the earliest evidence for protein quality control at the er came from observations that unassembled subunits of the t cell receptor were rapidly degraded in the cells ( lippincott - schwartz et al . ,
this degradation occurred independently of lysosomal proteases , leading to the proposal that the er itself would house some uncharacterized proteolytic activity toward misfolded proteins .
then a landmark study in yeast showed that the degradation of a short - lived misfolded er membrane protein was blocked in cells lacking ubc6 , a component of the ubiquitin conjugation machinery ( sommer and jentsch , 1993 ) .
the ubiquitin system mediates the covalent attachment of ubiquitin , a small 76amino acid protein , to target proteins in the cytoplasm by the sequential action of activating ( e1 ) , conjugating ( e2 ) , and ligase ( e3 ) enzymes ( pickart , 2001 ) .
proteasome system in er protein quality control was confirmed by studies on the degradation of mutant and wild - type cystic fibrosis transmembrane conductance regulator ( cftr ) , a large membrane protein with a complicated folding process ( jensen et al .
inhibition of proteasome function led to accumulation of cftr molecules , and interestingly , a significant fraction of these was detected as ubiquitin conjugates ( jensen et al .
soon after , it became clear that a similar mechanism could also account for the degradation of luminal misfolded proteins such as cpy * , a mutant version of the yeast vacuolar carboxypeptidase y and a prototype erad substrate ( hiller et al .
, these papers demonstrated that aberrant proteins in the lumen and membrane of the er are degraded in the cytoplasm where the components of the ubiquitin  proteasome system reside .
subsequent genetic and biochemical studies , primarily in budding yeast but also in mammalian cells , identified many erad components and led to a general understanding of the organization of the pathway .
an important realization was that the  one - size - fits - all  model does not apply to erad and that this pathway encompasses multiple branches with distinct specificity for different classes of misfolded proteins ( taxis et al .
, 2003 ; vashist and ng , 2004 ; carvalho et al . , 2006 ;
however , irrespective of the branch , the same sequence of events leads to the degradation of all erad substrates ( fig .
1 a ) . the first step is the recognition of a substrate in the crowded er environment .
then the substrate is transported across the er lipid bilayer back into the cytoplasm , a step known as retrotranslocation . on the cytosolic side of the er membrane , the substrate
subsequently , the ubiquitinated substrate is extracted from the membrane in an atp - dependent manner and released in the cytoplasm for degradation by the proteasome .
the execution of these steps is coordinated by a membrane - embedded protein complex named after the e3 ligase at its core .
the canonical e3 ligases involved in erad are themselves multispanning membrane proteins , in which the ring ( really interesting new gene ) domain responsible for the ligase activity is in the cytoplasm .
1 b and table 1 ) where doa10 ( swanson et al . , 2001 ) and hrd1 ( bordallo et al .
assemble into the doa10 and the hrd1 complexes , respectively , each responsible for the degradation of a class of erad substrates ( carvalho et al . , 2006 ) .
( a ) the events defining the erad of a generic luminal substrate with a misfolded domain ( red star )
. substrate recognition , retrotranslocation , and ubiquitination are coordinated by a membrane - embedded e3 ligase complex .
( b ) the e3 ligase complexes involved in erad in s. cerevisiae and their substrate specificities .
er proteins with a misfolded domain in the cytoplasm ( erad - c substrates ) are degraded via the doa10 complex .
proteins with luminal ( erad - l ) or intramembrane ( erad - m ) misfolded domains are degraded via the hrd1 complex . misfolded domains on proteins are indicated by a red star .
components of the yeast e3 ligase complexes and their mammalian counterparts based on the analysis of a few model substrates , the e3 ligase complex specificity appears to be determined by the location of the misfolded lesion on a substrate relative to the er membrane : proteins with misfolded domains in the cytoplasmic side of the membrane ( erad - c substrates ) are degraded via the doa10 complex ; proteins with luminal ( erad - l substrates ) or intramembrane ( erad - m substrates ) misfolded domains are targeted to the hrd1 complex ( fig .
, 2003 ; vashist and ng , 2004 ; carvalho et al . , 2006 ) .
factors involved in substrate recognition are unique to the e3 ligase complex and likely determine the substrate specificity of each erad branch . on the other hand , the components that act at late steps of erad , such as the cdc48 atpase complex ( p97 in mammals ) required for membrane extraction of ubiquitinated substrates ( bays et al .
, 2001b ; ye et al . , 2001 ; jarosch et al . , 2002 ;
2002 ) , are common to both e3 ligase complexes . in mammalian cells the best - studied e3 ligases are hrd1 and gp78 ( table 1 ) .
they are both homologous to yeast hrd1 but assemble distinct e3 ligase complexes that preferably target different substrates ( schulze et al . , 2005 ; mueller et al . , 2008
several more e3 ligases have been implicated in erad in mammalian cells ( such as rma1/rnf5 , trc8 , rfp2 , rnf170 , and rnf185 ) but these are still poorly characterized .
only few substrates are known for each ligase and a preference for particular erad substrate classes has been difficult to infer ( claessen et al . , 2012 ) .
the commitment to degradation by erad occurs at the level of substrate recognition ; therefore , this step needs to be tightly controlled .
