogenic cellular misfolding , we compared 
 the conformational stability and cellular trafficking measurements . 

u values for the folding of the pmp22 apoprotein against relative 
 degree of plasma membrane trafficking but found no apparent relationship 
 ( supplementary figure 4 ) .
in contrast , 
 a linear relationship is apparent between the energetic effects of 
 mutations on zn(ii)-mediated pmp22 folding ( gapp , total ) and the degree of plasma membrane 
 trafficking ( r = 0.73 , figure 5a ) .
a key feature of this correlation 
 is the differentiation of pathogenic phenotypes that arises from these 
 combined measurements : mutations that cause severe disease phenotypes 
 ( dss ) exhibit the highest degree of destabilization and the largest 
 reduction in cellular trafficking , while mutations that cause moderate 
 to mild phenotypes ( cmt1 and hnpp ) have more subtle effects .
this 
 correlation has critical implications for the role of metal ions as 
 pmp22 cofactors . to further examine the relationship between binding , 
 folding , and trafficking
, we corrected the apparent zn(ii ) binding 
 constants for differences in the stabilities of the variants ( supplemental theory ) .
simulations based on corrected 
 thermodynamic parameters confirm zn(ii ) binding represents a key mediator 
 of the folding equilibrium constant and cellular trafficking of pmp22 
 ( supplemental theory ) .
together these findings 
 strongly suggest that pathogenic misfolding arises as a result of 
 the impact of mutations on the zn(ii)-binding - linked conformational 
 stability of pmp22 .
values represent 
 the average of 
 three replicate population - averaged values ( 2,500 gfp - positive cells 
 per replicate ) , and errors reflect the standard deviation of the population - averaged 
 values .
we next sought to 
 determine whether these observations are also 
 consistent with the degree of dysmyelination arising from these mutations . 
 because myelin principally provides insulation for nerve axons to 
 enhance conductance , pathogenic dysmyelination in cmt1 and related 
 disorders reduce motor nerve conduction velocities ( ncv ) .
ncv values 
 for patients who are heterozygous for the mutations studied in this 
 work therefore represent a favorable metric for the severity of the 
 disease phenotype .
a striking linear correlation ( r = 0.88 ) is apparent between ncv values and the relative 
 concentrations pmp22 variants at the plasma membrane ( figure 5b ) , which strongly supports 
 the notion that the mistrafficking of pmp22 variants represents a 
 key pathogenic mechanism leading to dysmyelination .
furthermore , ncv 
 values are also correlated with the energetic effects of these mutations 
 on zn(ii)-mediated pmp22 folding ( r = 
 0.64 , figure 5c ) , suggesting 
 that the compromised stability of the zn(ii)-bound forms of these 
 variants is responsible for their aberrant trafficking in 
 vivo and , ultimately , for dysmyelination .
together , these 
 findings indicate that the conformational instability of pmp22 underlies 
 most neuropathies arising from pathogenic missense mutations in the pmp22 gene .
collectively , our results 
 suggest that metal ion - mediated folding 
 is a limiting reaction within the pmp22 biosynthetic pathway .
these 
 findings strongly suggest that conformational stability is a critical 
 variable in the proper production and cellular trafficking of integral 
 membrane proteins in the cell ; a finding with broad relevance to the 
 molecular basis of a number of diseases .
furthermore , we believe these results serve to unify a number of 
 observations involving pathogenic pmp22 variants and have implications 
 for the rational design of novel therapeutics for this class of hereditary 
 peripheral neuropathies .
we recently found that the positions of pathogenically 
 mutated side chains associated with severe disease phenotypes almost 
 all fall within the protein core of a structural model of the pmp22 
 apoprotein ( figure 1b , c ) , suggesting that pathogenic misfolding may principally arise 
 as a result of the disruption of tertiary contacts within the native 
 structure . while the findings of this 
 paper are generally consistent with this notion , the results also 
 highlight metal ion binding as a factor that likely contributes to 
 the net conformational stability of pmp22 under physiological conditions 
 ( figure 5a , c ) .
the 
 conformational stability of pmp22 in vivo should 
 therefore depend both on the intrinsic conformational stability of 
 the apoprotein and the metal ion binding affinity .
