Specificity and Regulation of the Endoplasmic Reticulum-Associated Degradation Machinery

Jessica Merulla1,2, Elisa Fasana1, Tatiana Soldà1 and Maurizio Molinari1,3*

1Institute for Research in Biomedicine, Protein Folding and Quality Control,

CH-6500 Bellinzona, Switzerland 2Graduate School for Cellular and Biomedical Sciences, University of Bern, Switzerland

3Ecole Polytechnique Fédérale de Lausanne, School of Life Sciences, CH-1015 Lausanne,

Switzerland

Correspondence to: maurizio.molinari@irb.usi.ch

Keywords Chemical and pharmacological chaperones Conformational diseases Defective ribosomal products (DRiPS) Endoplasmic reticulum ER-associated degradation (ERAD) ERAD tuning ER stress Hijacking by pathogens Misfolded proteins Proteostasis Ubiquitin proteasome system Unfolded protein response (UPR)

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/tra.12068

© 2013 John Wiley & Sons A/S

Abstarct

The endoplasmic reticulum-associated degradation (ERAD) machinery selects native and misfolded polypeptides

for dislocation across the ER membrane and proteasomal degradation. Regulated degradation of native proteins

is an important aspect of cell physiology. For example, it contributes to the control of lipid biosynthesis, calcium

homeostasis and ERAD capacity by setting the turnover rate of crucial regulators of these pathways. In contrast,

degradation of native proteins has pathologic relevance when caused by viral or bacterial infections, or when it

occurs as a consequence of dysregulated ERAD activity. The efficient disposal of misfolded proteins prevents

toxic depositions and persistent sequestration of molecular chaperones that could induce cellular stress and

perturb maintenance of cellular proteostasis.

In the first section of this review, we survey the available literature on mechanisms of selection of native and non-

native proteins for degradation from the ER and on how pathogens hijack them. In the second section, we

highlight the mechanisms of ERAD activity adaptation to changes in the ER environment with a particular

emphasis on the post-translational regulatory mechanisms collectively defined as ERAD tuning.

Introduction The cellular proteome is mostly synthesized by

cytosolic ribosomes to operate in the cytosol and,

upon appropriate targeting, in various intracellular

organelles or in the extracellular space. Cellular

compartments where protein folding occurs (e.g.,

the cytosol, mitochondria, the endoplasmic

reticulum (ER)) contain two classes of non-native

polypeptides: i) newly synthesized polypeptide

chains that must be assisted by folding chaperones

and enzymes to attain the native mono- or

oligomeric structure; ii) terminally misfolded

conformers that must efficiently be degraded to

prevent the formation of toxic deposits and the

persistent sequestration of chaperones that could

eventually inhibit the cellular protein folding capacity

and elicit stress (1-3) (Fig. 1A). The distinction

between the two classes of non-native chains, one

to be preserved, the second to be cleared from the

folding compartment is not an easy task for the

cellular quality control machineries. Selection for

disposal might be a stochastic process: the longer

the persistency of structural defects in the ER, the

greater is the probability to be selected for

destruction. Therefore, mutations that delay folding

may channel the polypeptide into destructive

pathways, even if they do not compromise the

function of the mutated protein.

In the ER, non-native, but also native proteins are

selected for degradation by components of the

ERAD machinery that deliver them at dislocation

sites embedded in the ER membrane. Dislocation

sites consist of a multitude of luminal and

membrane-bound specialized ER-resident proteins

as well as a number of cytosolic factors insuring

dislocation across the ER membrane, poly-

ubiquitylation and disposal of ERAD substrates by

26S proteasomes (Section A, Specificity of the

ERAD machinery). Dysregulated ERAD activity

caused by excessive luminal content of ERAD

factors may result in premature interruption of

ongoing folding attempts where proteins that would

have attained functional structures are

inappropriately destroyed thus causing loss-of-

function phenotypes or disorders (Fig. 1B). All in all,

balanced activity of the degradation machinery

crucially contributes to maintenance of protein

homeostasis (proteostasis) (Section B, Regulation

of the ERAD activity).

