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QUALITY CONTROL IN THE
ENDOPLASMIC RETICULUM
Lars Ellgaard and Ari Helenius
The endoplasmic reticulum (ER) has a quality-control system for ‘proof-reading’ newly
synthesized proteins, so that only native conformers reach their final destinations. Non-native
conformers and incompletely assembled oligomers are retained, and, if misfolded persistently,
they are degraded. As a large fraction of ER-synthesized proteins fail to fold and mature properly,
ER quality control is important for the fidelity of cellular functions. Here, we discuss recent
progress in understanding the conformation-specific sorting of proteins at the level of ER
retention and export.
Institute of Biochemistry,
Swiss Federal Institute of
Technology (ETH) Zürich,
Hönggerberg, CH – 8093
Zürich, Switzerland.
Correspondence to A.H.
e-mail:
[email protected]
doi:10.1038/nrm1052
In the assembly of a complex machine, such as a car,
every essential component must conform to carefully
defined specifications, and is therefore subject to stringent quality control (QC). In the cell something similar
occurs — there are QC systems for practically every
step that leads to the synthesis of DNA, RNA and protein molecules1–5. As a result, the number of accumulated errors in macromolecules that are ultimately
deployed by cells is extremely low.
For proteins, ‘proof-reading’ occurs at the level of
transcription, translation, folding and assembly. To pass
the final QC checkpoints, a protein must typically have
reached a correctly folded conformation. This is generally the so-called ‘native’ conformation that corresponds
to the most energetically favourable state. In the case of
proteins with several subunits, proper oligomeric
assembly is usually necessary.
If the folding and maturation process fails, a protein
molecule is not transported to its final destination in
the cell, and is eventually degraded. To distinguish
between native and non-native protein conformations,
the cell uses various sensor molecules. By definition, the
sensors include the molecular chaperones, because
these interact specifically with incompletely folded proteins. Molecular chaperones often have the dual role of
assisting the folding process and dispatching any improperly folded proteins for destruction. The conformationsensing system also includes enzymes that selectively
and covalently ‘tag’ misfolded proteins for recognition by the folding and degradation machinery. The
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best-known tags are ubiquitin, a small protein that is
attached to lysine side chains as a degradation signal6,
and glucose, which is added to the N-linked glycans of
glycoproteins as a retention signal in the endoplasmic
reticulum (ER)7,8.
In this review, we discuss the QC process that functions on newly synthesized proteins in the ER of
eukaryotic cells. The stringent distinction between
proteins that can or cannot be transported along the
secretory pathway secures the fidelity and functionality of proteins that are expressed in the extracellular
space, the plasma membrane and in the compartments that are involved in secretion and endocytosis.
ER QC also regulates the degradation of those proteins that are not correctly folded9. Disposal occurs by
a process called ER-associated degradation (ERAD);
misfolded proteins are retro-translocated from the ER
into the cytosol where they are ubiquitylated and subsequently degraded by proteasomes. As ERAD has
recently been reviewed in detail in this journal10 and
elsewhere11,12, we place our emphasis on aspects of
transport regulation.
Protein folding and quality control in the ER
The ER provides an environment that is optimized for
protein folding and maturation. Like the lumen of
other organelles of the secretory pathway (BOX 1), the ER
lumen is extracytosolic and is therefore topologically
equivalent to the extracellular space. Consequently, the
milieu of the ER differs from that of the cytosol with
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Box 1 | Secretory-pathway organelles that are involved in quality control
Lysosome/
vacuole
ER
COPI
vesicle
Golgi complex
d
c
ERAD
Translocon
complex
ER exit site
b
COPII
vesicle
a
Ribosome
Plasma
membrane
ERGIC
Nascent protein
chain
TGN
Cytosol
The endoplasmic reticulum (ER) is the site of synthesis and maturation of proteins
entering the secretory pathway (see figure, part a). It contains molecular chaperones and
folding factors that assist protein folding and retain non-native conformers. Terminally
misfolded proteins and unassembled oligomers are retro-translocated to the cytosol and
are degraded by the proteasome — a process referred to as ER-associated degradation
(ERAD). Once they are correctly folded, native conformers enter ER exit sites (see figure,
part b). Vesicles that are coated with the coatomer protein (COP)II coat bud off and
traffic through the ER–Golgi intermediate compartment (ERGIC) to the cis-face of the
Golgi complex. In certain cases, the retrieval of misfolded proteins from the Golgi
complex by COPI vesicles has been observed (see figure, part c). The Golgi complex does
not contain molecular chaperones and does not seem to support protein folding. Once
they have passed through the cis-Golgi, proteins proceed through the trans-Golgi
network (TGN) to the plasma membrane or beyond. In special cases, which have been
observed mainly in Saccharomyces cerevisiae, non-native proteins can be diverted from
the TGN to the lysosome/vacuole for degradation (see figure, part d).
respect to ions, redox conditions and the complement
of molecular chaperones. In the ER, several cotranslational and post-translational modifications take place
that do not occur in the cytosol, such as disulphidebond formation, signal-peptide cleavage, N-linked glycosylation and glycophosphatidylinositol (GPI)-anchor
addition. These covalent changes are important for correct protein folding. The rules for folding in the ER are
different from those that are applied in the cytosol.