inspection of the 
 folding data and binding data reveals that pathogenic mutations can 
 destabilize the protein by interfering with helical packing , metal 
 ion binding , or both ( table 1 ) .
regardless of the destabilization mechanisms , the net energetic 
 effect of these mutations on the formation of the fully folded , zn(ii)-bound 
 pmp22 is clearly important for its cellular trafficking ( figure 5a ) and the degree 
 of dysmyelination ( figure 5c ) .
the notion that metal ions play a critical role in pmp22 
 biogenesis is quite reasonable considering the essential roles metal 
 ions play in the nervous system .
zinc and copper are plentiful in 
 myelin , and depletion of either results in peripheral 
 neuropathy . moreover , compaction of pns myelin has been shown 
 to be mediated by zn(ii ) and other zn(ii)/cu(ii ) 
 binding proteins expressed in myelin such as the myelin basic protein . based on our results
, we hypothesize that the binding of metal ions 
 likely constitutes one of the decisive interactions in the biosynthetic 
 pathway of pmp22 .
such a role for metal ions in pmp22 biogenesis is 
 fully consistent with emerging perspectives on the influence of metabolites 
 and cofactors on the rate and efficiency of protein folding in the 
 cell .
correlation 
 of zn(ii ) binding energetics and cellular trafficking 
 of pmp22 variants to clinical phenotypes .
correlations between the 
 quantitative effects of mutations on the apparent free energy of zn(ii ) 
 binding ( gapp , total ) , the 
 quantitative effects of mutations on the cellular trafficking of pmp22 , 
 and the motor nerve conduction velocities of patients with the corresponding 
 mutations are shown .
( a ) the surface - immunostaining of mdck cells 
 expressing pmp22 variants were normalized relative to that of the 
 wt protein ( table 2 ) and plotted against the corresponding gapp , total values ( table 1 ) .
a linear fit of the data weighted according to the 
 experimental errors ( r = 0.73 ) is shown 
 ( dashed line ) .
( b ) motor nerve conduction 
 velocities ( ncv ) of patients harboring pathogenic mutations ( supplementary table 1 ) were plotted against the 
 relative surface immunostaining of mdck cells expressing the corresponding 
 pmp22 variant ( table 2 ) .
an unweighted linear fit of the data ( r = 0.88 ) is shown ( dashed line ) .
( c ) ncvs from patients 
 harboring pathogenic mutations in pmp22 ( supplementary 
 table 1 ) were plotted against the gapp , total values of the corresponding pmp22 variant ( table 1 ) .
an unweighted linear 
 fit of the data ( r = 0.64 ) is shown ( dashed 
 line ) .
this work confirms the long - held suspicion that the conformational 
 stability of pmp22 is intimately linked to its cellular fate . considering that passage through qc involves a large number of diverse 
 protein 
nevertheless , it should be noted that 
 a similar empirical linear relationship has previously been documented 
 between the conformational stabilities of mutant forms of a water - soluble 
 protein ( bovine pancreatic trypsin inhibitor ) and their secretion 
 efficiencies .
such relationships suggest that 
 the structural defects detected by qc are closely linked to conformational 
 stability .
differences in the folding and/or unfolding kinetics , for 
 example , may underlie differential recognition unstable variants . in this regard ,
it should be 
 noted that pmp22 folding , which requires hours to reach equilibrium in vitro , is accelerated roughly 
 30-fold in the presence of zn(ii ) ( supplementary 
 table 2 ) .
the increase of the folding rate of binding - competent 
 variants by metal ions may , in turn , prevent the formation of interactions 
 of between pmp22 and various folding sensors such as calnexin , which is known to form long - lived interactions 
 with destabilized pmp22 variants .
further studies are needed to gain mechanistic insight 
 into the key interactions that limit the folding and trafficking of 
 pmp22 in cells .
the apparent relationships between folding , trafficking , 
 and ncv values in patients harboring these mutations suggest the decreased 
 yield of folded pmp22 at the plasma membrane constitutes the key driver 
 of dysmyelination in the afflicted patients ( figure 5b , c ) .