A. Specificity of the ERAD machinery

ERAD of native proteins in healthy cells: select

examples

The regulated degradation of functional proteins is a

crucial aspect of cellular physiology and contributes,

for example, to set the level of lipid biosynthesis, to

adjust calcium homeostasis, to determine the

constitutive level of the ERAD activity and to adapt

it to variations in misfolded proteins load (4, 5).

The ERAD machinery controls the intracellular

content of the 3-hydroxy-3-methyl-glutaryl-CoA

reductase (HMG-CoA reductase), a rate-limiting

enzyme in the synthesis of sterols from acetyl-CoA.

The HMG-CoA reductase turnover is enhanced

when the cellular sterol level is high, a classical

example of feedback regulation (6-8). Likewise,

ERAD factors may select the lipid carrier ApoB (9,

10) or the IP3P receptor (11) for destruction when

lipid levels are low or when cytosolic levels of IP3

and calcium are high, respectively.

Poorly studied, but crucial to maintain cellular

proteostasis, are the constitutive and the regulated

degradation of ERAD factors (5). ERAD factors

turnover may directly be affected by the presence of

misfolded polypeptides in the ER lumen and

contributes in determining the rapid adaptation of

the overall ERAD activity to changes in the ER

folding environment as described in Section B.

Degradation of native proteins triggered by

pathogens: select examples

Viruses and bacteria may dysregulate ERAD

pathways of infected cells to enhance their chances

to survive in the host, to generate their progeny and

to escape immunosurveillance (12, 13). This

highlights the impact that ERAD modulation may

have on cellular physiology and on protein

production.

The human cytomegalovirus (HCMV): One of the

most revealing examples of manipulation of the

equilibrium existing in eukaryotic cells between

maturation and disposal of newly synthesized

polypeptides relates to cells infected with HCMV.

These cells produce the viral proteins US2 and

US11 that select for disposal newly synthesized

class I or class II MHC (14-16). The intervention of

US2 and US11 anticipates the assembly of

complexes that bind viral epitopes in the ER lumen

and transport them at the cell surface for activation

of the host immune response. In infected cells,

class I and class II molecules are cleared from the

ER with intervention of conventional ERAD factors

(thoroughly described below) such as SEL1L, derlin

1, VIMP, p97/VCP and the E3 ubiquitin ligase TRC8

(17-19). Infected cells may also express the viral

protein Pp65 to inhibit proteasomal activity (20), a

strategy that HMCV shares with several other

viruses (Epstein-Barr virus (21), human

immunodeficiency virus (HIV) (22), hepatitis B virus

(HBV) (23), Kaposi sarcoma herpes virus (24)) to

elude immunosurveillance.

HIV: Cells infected with HIV express the viral

protein Vpu causing the proteasome-mediated

degradation of the viral receptor CD4 thereby

preventing superinfection (25-28).

The murine γ-Herpes virus 68 and the rodent

herpes virus Peru (RHVP): The expression of viral

E3 ubiquitin ligases is yet another strategy used by

pathogens to specifically clear host cell proteins that

could activate the immune response. The viral E3

ligase mK3 expressed in cells infected with murine

γ-Herpes virus 68 binds to and uses the

TAP/Tapasin complex as an adaptor to specifically

polyubiquitylate and destroy the class I MHC heavy

chain (29-32). Interestingly, the ubiquitin ligase

activity of mK3 is directed to serine and threonine in

addition to the conventional lysine residues (33).

The viral pK3 expressed in cells infected with RHVP

promotes proteasome-mediated degradation of the

class I MHC heavy chain (34).

The hepatitis C (HCV) and HBV viruses: Another

example of manipulation of the host cell ERAD

activity during the viral life cycle is reported for cells

infected with HCV (35) or HBV (36). ERAD

enhancement resulting from the ER stress-induced

up-regulation of proteins of the EDEM family (37)

reduces the fraction of newly synthesized viral

glycoproteins able to attain the native structure in

due time. Consistent with data showing that ERAD

enhancement interferes with completion of ongoing

folding programs (38-41), folding and assembly of

viral spikes becomes inefficient. This is proposed to

maintain low production of viral particles thus

contributing to infection persistency.