Usually, proteins that have evolved to fold in the ER do
not fold correctly if targeted to the cytosol and vice
versa.
In mammalian cells, proteins are translocated into
the ER cotranslationally, and folding typically occurs in
three phases. First, cotranslational and cotranslocational
protein folding occurs in the context of the translocon
complex — a proteinaceous channel through which the
nascent chain enters the ER lumen or the ER membrane.
Second, post-translational folding takes place after the
completed chain has been released from the ribosome
and the translocon complex. Finally, oligomeric assembly takes place. This usually occurs when the subunits
have reached a conformation that is close to the final
folded state. Chaperones and folding enzymes (BOX 2)
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reside in the ER lumen in high concentrations, and
participate in all three stages of folding.
The presence of a strict QC system in the ER is essential for several reasons. By preventing the premature exit
of folding intermediates and incompletely assembled
proteins from the ER, it extends the exposure of the substrates to the folding machinery in the ER lumen and
thereby improves the chance of correct maturation;
downstream organelles in the secretory pathway do not
generally support protein folding13. Furthermore, ER
QC ensures that proteins are not dispatched to terminal
compartments when they are still incompletely folded
and therefore potentially damaging to the cell. For
example, it is essential that non-functional or partially
functional ion channels, transporters and receptors do
not reach the plasma membrane, where their presence
could be toxic14,15. Finally, cells use ER QC to regulate the
transportation and activation of specific proteins posttranslationally. These proteins are involved in processes
such as gene regulation and nutrient storage, and some
have extracellular carrier functions for ligands that have to
be loaded in the ER before the proteins become secretioncompetent16–18.
Primary quality control
The system that regulates the transport of proteins
from the ER to the Golgi apparatus is complicated and
sophisticated. It works at a general level (‘primary
QC’) that is applied to all proteins, regardless of their
origin and individual characteristics, and at a specific
level that is reserved for selected categories of proteins
(‘secondary QC’)19.
Primary QC is based on common structural and biophysical features that distinguish native from non-native
protein conformations. This means that, although many
cargo molecules acquire functionality while they are in
the ER, transport competence is not dependent on protein function. Important features for recognition include
the exposure of hydrophobic regions, unpaired cysteine
residues and the tendency to aggregate.
The molecular chaperones and folding sensors that
are used in primary QC are abundant in the ER. They
include BiP, calnexin, calreticulin, glucose-regulated
protein (GRP)94 and the thiol-disulphide oxidoreductases protein disulphide isomerase (PDI) and ERp57
(BOX 2). Frequently, even minor deviations from the
native conformation, because of incomplete folding or
misfolding, lead to a protein being bound by one or
more of these factors and therefore to its retention in the
ER20–22. In fact, there are various serious diseases that
derive from endogenous proteins that contain mutations and defects that affect folding and lead to protein
accumulation in the ER23–25. As no specific signals or
amino-acid sequence motifs are needed for primary
QC, it applies not only to endogenous wild-type proteins, but also to chimeric and recombinantly expressed
heterologous proteins.
A correlation with protein stability. Primary QC is a
retention-based system in which the incompletely folded
conformers and unassembled oligomers are recognized
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Box 2 | The main chaperone families of the endoplasmic reticulum
The endoplasmic reticulum (ER) contains chaperones that belong to several of the
classical chaperone families, such as heat shock protein (Hsp)40, Hsp70 and Hsp90,
with the two main exceptions being the Hsp104 and Hsp60/Hsp10 proteins. The ER
also contains chaperones and folding enzymes that are unique, such as calnexin and
calreticulin and the family of thiol-disulphide oxidoreductases.
Hsp70s
The main protein of this family is BiP, which takes part in many aspects of ER quality
control (QC). It binds to various nascent and newly synthesized proteins and assists
their folding. In addition, it is involved in the processes of ER-associated degradation
and the unfolded protein response. The functions of the second Hsp70 family member,
glucose-regulated protein (GRP)170, are relatively unexplored.
Hsp40s
Five ER proteins of the Hsp40 family (ERdj1–5) are known. They contain a luminally
exposed J-domain and can stimulate BiP ATPase activity in vitro.
Hsp90
The only known Hsp90 family member is GRP94. Despite being abundant in the ER, it
is not essential for cell viability and seems to limit its interactions to a small set of
substrates.
Peptidyl-prolyl isomerases
Peptidyl-prolyl isomerases (PPIases) from both of the two main PPIase families — the
cyclophilins and the FK506-binding proteins — are found in the ER. Catalysis of
cis/trans isomerization of peptidyl-prolyl bonds in vivo remains to be shown
conclusively for these proteins.