inefficient detection of misfolded proteins leads to their accumulation , ultimately affecting cell function ( travers et al . ,
, overactive erad would likely have its cost , with the degradation of significant amounts of folding intermediates .
this is a complex task considering the er environment , in which a complete spectrum of protein species coexist , from newly synthesized unfolded molecules to fully folded proteins .
proteins share structural similarities , for example the exposure of hydrophobic patches that are normally hidden once proteins acquire the native structure .
these molecules are kept in a soluble state by binding to chaperones such as the er hsp70 ( kar2 in yeast and bip in mammals ) , which are essential for the folding of newly synthesized polypeptides as well as for the disposal of misfolded proteins .
however , chaperones on their own do not appear to determine the fate of their clients . instead , recognition factors that are part of the e3 ligase complexes play a major role in erad substrate selection .
for example , the recognition of erad - l substrates requires the luminal components of the hrd1 complex hrd3 , kar2 and , in the case of glycosylated substrates , the lectin yos9 ( plemper et al . , 1997 , 1999 ; bhamidipati et al . , 2005
; kim et al . , 2005 ; szathmary et al . , 2005 ; carvalho et al . , 2006 ; denic et al
the yeast derlin der1 , a membrane protein of the hrd1 complex , might also be involved in erad - l substrate recognition ( gauss et al .
, 2006b ; stanley et al . , 2011 ) . moreover , certain erad - m substrates appear to be recognized directly by the e3 ligase hrd1 ( sato et al . , 2009 )
. however , the features recognized on the misfolded proteins by these erad factors are largely unknown .
an informative exception is the recognition of luminal misfolded n - linked glycoproteins in saccharomyces cerevisiae ( fig .
as they enter the er lumen , proteins are often modified at asparagine residues ( in the context of the n - x - s / t sequence ) with a well - defined , branched glycan moiety made up of three glucose , nine mannose , and two n - acetylglucosamine residues , glc3man9glcnac2 ( fig .
early glycan - processing enzymes such as glucosidases lead to the binding of lectins that facilitate the folding of the newly synthesized proteins .
in contrast , late acting enzymes , such as the mannosidase htm1 , trigger the binding of a different lectin that engages the protein in erad ( jakob et al .
this difference in the kinetics of the glycan - trimming enzymes provides an opportunity for newly synthesized proteins to acquire the native conformation and traffic beyond the er ( fig .
2 a ) . a long er residency , indicative of folding problems , results in the processing of the misfolded glycoproteins by htm1 , which generates a biochemical mark ( 1,6-linked mannose ) decoded by the lectin yos9 , an erad substrate recognition factor ( quan et al . , 2008 ; clerc et al . , 2009 )
if properly folded , the proteins leave the er . prolonged residency in the er , indicative of a persistent misfolded domain ( red star ) , leads to htm1-dependent exposure of an -1,6linked mannose residue ( red bar ) .
together , the misfolded domain and the terminal -1,6 mannose form the degradation signal recognized by hrd3/yos9 .
( b ) recognition of native mhc i heavy chain by the cytomegalovirus -encoded us2 adaptor in infected cells .
us2 binds to folded mhc i in the er membrane and delivers it to an e3 ligase complex containing the e3 trc8 and the signal - peptide peptidase spp resulting in mhc i degradation by erad .
( c ) sterol - dependent recognition of hmgr by insigs in mammalian cells . under low sterol levels
high sterol levels , in particular the accumulation of 24,25-dihydrolanosterol ( four - ringed structure in gray ) , cause insig to bind to hmgr and to deliver it to an e3 ligase complex that promotes hmgr degradation by erad .
both yeast htm1 and its mammalian counterpart edem are in complex with oxidoreductases ( pdi1 in yeast , erdj5 in mammals ) , required for the stability of htm1 and also for reducing disulfide bonds in misfolded proteins , which might affect subsequent erad steps ( ushioda et al . , 2008 ; clerc et al . ,
the binding of yos9 to the 1,6-linked mannose is not sufficient to trigger the degradation of the misfolded protein .
this processed glycan must be located in an unstructured polypeptide segment that is bound by hrd3 ( xie et al . , 2009 ) .
the delivery of substrates to hrd3 might be facilitated by the luminal chaperone kar2 that is essential for erad ( plemper et al .
xie et al . , 2009 ) . the dual recognition of a specific n - linked glycan by yos9 and an unstructured segment by hrd3
likely enhances the stringency of erad substrate recognition , perhaps by a kinetic proofreading mechanism ( fig .
the recognition mechanism of misfolded luminal n - linked glycoproteins is likely similar in mammalian cells because the yeast components are largely conserved in higher eukaryotes ( table 1 ) .
os-9 and xtp3-b , the mammalian homologues of yos9 , use their glycan - binding domain not only to interact with glycans in the misfolded protein but also with sel1 , the mammalian version of hrd3 , which is itself a glycoprotein ( christianson et al . , 2008 ) .
whether the interactions with sel1 and the substrates occur sequentially or correspond to different pools of os-9/xtp3-b is unclear . in either case ,
using the same domain to interact with a component and a substrate offers os-9/xtp3-b an additional mechanism to regulate recognition of n - glycosylated erad substrates .
recent work in yeast suggests that a different type of glycosylation , o - mannosylation , plays an important role in removing certain luminal proteins from futile folding cycles and thus favoring their degradation by erad after prolonged residency in the er ( goder and melero , 2011 ; xu et al . , 2013 ) .