Degradation of misfolded proteins: specificity of

the ERAD machinery

Genomic defects, transcriptional and translational

errors may alter the amino acid composition or the

length of the polypeptide sequence. This may

prevent attainment of the native structure or delay

the folding process such that selection for disposal

of the mutated chains precedes completion of the

folding program. Intra- or extracellular accumulation

of folding-defective polypeptides causes gain-of-

toxic-function diseases, for example hereditary lung

emphysema and liver disease caused by mutant

forms of α1-antitrypsin or many types of

neurodegenerative disorders. On the other hand,

clearance of the aberrant gene products can result

in loss-of-function diseases, for example cystic

fibrosis or lysosomal storage diseases (thoroughly

reviewed elsewhere (42)). The development of

appropriate therapeutic approaches to contrast the

onset and the course of conformational (or protein

misfolding) diseases is of great interest. Such

diseases can individually be treated with

pharmacologic chaperones that specifically

enhance the folding-efficiency of a mutated gene

product (43). Alternatively, loss-of-function defects

could be rescued by targeting with therapeutic

agents the ER quality control machinery so that, as

one example, mutated polypeptides are offered

wider time windows to eventually attain the native

structure. Such approaches would greatly benefit of

the capacity to predict for each disease-causing

polypeptide, which chaperones and pathways will

be engaged for ER-retention, quality control and/or

disposal.

The presence of N-linked oligosaccharides

The oligosaccharyl transferase complex located at

the entry site of the ER transfers pre-assembled

glucose3-mannose9-N-acetyl glucosamine2-

oligosaccharides onto nascent polypeptide chains

entering the ER lumen (44). Normally, two terminal

glucose residues are rapidly removed by the

sequential intervention of the α-glucosidases I and

II. This generates a mono-glucosylated protein-

bound oligosaccharide that engages the lectin

chaperones calnexin and calreticulin and exposes

newly synthesized polypeptides to a folding

environment comprising the oxidoreductase ERp57

and the peptidyl-prolyl isomerase CypB. ERp57

promotes formation of the native set of disulfide

bonds, CypB the appropriate structuring of peptidyl-

prolyl bonds in the cis or in the trans configuration

(44).

Some nascent polypeptides rapidly enter off-

pathways of the folding program. This may inhibit

the action of the α-glucosidase II and results in the

inappropriate persistence of the di-glucosylated

form of protein-bound oligosaccharides. Aberrantly

structured newly synthesized polypeptides

displaying di-glucosylated oligosaccharides are

sequestered by a recently characterized membrane

anchored ER lectin, malectin (45). Malectin plays a

crucial role in retention of folding-defective

glycoproteins in the ER and acts in concert with

ribophorin I, a subunit of the ER glycosylation

machinery with proposed molecular chaperone

function (46, 47).

Persistent ER retention of misfolded conformers

elicits intervention of ER-resident mannosidases of

the glycosyl hydrolase 47 family (48) comprising the

ERManI, EDEM1, EDEM2, EDEM3. These directly

or indirectly enhance removal of α1,2-bonded

mannose residues (49-57) to extract misfolded

polypeptides from the calnexin chaperone system

(58) and to generate disposal signals decoded by

mannose-binding ERAD lectins such as OS-9 and

XTP3-B variants (50, 51, 59-62). The ERAD lectins,

which are interchangeable in function at least for

soluble ERAD substrates (ERAD-LS) (63), shuttle

misfolded polypeptides from the ER lumen to

dislocation sites in the ER membrane (Fig. 2). Both

the mannose-cleaving and the mannose-binding

ERAD factors also bind non-glycosylated proteins

(60, 61, 64-66) possibly as components of

supramolecular complexes containing general

chaperones such as BiP and GRP94 that direct

their action towards terminally misfolded proteins

(67).

The position of the folding defect and the

topology of the misfolded protein

E3 ubiquitin ligases embedded in the ER membrane

participate in supramolecular complexes containing

membrane-anchored and peripherally associated

factors both at the luminal and at the cytosolic side

of the bilayer. These multimeric complexes, the

dislocons, deliver misfolded proteins from the ER

lumen into the cytosol for proteasome-mediated

degradation (Fig. 2).