however , the etiology of cmt1 and related 
 peripheral neuropathies is believed to stem from a variety of biochemical 
 effects of pathogenic pmp22 mutations including the enhanced retention 
 of pathogenic variants in the early secretory pathway , wild - type / mutant heterodimerization , the formation of intracellular aggregates , and a diminished 
 capacity of the cellular proteostasis network .
for instance , the enhanced retention 
 of pathogenic variants in the secretory pathway and the concomitant 
 decrease in the yield of pmp22 at the plasma membrane likely arise 
 as a result of enhanced interactions between misfolded variants and 
 er chaperones / folding sensors .
formation of non - native dimers , in 
 turn , may form as a consequence of the buildup of misfolded variants 
 in the endoplasmic reticulum .
polyubiquitination and extrusion of 
 misfolded variants from the er eventually result in the buildup of 
 large , insoluble aggresomes in the cytosol that are engaged by proteasomal 
 and lysosomal machinery .
the association of 
 chaperones , proteasomes , and lysosomes with aggresomes coincides with 
 decreases in the buffering capacity of the cellular proteostasis network , which may in turn facilitate the secondary aggregation of unrelated 
 proteins that rely on common chaperones .
thus , pharmacological strategies 
 aimed at suppressing the misfolding of pathogenic pmp22 variants could 
 potentially abrogate the chain of events collectively responsible 
 for dysmyelination .
this perspective provides a source of optimism 
 regarding the potential utility of small molecule 
pharmacological 
 chaperones  that suppress misfolding by specifically binding 
 the native fold relative to misfolded conformations .
such pharmacological chaperones have been reported for 
 a number of other disease - linked membrane proteins including the cystic 
 fibrosis transmembrane conductance regulator ( cftr ) and various g 
 protein - coupled receptors .
indeed , the coupling 
 of zn(ii ) binding and folding observed for pmp22 in this work demonstrates 
 the feasibility of ligand - mediated pmp22 stabilization .
collectively , 
 these findings indicate that pmp22 represents a promising candidate 
 for the development of pharmacological chaperones .
studies of the biophysical basis of membrane protein 
 misfolding are in an early stage of development .
our findings suggest 
 that the intrinsic thermodynamic preference for the native conformation 
 over a competing ensemble of disordered conformations represents a 
 decisive factor in pmp22 biogenesis .
a long line of evidence suggests 
 these principles may extend to a number of other membrane proteins 
 implicated in disease .
first , as is true for pmp22 and various other 
 membrane proteins known to undergo misfolding including rhodopsin , cftr , and the vasopressin 
 v2 receptor , pathogenic mutations generally 
 appear to be evenly distributed throughout the primary sequence .
this 
 suggests that pathogenic loss of function most often occurs as a result 
 of misfolding rather than disruption of specific functional elements .
additionally , many of these mutations involve 
 nonconservative substitutions that would be expected to perturb native 
 conformations .
for example , pathogenic mutations often involve the 
 introduction of helix - breaking residues , polar residues , or charged 
 residues within tm helices .
the putative destabilization 
 imparted by such pathogenic substitutions has been previously suggested 
 based on various computational studies and from biophysical studies of isolated tm helices .
this work represents the first definitive evidence directly linking 
 conformational destabilization of a human membrane protein to pathogenic 
 cellular misfolding , which opens the door for additional mechanistic 
 investigations of this phenomenon in other related disease - linked 
 integral membrane proteins .
despite 
 growing appreciation of protein misfolding as the molecular 
 basis of for numerous diseases , we are just beginning to understand 
 the critical factors that prompt the misassembly of nascent membrane 
 proteins .
the linkage documented herein between the conformational 
 stability of an -helical membrane protein and its passage through 
 cellular qc is likely to be generally applicable to many other membrane 
 proteins implicated in disease and provides fundamental insight into 
 the logic of membrane protein qc in the er . our results also strongly 
 suggest a critical role for divalent metal ions in pmp22 biogenesis . 
 finally , pmp22 appears to be a suitable target for the development 
 of pharmacological chaperones to treat certain forms of cmt1 and related 
 neuropathies .