the enzymes involved in protein o - mannosylation physically associate with erad machinery , but how o - mannosylated proteins are captured by the erad components is still unclear ( goder and melero , 2011 ) .
therefore , o - mannosylation is another appealing mechanism for generating an irreversible biochemical mark on proteins displaying folding problems .
a common feature between erad substrate recognition by n - glycan trimming and o - mannosylating enzymes is that both appear to be slow processes , requiring substrates to stay for relatively prolonged periods in the er . whether other mechanisms involved in recognition of misfolded proteins by erad also require a lag period in the er is not known .
nevertheless , it is curious that newly synthesized proteins were shown to be protected from degradation for a period of time , even under conditions that favor their misfolding ( vabulas and hartl , 2005 ) .
therefore , recognition of misfolded proteins might have evolved as an intrinsically slow process , perhaps to spare some folding intermediates from prematurely engaging in erad .
the degradation of specific folded proteins by erad is mediated by the same general machinery , but the recognition of these substrates involves distinct factors . a simple mechanism to target a native protein to erad is by a substrate - specific adaptor . for example , the human cytomegalovirus encodes er membrane adaptor proteins , us2 and us11 , which bind independently to newly synthesized mhc i molecules and deliver them to erad components for degradation . as a consequence , infected cells display less mhc i complex at their surface and escape detection by the immune system ( wiertz et al . , 1996a ) . despite this common outcome ,
us11 uses its transmembrane domain to recruit mhc i into a complex which contains derlin-1 as well as sel1l , the p97 atpase complex and its membrane adaptor ubxd8 ( lilley and ploegh , 2004 ; ye et al .
intriguingly , the e3 ligase required for us11-mediated mhc i degradation is not known and both hrd1 and gp78 e3 ligases , which are found in complex with derlin-1 , appear to be dispensable .
us2 , on the other hand , delivers its substrate to the erad ligase complex containing the e3 trc8 and spp , the signal peptide peptidase ( fig .
the precise function of spp in erad is not known and it is not even clear whether it involves its proteolytic activity . despite these differences , both us2 and us11 act as adaptors to deliver a specific erad substrate , mhc i heavy chain , to the e3 ligase complexes that promote its degradation .
a similar mechanism targets cd4 for degradation in cells expressing the hiv - encoded er membrane protein vpu . in this case
, vpu works not only as the substrate adaptor for cd4 but also as a scaffold to recruit a cytosolic e3 ligase complex , scf , required for cd4 ubiquitination ( fujita et al .
, 1997 ; schubert et al . , 1998 ) . in vitro reconstitution of vpu - mediated cd4 ubiquitination revealed that the specificity of adaptor - mediated substrate selection can be further increased at the level of substrate ubiquitination , which can be counteracted by the activity of de - ubiquitinating enzymes ( zhang et al . , 2013 ) .
the strategy of using a substrate - specific adaptor is not exclusive to viral encoded proteins . both in drosophila and in mammalian cells , the derlin - related irhom proteins function as adaptors in the erad - mediated degradation of egfr ligands as they traffic through the er ( zettl et al . , 2011 ) .
elegant genetic experiments in flies showed that this mode of regulated erad was important to control sleeping behavior that requires egfr signaling ( zettl et al . , 2011 ) .
it is likely that more substrate - specific adaptors will be identified as our knowledge of the mechanisms of regulated erad expands .
a substrate - specific adaptor also functions in the regulated erad of hmgr , a key enzyme of the sterol biosynthetic pathway . in this case
the adaptor , either insig-1 or insig-2 , does not bind constitutively to hmgr ( song et al . ,
instead , the interaction only occurs in the presence of 24,25-dihydrolanosterol , an intermediate metabolite in sterol biosynthesis . under low sterols levels hmgr
2 c ) . on the other hand , high sterol synthesis leads to a rise in 24,25-dihydrolanosterol concentration , which favors the binding of hmgr to one of the insig proteins , its delivery to an e3 ligase complex , and consequently its degradation by the proteasome ( fig .
2 c ) . whereas gp78 and the trc8 were originally implicated in hmgr regulated erad
, recent data suggest that additional e3 ligases might also be involved ( song et al .
, 2005 ; lee et al . , 2010 ; jo et al . , 2011
degradation of hmgr by erad results in reduced flux through the sterol biosynthetic pathway and reestablishment of membrane lipid homeostasis .
interestingly , insig-1 ( but not insig-2 ) is itself subjected to reciprocal sterol - regulated erad ( lee et al . , 2006 ) .
conversely , if sterol levels are high insig-1 binds to scap , another key er membrane protein required for sterol homeostasis , leading to a much longer insig-1 half - life .
these data illustrate the complex interplay between the opposing effects of sterols on the stability of the hmgr enzyme and one of its adaptors for regulated degradation by erad . in yeast ,
sterol homeostasis also involves negative feedback of an hmgr homologue , hmg2 ( hampton et al . , 1996 ) .
like in mammals , degradation of yeast hmg2 is controlled by the insig - like proteins nsg1 and nsg2 and requires the e3 ligase hrd1 ( bays et al .
in fact , hrd1 was originally identified in a genetic screen for mutants defective in hmgr degradation ( hrd genes ; hampton et al . , 1996 ) .
the binding of hmg2 to nsg1 is also modulated by sterol levels ( theesfeld and hampton , 2013 ) .
however , in contrast to mammalian cells , the binding of nsg1 promotes hmg2 stability , indicating that the recognition of this substrate is mechanistically different in the two systems ( flury et al .