In S. cerevisiae, dislocons are built around two E3

ubiquitin ligases, Hrd1p and Doa10p. The luminal

(ERAD-L), transmembrane (ERAD-M) or cytosolic

(ERAD-C) position of the folding defect may

determine the engagement of either one of them.

Hrd1p preferentially assists ERAD-L and ERAD-M

substrates (68-72); Doa10p assists ERAD-C

substrates (68, 69, 73). The two yeast E3 ligases

have redundant function in assisting polytopic,

folding-defective polypeptides such as the ΔF508

cystic fibrosis transmembrane regulator (CFTR)

(74).

The mammalian ER membrane contains at least 24

RING-finger E3 ubiquitin ligases (75), some of

which (e.g., HRD1, gp78, RNF5, TEB4, RFP2,

TRC8) have a documented role in ERAD (76). The

ERAD system in Metazoa is therefore much more

complex and redundant than in Fungi. Only very

few attempts have been performed to systematically

establish how changes in the biophysical features of

misfolded polypeptides determine the engagement

of specific dislocation factors. For example,

misfolded luminal modules (ERAD-LS substrates)

strongly depend on the supramolecular complex

built around HRD1, comprising the associated

adaptor SEL1L and the two interchangeable ERAD

lectins OS-9 and XTP3-B that deliver the misfolded

polypeptides from the ER lumen to the dislocation

site. The same misfolded modules tethered at the

ER membrane (ERAD-LM substrates) are efficiently

cleared from the ER even in cells with non-

functional HRD1 dislocons, possibly because lateral

diffusion in the lipid bilayer facilitates delivery to

alternative machineries (Fig. 2) (63, 77).

The disposal of orphan/misfolded membrane

proteins: select examples

The study of the fate of orphan subunits of

oligomeric complexes is of historical importance.

Klausner and colleagues crucially reported that

unassembled subunits of the T cell receptor (TCR)

are degraded in a pre-lysosomal compartment

being part or closely related to the ER (78). Since

then, several TCR subunits, namely TCRα, TCRβ

and CD3δ have become model substrates to

investigate the mechanisms of protein disposal from

the ER (79-86). More recently, CD147 a component

of several membrane transporters, has been

identified as an endogenous ERAD substrate

whose turnover may regulate expression of the

corresponding functional complexes (87). The

peculiarity of all these proteins is the presence of

charged residues in the intramembrane space.

These residues promote the assembly of functional

complexes by pairing with residues of opposite

charge present in the transmembrane domains of

partner polypeptides in oligomeric complexes.

When oligomerization fails, the intramembrane

domains displaying charged residues might

facilitate the rapid degradation of the orphan

subunits by maintaining them in a monomeric state

and by recruiting specific ERAD factors (86, 88-90).

They may also serve, in some specific case, as

intramembrane cleavage sites that may facilitate

extraction from the ER membrane of the orphan

subunit to be degraded (91). TCRα and CD3δ are

both type I glycopolypeptides with charged residues

in the intramembrane space. Yet, their requirements

for efficient degradation differ quite extensively, thus

showing how difficult it may be to predict factors

and pathways engaged during specific polypeptides

quality control. For instance, only degradation of

CD3δ requires extensive N-glycans de-

mannosylation and only the cytosolic dislocation of

CD3δ is coupled with proteasomal activity (82).

Significantly, the cytosolic domain of TCRα is poly-

ubiquitylated on serine residues (85), rather than on

the more conventional lysine residues. Serine,

cysteine and threonine residues as well as the

polypeptide N-terminus are alternative ubiquitin

acceptor sites (33, 92-94).

The evolution of transmembrane domains

displaying destabilizing charged residues is one of

the strategies developed by viruses to co-opt the

mammalian dislocation machinery during the

infection cycle. This has been reported for simian

virus 40, where a charged residue in the

membrane-inserted domain of the VP2 protein,

which is conserved in several other polyomaviruses,

engages the ERAD factor BAP31 to possibly

promote dislocation of the entire virion across the

ER membrane (90).