quickchange mutagenesis was used to create 
 mutants of pmp22 within the pah13 construct , which has been previously 
 described for the expression of pmp22 in escherichia 
 coli . for transient expression of pmp22 
 in mammalian cells ,
this construct expresses pmp22 and egfp off of the same transcript , 
 which enables facile identification of transformed cells expressing 
 pmp22 . due to the lack of high - quality monoclonal antibodies for pmp22 , 

we used quickchange mutagenesis to insert a myc epitope into the second 
 extracellular loop of pmp22 within the pires2 constructs to enable 
 immunological detection .
it has previously been shown that this modification 
 does not impact the trafficking or turnover of the protein .
quickchange mutagenesis was also used to introduce point mutations 
 into the pires2 vectors prior to purification of each vector using 
 a perfectprep endofree plasmid maxi kit ( 5prime , gaithersburg , md ) .
expression and purification of mutant proteins 
 were also performed as previously described for the wt protein , except 
 that the mutant proteins were separated from the cleaved expression 
 tag and protein aggregates by cation exchange chromatography using 
 a 1 ml hitrap sp sepharose column and/or size exclusion chromatography 
 using a 24 ml superpose 6 column on an kta fplc ( ge healthcare , 
 piscataway , nj ) when necessary .
we previously demonstrated that 
 wt pmp22 is only 50% folded ( gf  u  0 kcal / mol ) at equilibrium in dpc micelles , which implies the fraction of folded protein 
 ( ffold ) should be sensitive to the effects 
 of mutations on the conformational stability under these conditions . 

therefore , we directly assessed the ffold of each variant in dpc micelles using near - uv cd spectroscopy in 
 order to determine the corresponding free energy of folding ( gf  u ) . to measure the ffold for each variant , we assessed the mean residue ellipticity 
 at 299 nm ( [ ]299 ) , which serves as a reliable probe 
 for the degree of tertiary structure formation , of each variant in nondenaturing dpc micelles and subtracted the 
 signal of the denatured protein in mixed micelles containing dpc and 
 a high mole fraction of the denaturing detergent n - lauroyl sarcosine ( ls ) ( xls ) and then 
 divided this quantity by the signal of the fully folded protein as 
 described below .
each purified variant protein was equilibrated 
 at room temperature in 25 mm sodium acetate ( ph 5.5 ) containing 150 
 mm nacl , 1.0 mm tcep , and 28 mm dpc ( 0.00 xls ) prior to acquisition of the near - uv cd spectrum of the protein 
 under nondenaturing conditions using a jasco j-810 spectropolarimeter 
 ( jasco , easton , md ) .
purified variants were also equilibrated at room 
 temperature in 25 mm sodium acetate ( ph 5.5 ) containing 150 mm nacl , 
 1.0 mm tcep , 28 mm dpc , and 28 mm ls ( 0.50 xls ) , a condition under which the tertiary structure of the 
 wt protein is known to be denatured .
cd spectra 
 for both the folded protein and the denatured protein were corrected 
 by subtracting the spectra of 25 mm sodium acetate ( ph 5.5 ) containing 
 150 mm nacl , 1.0 mm tcep , and 28 mm dpc ( 0.00 xls ) ( nondenaturing conditions ) or of 25 mm sodium acetate ( ph 
 5.5 ) containing 150 mm nacl , 1.0 mm tcep , 28 mm dpc , and 28 mm ls 
 ( 0.50 xls ) ( denaturing conditions ) , respectively . 
 to minimize the influence of slight deviations in the spectral baselines , 

all spectra were empirically normalized based on the average signal 
 between 320 and 330 nm , which was observed to be close to zero in 
 all cases . to determine the signal of the folded variant protein from 
 these spectra , the average [ ]299 value from three 
 replicate cd spectra of the denatured protein ( at 0.50 xls ) , which represents the baseline signal of the fully 
 unfolded protein , was subtracted from the average [ ]299 value of the protein under nondenaturing conditions ( 0.00 xls ) . to determine the ffold value of the variant , this corrected signal
was then divided 
 by 105  2 deg cm dmol res , which is the average signal of the fully 
 folded protein as determined from the fitting of three replicate wt 
 pmp22 equilibrium unfolding transitions measured in the presence of 
 15% glycerol using a two - state equilibrium model ; see ref ( 21 ) .