, it has been proposed that nsg1 and nsg2 work as hmg2-specific chaperones and that in their absence hmg2 presents sufficient conformation instability to engage in erad as a misfolded protein ( flury et al .
this degradation is further accelerated by high concentrations of an early sterol - intermediate metabolite , geranylgeranyl pyrophosphate ( theesfeld and hampton , 2013 ) .
therefore , a change in the affinity to a binding partner is another strategy to target a protein for erad in a signal - dependent manner .
squalene monooxygenase ( sqle ) , another enzyme of the sterol biosynthetic pathway , is also targeted by regulated erad both in yeast and in mammals ( foresti et al . , 2013 ) .
sqle degradation requires the yeast doa10 e3 ligase complex or its mammalian homologue teb4 , indicating that two independent branches of erad control distinct steps in sterol biosynthesis ( foresti et al . , 2013 ) .
although the mechanism for the recognition of sqle by erad machinery is still not known , it is clear that insigs are dispensable for this recognition , both in mammals and in yeast ( gill et al . , 2011 ; foresti et al . , 2013 ) .
in mammals , the n - terminal domain of sqle is necessary and sufficient for cholesterol - dependent degradation ( gill et al . , 2011 ) .
whether this domain binds directly to cholesterol or interacts with an erad - specific adaptor is not known .
the mechanism for sqle recognition by erad in yeast is likely to be different because this n - terminal domain is only conserved among certain animals .
based on these few examples , it is clear that signal - mediated erad depends on the ability of cells to sense the concentration of some lipids in their membranes and on specific adaptors to selectively degrade key enzymes
. our knowledge on the mechanisms by which other classes of regulated erad substrates are recognized will grow as more of these are identified .
the commitment to degradation by erad occurs at the level of substrate recognition ; therefore , this step needs to be tightly controlled .
inefficient detection of misfolded proteins leads to their accumulation , ultimately affecting cell function ( travers et al . ,
, overactive erad would likely have its cost , with the degradation of significant amounts of folding intermediates .
this is a complex task considering the er environment , in which a complete spectrum of protein species coexist , from newly synthesized unfolded molecules to fully folded proteins .
proteins share structural similarities , for example the exposure of hydrophobic patches that are normally hidden once proteins acquire the native structure .
these molecules are kept in a soluble state by binding to chaperones such as the er hsp70 ( kar2 in yeast and bip in mammals ) , which are essential for the folding of newly synthesized polypeptides as well as for the disposal of misfolded proteins .
however , chaperones on their own do not appear to determine the fate of their clients . instead , recognition factors that are part of the e3 ligase complexes play a major role in erad substrate selection .
for example , the recognition of erad - l substrates requires the luminal components of the hrd1 complex hrd3 , kar2 and , in the case of glycosylated substrates , the lectin yos9 ( plemper et al . , 1997 , 1999 ; bhamidipati et al . , 2005
; kim et al . , 2005 ; szathmary et al . , 2005 ; carvalho et al . , 2006 ; denic et al
the yeast derlin der1 , a membrane protein of the hrd1 complex , might also be involved in erad - l substrate recognition ( gauss et al .
, 2006b ; stanley et al . , 2011 ) . moreover , certain erad - m substrates appear to be recognized directly by the e3 ligase hrd1 ( sato et al . , 2009 )
. however , the features recognized on the misfolded proteins by these erad factors are largely unknown .
an informative exception is the recognition of luminal misfolded n - linked glycoproteins in saccharomyces cerevisiae ( fig .
as they enter the er lumen , proteins are often modified at asparagine residues ( in the context of the n - x - s / t sequence ) with a well - defined , branched glycan moiety made up of three glucose , nine mannose , and two n - acetylglucosamine residues , glc3man9glcnac2 ( fig .
early glycan - processing enzymes such as glucosidases lead to the binding of lectins that facilitate the folding of the newly synthesized proteins .
in contrast , late acting enzymes , such as the mannosidase htm1 , trigger the binding of a different lectin that engages the protein in erad ( jakob et al .
this difference in the kinetics of the glycan - trimming enzymes provides an opportunity for newly synthesized proteins to acquire the native conformation and traffic beyond the er ( fig .
2 a ) . a long er residency , indicative of folding problems , results in the processing of the misfolded glycoproteins by htm1 , which generates a biochemical mark ( 1,6-linked mannose ) decoded by the lectin yos9 , an erad substrate recognition factor ( quan et al . , 2008 ; clerc et al . , 2009 )
if properly folded , the proteins leave the er . prolonged residency in the er , indicative of a persistent misfolded domain ( red star ) , leads to htm1-dependent exposure of an -1,6linked mannose residue ( red bar ) .
together , the misfolded domain and the terminal -1,6 mannose form the degradation signal recognized by hrd3/yos9 .
( b ) recognition of native mhc i heavy chain by the cytomegalovirus -encoded us2 adaptor in infected cells .
us2 binds to folded mhc i in the er membrane and delivers it to an e3 ligase complex containing the e3 trc8 and the signal - peptide peptidase spp resulting in mhc i degradation by erad .