The multispanning CFTR protein has also

extensively been described. It is characterized by

low folding-efficiency, about 25%, which is further

decreased upon gene mutations (95-97). The

ΔF508CFTR mutant form was the first reported

example of involvement of cytosolic programs in the

clearance of folding-defective polypeptides from the

mammalian ER (98, 99). This cystic fibrosis-causing

defective gene product is a paradigm for all mutant

proteins that maintain their function (100), yet are

rapidly destroyed from the ER (98, 99) or from the

cell surface (101) because structurally unstable.

This inevitably results in a loss-of-function protein

folding disease. Such mutants, or the quality control

pathways that they engage, are or may become

targets of therapeutic intervention based on the use

of chemical or pharmacologic chaperones as well

as proteostasis modifiers (95, 102). This alone

justifies the importance of deciphering the

mechanisms regulating protein quality control at the

molecular level.

The needs for unfolding

Many models (and at least as many candidates for

a channel in the ER membrane (103-105)) have

been proposed to explain how misfolded

polypeptides, tightly folded domains, catalytic

subunits of AB toxins (e.g., ricin, cholera, Shiga-

like) and even entire viral particles are dislocated

across the ER membrane. This remains poorly

understood but it relies on the intervention of

several luminal, cytosolic and membrane bound

factors including BiP, BAP31 and BAP29 (90, 106),

EDEM1 (107, 108), SEL1L (109), derlins (110-112),

HRD1 (113, 114), gp78 (114), p97/VCP (115, 116)

and many others.

Seemingly contrasting data are available on the

requirement for polypeptides unfolding to facilitate

transport across the lipid bilayer. For example, it

has been shown that the tightly folded DHFR

protein does not affect dislocation when appended

to class I MHC in US2 and US11-expressing cells

(117, 118). Moreover, compelling data do exist

showing that virions can dislocate across the ER

membrane and that misfolded glycopolypeptides,

which display bulky, highly hydrophilic

oligosaccharides bound to the polypeptide

backbone can efficiently pass the lipid bilayer to be

exported into the cytosol. However, other data imply

that a certain degree of unfolding is required for

efficient dislocation. For example, in contrast to

reference (117), DHFR appended to ERAD

substrates in non-infected cells may substantially

impair dislocation (119). Moreover, several

evidence highlight the involvement of members of

the protein disulfide isomerase (PDI) family in the

preparation of misfolded polypeptides for dislocation

across the ER membrane and in transport across

the bilayer of virions and of catalytic subunits of

toxins. For example, intra- (120) and inter-molecular

disulfide bonds (121) in ERAD substrates as well as

interchain disulfides linking the regulatory and

catalytic subunits of bacterial toxins are reduced

with the intervention of PDI (122-126) and/or PDI

family members (e.g., ERdj5 (127-129), ERp57

(106), ERp72, ERp29 (130), TMX1 (131), ERFAD-

ERp90 (132) and ERO1 (133)).

Recent work from our lab showed that the

conversion of cis into trans peptidyl-prolyl bonds by

the peptidyl-prolyl cis/trans isomerase CyPB may

facilitate transport of misfolded polypeptides

through the elusive transmembrane dislocation

channel by unfolding the polypeptide chain (134).

Finally, a role of rhomboid proteases and

pseudoproteases in ERAD substrates extraction

from the ER has been proposed and relates to their

capacity to cleave intramembrane anchors or to

cause bilayer thinning, respectively (91, 135, 136).

B. Regulation of the ERAD activity

Physiologic and pathologic destruction of

nascent chains

Contrasting data are available on the overall

fraction of nascent chains degraded before

attainment of the native structure, i.e., of defective

ribosomal products (DRiPS). Values range from

more than 30% (137) to substantially less (138).

DRiPs include polypeptides that have failed the

folding program. In this context, individual

mammalian polypeptides are characterized by

specific folding efficiencies (as low as 25% for the

CFTR, approaching the 90% for α1AT (60)). DRiPs

may also derive from defective mRNA or may be

characterized by errors in the primary sequence

that occur when the genetic information is

converted into proteins and most frequently result

from tRNA-aminoacid mis-acylation. If strictly

monitored by quality control programs, the relative

inefficiency of the protein biogenesis pathways is

fully compatible with life as it warrants production of

sufficient amount of functional proteins that keep

cells, tissues and organisms running. It probably

offers evolutionary advantages; DRiPs have been

proposed to provide ligands for class I MHC

presentation, thus enabling the immune system to

rapidly detect alterations in cellular gene expression

possibly caused by pathogen infection or

tumorogenic deviance (139).