the ffold values were then used to calculate equilibrium constants 
 and the free energy of folding ( gf  u ) assuming a two - state equilibrium , which was previously shown to 
 adequately describe the behavior of the wt protein under these conditions 
 at equilibrium .
briefly , purified pmp22 variants were equilibrated at a 
 protein concentration of 1 m at room temperature in 25 mm sodium 
 acetate ( ph5.5 ) containing 150 mm nacl and 28 mm dpc for at least 
 1 h prior to binding measurements .
the solution was then equilibrated 
 in a quartz cuvette containing a stir bar at 25 c for at least 
 15 min prior to serial titration with a stock zn(ii ) solution containing 
 25 mm sodium acetate ( ph 5.5 ) , 150 mm nacl , and 550 mm zncl2 using a hamilton syringe ( reno , nv ) .
final concentrations ranging 
 from 40 to 80 mm zncl2 were achieved , depending on the 
 variant , in order to ensure saturation of both binding pockets .
the 
 binding reaction was allowed to proceed for 5 min after each addition , 
 which was judged to be sufficient for the binding kinetics of wt and 
 mutant pmp22 ( supplementary table 2 ) .
the 
 extent of binding was then monitored with a 20 s average of the fluorescence 
 emission intensity at 345 nm with a jobin yvonne fluoromax spex-3 
 ( horiba scientific , edison new jersey , nj ) .
the signal was then corrected 
 according to the dilution factor , and the gain in the fluorescence 
 intensity was normalized relative to the fluorescence intensity of 
 pmp22 in the absence of zncl2 .
the background fluorescence 
 was judged to be negligible according to titrations performed in the 
 absence of pmp22 .
binding isotherms were best described by a two - site 
 binding model as judged by the residuals of the fits of the data to 
 single- and two - site binding models . to reduce the influence of random 
 errors on the fitted kd values ,
three 
 replicate binding isotherms were globally fitted to determine the two kd values .  
mdck cells , which have proven to be 
 robust in previous studies of the cellular trafficking of integral 
 membrane proteins , were grown at 37 c 
 and 5% co2 in f-12 dmem containing 10% fetal bovine serum 
 ( fbs ) , penicillin , and streptomycin ( life technologies , grand island , 
 ny ) to 2050% confluency in 6 cm culture dishes .
the cells 
 were then transfected in optimem media ( life technologies , grand island , 
 ny ) for 24 h with 1 g of plasmid per dish using effectine transfection 
 reagent ( qiagen , venlo , nl ) .
the transfection media was then removed , 
 and the cells were allowed to grow in f-12 dmem containing 10% fbs , 
 penicillin , and streptomycin for an additional 24 h. transfected cells 
 were trypsinized and prepared for facs analysis using the fix & 
 perm kit in accordance with the manufacturer s instructions 
 ( life technologies , grand island , ny ) .
briefly , half of the cells 
 from a confluent 6 cm culture dish ( ca .
1.6  10 cells ) 
 were suspended in 100 l of culture media , and a pe - labeled 
 monoclonal anti - myc antibody ( clone 9e10 ) ( cell signaling technology , 
 danvers , ma ) was added to the solution to a final concentration of 
 0.75 g / ml to immunostain pmp22 on the surface of the cell . 

the cells were then incubated in the dark at room temperature for 
 30 min .
100 l of the fixation solution was then added to the 
 media , and the cells were incubated for 15 min in the dark at room 
 temperature .
the cells were then rinsed and pelleted by centrifugation 
 twice with 3 ml of pbs containing 5% fbs and 0.1% nan3 ( rinse solution ) . 

the cells were then suspended in 100 l of the permeabilization 
 solution , and an alexa fluor 647-labeled monoclonal anti - myc antibody 
 ( clone 9e10 ) ( cell signaling technology , danvers , ma ) was added to 
 the solution to a final concentration of 0.75 g / ml to label 
 intracellular pmp22 . after a 30 min incubation in the dark at room 
 temperature ,
the cells were again rinsed and pelleted by centrifugation 
 twice using rinse solution then resuspended in 300 l of the 
 rinse solution prior to facs analysis .
immunostained cells were 
 analyzed with a facs canto ii flow cytometer ( bd bioscience , san jose , 
 ca ) .