( c ) sterol - dependent recognition of hmgr by insigs in mammalian cells . under low sterol levels
high sterol levels , in particular the accumulation of 24,25-dihydrolanosterol ( four - ringed structure in gray ) , cause insig to bind to hmgr and to deliver it to an e3 ligase complex that promotes hmgr degradation by erad .
both yeast htm1 and its mammalian counterpart edem are in complex with oxidoreductases ( pdi1 in yeast , erdj5 in mammals ) , required for the stability of htm1 and also for reducing disulfide bonds in misfolded proteins , which might affect subsequent erad steps ( ushioda et al . , 2008 ; clerc et al . ,
the binding of yos9 to the 1,6-linked mannose is not sufficient to trigger the degradation of the misfolded protein .
this processed glycan must be located in an unstructured polypeptide segment that is bound by hrd3 ( xie et al . , 2009 ) .
the delivery of substrates to hrd3 might be facilitated by the luminal chaperone kar2 that is essential for erad ( plemper et al .
xie et al . , 2009 ) . the dual recognition of a specific n - linked glycan by yos9 and an unstructured segment by hrd3
likely enhances the stringency of erad substrate recognition , perhaps by a kinetic proofreading mechanism ( fig .
the recognition mechanism of misfolded luminal n - linked glycoproteins is likely similar in mammalian cells because the yeast components are largely conserved in higher eukaryotes ( table 1 ) .
os-9 and xtp3-b , the mammalian homologues of yos9 , use their glycan - binding domain not only to interact with glycans in the misfolded protein but also with sel1 , the mammalian version of hrd3 , which is itself a glycoprotein ( christianson et al . , 2008 ) .
whether the interactions with sel1 and the substrates occur sequentially or correspond to different pools of os-9/xtp3-b is unclear . in either case ,
using the same domain to interact with a component and a substrate offers os-9/xtp3-b an additional mechanism to regulate recognition of n - glycosylated erad substrates .
recent work in yeast suggests that a different type of glycosylation , o - mannosylation , plays an important role in removing certain luminal proteins from futile folding cycles and thus favoring their degradation by erad after prolonged residency in the er ( goder and melero , 2011 ; xu et al . , 2013 ) .
the enzymes involved in protein o - mannosylation physically associate with erad machinery , but how o - mannosylated proteins are captured by the erad components is still unclear ( goder and melero , 2011 ) .
therefore , o - mannosylation is another appealing mechanism for generating an irreversible biochemical mark on proteins displaying folding problems .
a common feature between erad substrate recognition by n - glycan trimming and o - mannosylating enzymes is that both appear to be slow processes , requiring substrates to stay for relatively prolonged periods in the er . whether other mechanisms involved in recognition of misfolded proteins by erad also require a lag period in the er is not known .
nevertheless , it is curious that newly synthesized proteins were shown to be protected from degradation for a period of time , even under conditions that favor their misfolding ( vabulas and hartl , 2005 ) .
therefore , recognition of misfolded proteins might have evolved as an intrinsically slow process , perhaps to spare some folding intermediates from prematurely engaging in erad .
the degradation of specific folded proteins by erad is mediated by the same general machinery , but the recognition of these substrates involves distinct factors . a simple mechanism to target a native protein to erad is by a substrate - specific adaptor . for example , the human cytomegalovirus encodes er membrane adaptor proteins , us2 and us11 , which bind independently to newly synthesized mhc i molecules and deliver them to erad components for degradation . as a consequence , infected cells display less mhc i complex at their surface and escape detection by the immune system ( wiertz et al .
us11 uses its transmembrane domain to recruit mhc i into a complex which contains derlin-1 as well as sel1l , the p97 atpase complex and its membrane adaptor ubxd8 ( lilley and ploegh , 2004 ; ye et al . , 2004 ; mueller et al . , 2008
intriguingly , the e3 ligase required for us11-mediated mhc i degradation is not known and both hrd1 and gp78 e3 ligases , which are found in complex with derlin-1 , appear to be dispensable .
us2 , on the other hand , delivers its substrate to the erad ligase complex containing the e3 trc8 and spp , the signal peptide peptidase ( fig .
the precise function of spp in erad is not known and it is not even clear whether it involves its proteolytic activity . despite these differences , both us2 and us11 act as adaptors to deliver a specific erad substrate , mhc i heavy chain , to the e3 ligase complexes that promote its degradation .
a similar mechanism targets cd4 for degradation in cells expressing the hiv - encoded er membrane protein vpu . in this case
, vpu works not only as the substrate adaptor for cd4 but also as a scaffold to recruit a cytosolic e3 ligase complex , scf , required for cd4 ubiquitination ( fujita et al . , 1997 ; schubert et al . ,
1998 ) . in vitro reconstitution of vpu - mediated cd4 ubiquitination revealed that the specificity of adaptor - mediated substrate selection can be further increased at the level of substrate ubiquitination , which can be counteracted by the activity of de - ubiquitinating enzymes ( zhang et al . , 2013 ) .
the strategy of using a substrate - specific adaptor is not exclusive to viral encoded proteins . both in drosophila and in mammalian cells , the derlin - related irhom proteins function as adaptors in the erad - mediated degradation of egfr ligands as they traffic through the er ( zettl et al . , 2011 ) .