A dysregulated ERAD activity may substantially

contribute to shift the physiological inefficiency of

protein folding to a pathological condition. A

constitutive ERAD activity surveys the normal

production of by-products of protein biogenesis

thereby contrasting their toxic accumulation. If the

constitutive ERAD activity is insufficient, folding-

defective polypeptides may remain in the ER lumen

thereby eventually interfering with protein

biogenesis and secretion upon, as one example,

persistent sequestration of folding chaperones (41,

140, 141). In contrast, excessive ERAD activity

would drastically reduce the time allocated to

nascent chains for maturation, thus causing loss-of-

function phenotypes and diseases (19, 34, 35, 38-

40, 142-149).

Understanding ERAD activity regulation is one of

the fascinating questions addressed in several

laboratories working on cellular proteostasis. The

transcriptional/translational induction of ERAD

factors upon activation of unfolded protein response

(UPR) programs is the best studied mechanism

operating in eukaryotic cells to adapt ERAD (and

folding) activity to changes in cellular needs (150).

UPR activation may optimally respond to long-

lasting (chronic) ER stresses. It seems however

inappropriate to respond to transient, acute and/or

periodic fluctuations in ER cargo load. In fact, onset

of these programs has a latency of several hours (2,

151). Moreover, the recovery from a stress requires

activation of elusive mechanisms that must reduce

the size of the ER after the UPR-triggered

expansion and must remove the excess of

chaperones produced during the ER stress phase.

These might be very slow processes because ER

stress-induced chaperones and enzymes such as

PDI family members, calnexin, calreticulin, BiP have

half-lives above the 24 h (152).

We propose that in healthy eukaryotic cells, the

constitutive ERAD that must cope with the

physiological production of by-products of protein

biogenesis without interfering with ongoing folding

programs is controlled by mechanisms collectively

defined as ERAD tuning. These mechanisms

survey the turn-over, the segregation from the ER

and the engagement of ERAD factors in

supramolecular functional complexes (5).

The concept of ERAD tuning

The concept of ERAD tuning is based on three

observations: i) unlike conventional ER-resident

chaperones and enzymes, several ERAD factors

(e.g., ERManI (153, 154), EDEM1 (38, 155-157),

OS-9 (149), XTP3-B (59), HERP (158, 159), SEL1L

(160, 161), the E3 ligases SMURF1 (162, 163) and

gp78 (164, 165), JAMP (166), ataxin-3 (167, 168))

are subjected to fast turnover, which may involve

the ubiquitin proteasome system, autophagy or

autophagy-like pathways; ii) select ERAD factors

are constitutively segregated from the ER (149,

169); iii) luminal expression of misfolded

polypeptides may delay the turnover of ERAD

factors, may retain them in the ER by interfering

with their vesicle-mediated segregation from the

compartment and may directly affect the

composition of supramolecular ERAD complexes

thereby setting their activity (149, 170, 171) (Fig. 3).

The concept of ERAD tuning proposes that

interactions with misfolded proteins synthesized in

the ER directly regulate (tune) the abundance, the

localization and the activity of components of the

ERAD machinery. Misfolded polypeptides may

support the assembly (or inhibit the disassembly) of

active supramolecular ERAD complexes as

reported, for example, for the association of the

adaptor protein SEL1L with the ER membrane-

embedded HRD1 dislocation machinery (149).

Moreover, since E3 ligases may ubiquitylate

themselves (164, 165, 172-174) and other

components of the ERAD machinery that are

engaged with them in supramolecular complexes

(158-160, 164, 165, 168), a possible scenario is that

folding-defective polypeptides serve as preferred

acceptors for the ubiquitylating activity of the E3

ligases thereby protecting the ERAD machinery

from self-destruction (Fig. 3).