2500 transfected cells expressing pmp22 were analyzed 
 from each sample by gating on gfp - positive cells ( excited with a 488 
 nm laser , detected with 515545 nm emission filter ) .
the single - cell 
 pe intensity ( surface pmp22 , excited with a 488 nm laser , detected 
 with 564606 nm emission filter ) and alexa fluor 647 intensity 
 ( internal pmp22 , excited with a 633 nm laser , detected with 650670 
 nm emission filter ) signals were corrected for nonspecific binding 
 by subtracting the average intensities of untransfected , gfp - negative 
 cells within each sample . to correct for the difference in the fluorescence 
 intensity of the two antibodies ,
cells expressing wt pmp22 were stained 
 with either the pe - labeled antibody or the alexa fluor 647-labeled 
 antibody prior to facs analysis , and the ratio of the average intensities 
 of these cells was used to normalize the two signals .
single - cell 
 trafficking efficiency values were then calculated from the ratio 
 of the corrected pe signal of a given cell over the sum of its corrected 
 alexa fluor 647 and pe signals .
average trafficking efficiency values 
 calculated in this fashion were found to be similar to those determined 
 by a comparison of the population - averaged intensities of intact ( surface 
 pmp22 ) and permeabilized ( total pmp22 ) cells stained with the same 
 concentration of the same fluorescently labeled antibody .
single - cell 
 fluorescence intensity values below the background intensity were 
 assigned an intensity of 0 .
cells for which this correction or for 
 which extremely low intensity values resulted in a nonreal or artificially 
 inflated efficiency ratio ( > 50% efficiency , typically less than 
 1% 
 of the population ) were discarded from the analysis .
results were 
 analyzed and visualized using flowjo x software ( treestar inc . , ashland , 
 or ) .
from titrations of both intact and permeable cells expressing 
 wt 
 pmp22 with fluorescently labeled antibodies , we found the average 
 fluorescence intensity to be linearly dependent upon the antibody 
 concentration .
this confirms that fluorescence intensity values fall 
 within the linear range of the detectors .
moreover , this ensures that 
 the observed trafficking efficiency values are independent of the 
 chosen antibody concentration .
compensation for spillover of the fluorescence 
 signals between the channels utilized for the analysis as well as 
 the gates for the selection of single cells , gfp - positive cells , and 
 gfp - negative cells was initially set manually but was kept consistent 
 for the collection of all data sets obtained thereafter .
 despite broad biochemical 
 relevance , our understanding of the physiochemical 
 reactions that limit the assembly and cellular trafficking of integral 
 membrane proteins remains superficial . in this work , 
 we report the 
 first experimental assessment of the relationship between the conformational 
 stability of a eukaryotic membrane protein and the degree to which 
 it is retained by cellular quality control in the secretory pathway . 
 
 we quantitatively assessed both the conformational equilibrium and 
 cellular trafficking of 12 variants of the -helical membrane 
 protein peripheral myelin protein 22 ( pmp22 ) , the intracellular misfolding 
 of which is known to cause peripheral neuropathies associated with 
 charcot  marie  tooth disease ( cmt ) . 
 we show that the 
 extent to which these mutations influence the energetics of zn(ii)-mediated 
 pmp22 folding is proportional to the observed reduction in cellular 
 trafficking efficiency . 
 strikingly , quantitative analyses also reveal 
 that the reduction of motor nerve conduction velocities in affected 
 patients is proportional to the extent of the mutagenic destabilization . 
 
 this finding provides compelling evidence that the effects of these 
 mutations on the energetics of pmp22 folding lie at the heart of the 
 molecular basis of cmt . 
 these findings highlight conformational stability 
 as a key factor governing membrane protein biogenesis and suggest 
 novel therapeutic strategies for cmt . 
Introduction
Results
Discussion
Conclusion
Experimental Section