elegant genetic experiments in flies showed that this mode of regulated erad was important to control sleeping behavior that requires egfr signaling ( zettl et al . , 2011 ) .
it is likely that more substrate - specific adaptors will be identified as our knowledge of the mechanisms of regulated erad expands .
a substrate - specific adaptor also functions in the regulated erad of hmgr , a key enzyme of the sterol biosynthetic pathway . in this case the adaptor , either insig-1 or insig-2 , does not bind constitutively to hmgr ( song et al . ,
instead , the interaction only occurs in the presence of 24,25-dihydrolanosterol , an intermediate metabolite in sterol biosynthesis . under low sterols levels hmgr
2 c ) . on the other hand , high sterol synthesis leads to a rise in 24,25-dihydrolanosterol concentration , which favors the binding of hmgr to one of the insig proteins , its delivery to an e3 ligase complex , and consequently its degradation by the proteasome ( fig .
2 c ) . whereas gp78 and the trc8 were originally implicated in hmgr regulated erad
, recent data suggest that additional e3 ligases might also be involved ( song et al . , 2005 ;
degradation of hmgr by erad results in reduced flux through the sterol biosynthetic pathway and reestablishment of membrane lipid homeostasis .
interestingly , insig-1 ( but not insig-2 ) is itself subjected to reciprocal sterol - regulated erad ( lee et al . , 2006 ) .
conversely , if sterol levels are high insig-1 binds to scap , another key er membrane protein required for sterol homeostasis , leading to a much longer insig-1 half - life .
these data illustrate the complex interplay between the opposing effects of sterols on the stability of the hmgr enzyme and one of its adaptors for regulated degradation by erad . in yeast ,
sterol homeostasis also involves negative feedback of an hmgr homologue , hmg2 ( hampton et al . ,
is controlled by the insig - like proteins nsg1 and nsg2 and requires the e3 ligase hrd1 ( bays et al .
in fact , hrd1 was originally identified in a genetic screen for mutants defective in hmgr degradation ( hrd genes ; hampton et al . ,
the binding of hmg2 to nsg1 is also modulated by sterol levels ( theesfeld and hampton , 2013 ) .
however , in contrast to mammalian cells , the binding of nsg1 promotes hmg2 stability , indicating that the recognition of this substrate is mechanistically different in the two systems ( flury et al . , 2005 ;
, it has been proposed that nsg1 and nsg2 work as hmg2-specific chaperones and that in their absence hmg2 presents sufficient conformation instability to engage in erad as a misfolded protein ( flury et al . , 2005 ;
this degradation is further accelerated by high concentrations of an early sterol - intermediate metabolite , geranylgeranyl pyrophosphate ( theesfeld and hampton , 2013 ) .
therefore , a change in the affinity to a binding partner is another strategy to target a protein for erad in a signal - dependent manner .
squalene monooxygenase ( sqle ) , another enzyme of the sterol biosynthetic pathway , is also targeted by regulated erad both in yeast and in mammals ( foresti et al . , 2013 ) .
sqle degradation requires the yeast doa10 e3 ligase complex or its mammalian homologue teb4 , indicating that two independent branches of erad control distinct steps in sterol biosynthesis ( foresti et al . , 2013 ) .
although the mechanism for the recognition of sqle by erad machinery is still not known , it is clear that insigs are dispensable for this recognition , both in mammals and in yeast ( gill et al .
the n - terminal domain of sqle is necessary and sufficient for cholesterol - dependent degradation ( gill et al . , 2011 ) .
whether this domain binds directly to cholesterol or interacts with an erad - specific adaptor is not known .
the mechanism for sqle recognition by erad in yeast is likely to be different because this n - terminal domain is only conserved among certain animals .
based on these few examples , it is clear that signal - mediated erad depends on the ability of cells to sense the concentration of some lipids in their membranes and on specific adaptors to selectively degrade key enzymes .
our knowledge on the mechanisms by which other classes of regulated erad substrates are recognized will grow as more of these are identified .
after being selected , erad substrates are retrotranslocated across the er membrane back into the cytoplasm . in the case of misfolded luminal proteins the complete polypeptide needs
to be retrotranslocated , whereas for membrane substrates this step requires the transport of only certain domains .
as a consequence , ubiquitination of luminal substrates only occurs at late stages of retrotranslocation , once a portion of the substrate has been exposed to the cytoplasm .
in contrast , ubiquitination of most , but not all ( burr et al . , 2013 ) , membrane substrates is coupled to their retrotranslocation . in analogy to the transport of newly synthesized proteins into the er or mitochondria , it has been postulated that retrotranslocation occurs through a protein - conducting channel
. however , the identity of the retrotranslocation channel has been at the center of an intense debate that is almost as old as the research in this field . over the years , several channel candidates have been proposed but it has been difficult to gather definitive evidence in support of any of them .
the sec61 translocon used for protein import into the er was the first proposed retrotranslocation channel ( wiertz et al . , 1996b ) .
sec61 was found to interact with erad substrates both in yeast and in mammalian cells as well as with the yeast proteasome ( wiertz et al .