The concept of ERAD tuning implies that misfolded

proteins may directly enhance the activity of the

ERAD machinery by a sort of autocrine regulatory

mechanism(s) that controls turnover, localization

and assembly of ERAD components (Fig. 3). In this

scenario, rapid adaptation, i.e., enhancement and

inhibition of ERAD activity, does not require signal

transduction from the ER to the nucleus, as it is the

case when the UPR must be activated. All in all, the

ERAD tuning pathways may offer rapid and readily

reversible adaptation responses to deal with

transient problems that may arise in the folding

compartment.

Interestingly, pathogens such as the mouse

hepatitis virus have been shown to hijack ERAD

tuning pathways to co-opt ER-derived vesicles

containing segregated ERAD factors as platforms

for replication of the viral genome (149, 156).

Conclusion The capacity to distinguish between non-native

polypeptides to be retained in the folding

compartment to complete the maturation program

and non-native polypeptides that must be removed

from the ER to prevent their toxic accumulation is

crucial to maintain cellular proteostasis.

Dysregulated ERAD may lead to inappropriate,

premature selection of folding-competent

conformers for destruction (too high activity) or to

an equally inappropriate and harmful tolerance of

misfolded conformers in the folding-compartment

(too weak activity). As these conditions inevitably

affect maintenance of cellular and organisms

proteostasis, the molecular characterization of the

processes covered by this review is of daunting

importance. What are the mechanisms that

maintain the fragile equilibrium between the activity

of the folding and the degradation machineries

operating in the ER and in other cellular

compartments such as the cytosol and the

mitochondria where not-yet-native proteins must

attain their functional conformations, yet misfolded

conformers must efficiently be cleared? How is this

equilibrium maintained upon the frequent

modifications of the environmental conditions cells

are subjected to? How do cells revert situations of

disequilibrium that may intervene upon pathogen

attack, nutrient deprivation, variations in protein

synthesis levels, transient failure of folding

programs? How do pathogens actually exploit the

host protein folding and degradation machineries to

enhance their survival and replication chances?

How can we intervene to contrast their infection

cycles? These are few of the challenging questions

remaining for future research.

Acknowledgments Members of the Molinari’s lab are acknowledged for

discussions and suggestions. M.M. is supported by

grants from the Foundation for Research on

Neurodegenerative Diseases, the Swiss National

Science Foundation, the Association Française

contre les Myopathies, the Gabriele Foundation.

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Figure Legends

Fig. 1 Role of ERAD in cellular proteostasis. A Nascent polypeptide chains are co-translationally translocated

into the ER. Most nascent chains attain the native structure (step 1) and are exported from the folding

compartment (step 2). A variable percentage of the newly synthesized polypeptides enter off pathways of the

folding programs (step 3) and is removed from the ER by constitutive ERAD (step 4). In healthy cells, folding

intermediates are protected from recognition by ERAD factors and have sufficient time to complete the folding

process. B Dysregulated ERAD as a consequence of elevated luminal levels of ERAD factors may cause

inappropriate disposal of intermediates of ongoing folding programs (step 5) thereby reducing production of

native polypeptides (steps 1 and 2).

Fig. 2 Substrate-specific ERAD pathways. The same misfolded module shows different requirements for

efficient clearance from the ER when anchored to (ERAD-LM substrate) and when detached from the membrane

(ERAD-LS substrate), the latter showing stronger dependency on ERAD lectins and HRD1 dislocons.

Consistently, OS-9 and XTP3-B are depicted in lighter colors for ERAD-LM substrates and red arrows show that

ERAD-LM substrates can diffuse in the lipid bilayer to enter HRD1 or alternative dislocation machineries.

Fig. 3 ERAD regulation by misfolded proteins. A In healthy cells, unused dislocation machineries are

disassembled. E3 ubiquitin ligases may for example poly-ubiquitylate themselves or components of the

supramolecular complexes thereby resulting in their degradation. Orphan components may also be segregated

from the ER lumen as shown by the vesicle-mediated release of SEL1L, EDEM1 and OS-9 (149). B Misfolded

proteins engage ERAD factors thereby preserving ERAD complexes, delaying ERAD factors turnover and

segregation from the ER.