, 1996b ; kalies et al . , 2005 ; scott and schekman , 2008 ) . however , the significance of these associations is not clear .
recent work showed that proteins engaging the sec61 translocon aberrantly or persistently in their way into the er become substrates of the hrd1 ligase complex , which might explain the interaction between sec61 and some erad substrates ( rubenstein et al . , 2012 ) .
in addition , certain yeast sec61 mutants displayed defects in degrading model erad substrates , even under conditions in which general  forward  translocation appeared not to be dramatically affected ( pilon et al . , 1997 ;
it remains to be determined whether this phenotype is caused by a specific impairment in retrotranslocation .
the e3 ligase complexes interact with erad substrates immediately before and after their retrotranslocation , indicating this step occurs in their immediate vicinity . therefore , multispanning membrane proteins within the e3 ligase complexes have also been seen as good candidates to mediate retrotranslocation ( ye et al . , 2001 ; lilley and ploegh , 2004 ; kreft et al . , 2006 ; horn et al . , 2009 ; carvalho et al . , 2010 ;
these include the e3 ligases themselves as well as proteins of the derlin family ( der1 in yeast ) , which are essential for the degradation of all luminal erad substrates but whose function has remained elusive . in vitro experiments using mammalian - derived microsomes loaded with the yeast erad substrate mutant pro--factor indicate that derlin might be involved in retrotranslocation ( wahlman et al . , 2007 ) . in this simplified system , in which synthesis and retrotranslocation were uncoupled , the substrate cross - linked to derlin but not to sec61 .
moreover , its retrotranslocation was blocked by anti - derlin antibodies whereas antibodies directed to sec61 had no effect ( wahlman et al . , 2007 ) .
it should be noted that mutant pro--factor is a noncanonical substrate because its retrotranslocation does not require ubiquitination .
interactions between the yeast der1 and the prototype erad - l substrate cpy * were also detected by site - specific photocrosslinking in yeast ( carvalho et al .
these cross - links were seen even in cells lacking the substrate recognition factors hrd3 and yos9 , and were increased if conserved polar residues in the membrane domain of der1 were mutated .
an interpretation of these results is that der1 mediates the transfer of substrates from the recognition factors hrd3/yos9 to the hrd1 ligase inside the membrane ( mehnert et al . , 2014 ) .
a compelling piece of evidence for a function during substrate retrotranslocation was reported for the e3 ligase hrd1 ( fig .
although commonly working in the context of the hrd1 complex , overexpressed hrd1 can mediate the degradation of erad - l substrates even in the absence of its membrane partners hrd3 , der1 , and usa1 ( plemper et al .
hrd1 selectivity for misfolded proteins is lost , suggesting that the other subunits of the complex are critical to control hrd1 activity and substrate specificity ( denic et al . , 2006 ) .
hrd1 interacts with a sizeable region of a modified version of cpy * during the early stages of retrotranslocation , as assayed by site - specific photocrosslinking ( carvalho et al . ,
importantly , the interaction likely occurs inside of the er bilayer because it is lost if substrate recognition is blocked or if the transmembrane segments of hrd1 are mutated .
hrd1 contains only six transmembrane segments ; therefore , erad - l substrate retrotranslocation requires hrd1 oligomerization , which normally is facilitated by usa1 but can occur spontaneously upon hrd1 overexpression ( horn et al . , 2009 ;
all these data make a strong case for a direct role of hrd1 in the retrotranslocation of erad - l substrates , but the possibility that it works with some partner(s ) , such as der1 , sec61 , or other unknown factors can not be excluded at this point .
moreover , it is not known whether this is a unique feature of hrd1 or whether the membrane domains of other e3 ligases also participate in the retrotranslocation of other classes of erad substrates . a working model for hrd1-mediated retrotranslocation of a luminal misfolded glycoprotein .
upon recognition ( not depicted ) , the misfolded protein ( gray ) is transferred to hrd1 .
the binding can occur either to hrd1 monomers or to usa1-mediated hrd1 dimers ( 1 ) .
substrate - bound hrd1 dimer self - ubiquitinates ( 2 ) , which leads to the recruitment of the cdc48 atpase complex .
atp hydrolysis by cdc48 induces a conformational change in hrd1 dimer that facilitates the early stages of substrate retrotranslocation ( 3 ) .
once exposed to the cytoplasm , the substrate is ubiquitinated by hrd1 and recognized by the cdc48 complex ( 4 ) , which uses its atpase activity to complete substrate retrotranslocation ( 5 ) .
after retrotranslocation , the ubiquitinated substrate is released in the cytosol for degradation by the proteasome ( 6 ) .
ubiquitin is depicted as small circles . in most of these cross - linking experiments the retrotranslocation of cpy
* was dampened by fusing it to a very tightly folded domain , indicating that erad - l substrates need to be unfolded before this step ( bhamidipati et al .
2010 ) . whether unfolding is also a prerequisite for the retrotranslocation of other classes of erad substrates is not settled yet ( fiebiger et al . , 2002 ; tirosh et al . ,
, substrates are ubiquitinated , a modification that allows their recognition by the cdc48/p97 atpase complex composed of a homohexamer of cdc48/p97 and by the cofactors ufd1 and npl4 ( bays et al . , 2001b ; ye et al . , 2001 , 2003 ;
the recruitment of this atpase complex to the er membrane is facilitated by ubiquitin regulatory x ( ubx ) domain  containing proteins , ubx2 in yeast and ubxd8 in mammals ( neuber et al .
, 2005 ; schuberth and buchberger , 2005 ; mueller et al . , 2008 ) .
the atpase activity of the cdc48/p97 complex provides the driving force to move ubiquitinated substrates out of the membrane into the cytosol ( ye et al . , 2003 ) .
although the role of cdc48/p97 in this process is well established , the mechanism that couples atp hydrolysis to membrane extraction of the substrate is still not understood .
in addition to the cdc48/p97 complex , the atpase subunits of the proteasome regulatory particle were also shown to play a role in retrotranslocation of some erad substrates ( lipson et al . , 2008 ) .
the driving force for the retrotranslocation of the few non - ubiquitinated substrates , like the cholera toxin or pro--factor , is not known ( kothe et al . , 2005 ;
the cdc48/p97 complex also serves as a platform for other ubiquitin - modifying enzymes such as de - ubiquitinating enzymes ( dubs ; rumpf and jentsch , 2006 ; jentsch and rumpf , 2007 ; ernst et al .
although the role of some of these cdc48/p97-binding factors is not clear yet , it was shown that interfering with the dubs yod1 and ataxin-3 affected substrate retrotranslocation ( wang et al . , 2006 ; ernst et al . , 2009 ) .
the requirement for dub activity during retrotranslocation suggests that the process involves cycles of ubiquitination and de - ubiquitination .
in some cases these cycles might be important to increase the specificity of substrate recognition , as was shown for the vpu - mediated degradation of cd4 ( zhang et al . , 2013 ) .
interestingly , the retrotranslocation of some noncanonical substrates like cholera toxin , which are not ubiquitinated , is also affected by manipulation of both e3 ligase and dub activities ( hassink et al . , 2006 ;
these results suggest that retrotranslocation requires the ubiquitination of some factors other than the substrates .
future studies should test some obvious candidates such as the components of the e3 ligase complexes themselves .
there is some recent evidence that the yeast cdc48 complex might be acting also much earlier , during the initial stages of retrotranslocation of an erad - l substrate , before it is exposed to the cytoplasm ( fig .
3 ) . using a cross - linking strategy , it was shown that the interaction between hrd1 and an early retrotranslocation intermediate was lost in mutants of the cdc48 complex ( carvalho et al . , 2010 ) .
interestingly , a similar defect was detected in hrd1 mutants impaired for e3 ligase activity ( carvalho et al . ,
it was proposed that in early stages of erad - l substrate retrotranslocation , hrd1 induces the ubiquitination of an erad component , perhaps hrd1 itself .
this ubiquitination signals the recruitment of the cdc48 complex , which upon atp hydrolysis would induce changes in the conformation or in the oligomeric status of hrd1 , resulting in substrate retrotranslocation .
this model is consistent with the well - established role of cdc48/p97 in the disassembly and remodeling of protein complexes ( jentsch and rumpf , 2007 ) .
once the substrate emerges on the cytosolic side and is ubiquitinated by hrd1 , the atpase complex binds to it and promotes the final stages of its retrotranslocation .
although many aspects of this model still wait for experimental support , it would provide a unifying role for the cdc48/p97 atpase as the driving force for substrate retrotranslocation in erad ( fig .
substrates are kept soluble and transferred to the proteasome by cytosolic chaperones such as the bag6 complex ( claessen and ploegh , 2011 ; wang et al . , 2011 ; xu et al . , 2012 ) or shuttle factors like rad23 and dsk2 ( medicherla et al . , 2004 ) .
the identification of most of the components involved in this process and how these are pieced together and organized in the different erad branches were important achievements .
a major challenge for the future is to reveal the mechanistic aspects of the pathway .
such developments should , for example , help in discerning the basis by which misfolded proteins are recognized in each of the different erad branches
. the mechanisms of substrate retrotranslocation and the roles played by the different components , such as the e3 ligases and the cdc48/p97 complex , will certainly be another interesting area to follow .
however , progress on these topics might require the development of new approaches , such as in vitro systems with purified components recapitulating individual erad steps .
in early days , erad was perceived as a process mainly dedicated to er protein quality control .
the picture now emerging places erad closer to other ubiquitination systems in the cytoplasm and nucleus , which control the turnover of specific proteins to achieve a certain physiological state .
therefore , another major challenge for the coming years is the detailed characterization of the roles of erad beyond quality control .
although the central role of erad in sterol homeostasis is unequivocal , it will be important to clarify whether erad has a more general role in the regulated degradation of folded er proteins and in that way modulates other er - related functions .
a systematic and rigorous identification of regulated erad substrates should help in addressing these issues .
uncovering the intersections of erad with other cellular pathways will provide important insights into the mechanisms of er and cellular homeostasis .
 even with the assistance of many cellular factors , a significant fraction of newly synthesized proteins ends up misfolded . 
 cells evolved protein quality control systems to ensure that these potentially toxic species are detected and eliminated . 
 the best characterized of these pathways , the er - associated protein degradation ( erad ) , monitors the folding of membrane and secretory proteins whose biogenesis takes place in the endoplasmic reticulum ( er ) . 
 there is also increasing evidence that erad controls other er - related functions through regulated degradation of certain folded er proteins , further highlighting the role of erad in cellular homeostasis . 