Calnexin and calreticulin
These two lectin chaperones interact with and assist the folding of proteins that carry
monoglucosylated N-linked glycans.
Thiol-disulphide oxidoreductases
This large family of enzymes, of which protein disulphide isomerase is the best known,
catalyses the oxidation, isomerization and reduction of disulphide bonds.
CONFORMATIONAL STABILITY
The conformational stability of a
protein is defined as the free
energy change, ∆G, for the
conversion of its unfolded
(denatured) form to its folded
(native) form.
and selectively retained. Folded conformers are not
detected and are therefore free to leave.
Which factors influence the efficiency of secretion
for a given protein? When Wittrup and colleagues26,27
measured the secretion efficiency of a series of folding
mutants of bovine pancreatic trypsin inhibitor (BPTI)
in Saccharomyces cerevisiae, they found that there was
a clear correlation with the in vitro measured thermal
stability of the mutant proteins (that is, an increased
thermal stability resulted in an increased secretion
efficiency). This correlation was observed even when
the melting temperatures for all of the mutants were
well above the temperature used for protein expression. Therefore, an important parameter that determines the efficiency of secretion is the CONFORMATIONAL
STABILITY of the folded protein, that is, the free energy of
folding.
The generality of this observation has been confirmed in several systems. The introduction of point
mutations in insulin and T-cell-receptor chains, which
were designed to make these proteins more stable than
the respective wild-type proteins, were shown to result
in higher levels of secretion28,29. When mutants of BPTI
were screened and those that had an increased secretion
efficiency selected, all of the mutants showed a higher
thermal stability than the wild-type protein30. Together
with studies using the yeast surface display system (for
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an example, see REF. 31), these observations confirm that
the lower the free-energy barrier between native and
non-native structures, the larger the fraction of protein
that is retained in the ER and then degraded.
These findings indicate that QC in the ER might
depend on dynamic and kinetic properties, that is, on
structural fluctuations and not merely on the timeaveraged structure. Even when completely folded, both
wild-type and mutant proteins might unfold transiently
and expose chaperone-binding, misfolded conformations when they are in the ER. The lower the overall stability of a protein, the more frequently this occurs. The
longer the period of time a protein spends in non-native
conformations, the lower its chances of leaving the ER.
The above ideas are summarized in FIG. 1. The conformations of a protein are shown to interchange
between incompletely folded free forms (I), incompletely folded chaperone-bound forms (I–C) and the
native conformation (N). Although a few exceptions are
known32,33, exit from the ER to the Golgi complex is, in
this model, limited to proteins that have reached the
native conformation, whereas misfolded forms are
retro-translocated and degraded in the cytosol. The
higher the stability of the protein’s native form, the
more efficient its export and the smaller the degraded
fraction.
Inherent to this model is the idea that, although the
ER environment promotes and supports the folding of
proteins, it might also induce unfolding. That this is
possible was illustrated by experiments with the tsO45
folding mutant of the vesicular stomatitis virus (VSV)
G-protein: the VSV G-protein is partially unfolded by a
rise in temperature, but only when it is located in the
ER13,34. Furthermore, the unfolding power of the ER is
used by cholera toxin to gain access to the cytosol by
retro-translocation35,36. In addition, at least partial
unfolding by chaperones is probably needed before the
retro-translocation of misfolded proteins from the ER
to the cytosol, through the translocon channel, for
degradation10,36.
Molecular properties that determine ER retention.
Although the stability argument provides an explanation for the observation that proteins differ widely in
their secretion efficiency, the molecular cues that direct
folding sensors to non-native conformers remain
largely unclear. However, some information, albeit
incomplete, is available for BiP and also for the calnexin/calreticulin system, which is discussed below.
In vitro, BiP binds heptapeptides that have aliphatic
amino-acid side chains in alternating positions37,38.
Although such sequence determinants are present in
most proteins, only a limited set are actually used by BiP
as binding sites during protein folding22. Part of the reason might be that many segments are buried rapidly as
a protein folds. It might also be that, although exposed,
the sequences are sterically inaccessible to the peptidebinding site in BiP39. In the same way that the efficiency
of proteolytic cleavage at exposed sites on the surface of
native and partly folded proteins correlates with the
flexibility of the polypeptide segment40, the binding of
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ER
Chaperone
ERAD
Golgi
Translocon
complex
I–C
I
N
Cytosol
Figure 1 | The effects of the endoplasmic reticulum environment on folding equilibria.
The equilibrium between the native (N) and incompletely folded (I) forms of a protein (green) in the
endoplasmic reticulum (ER) is affected by the ER environment, in particular by the presence of
molecular chaperones and folding enzymes. Incompletely folded conformers are bound by
chaperones (I–C), which transiently stabilize them and protect them from aggregation.
Chaperones also directly promote folding and, in many cases, this process occurs by sequential
or even simultaneous interactions with different chaperones. In addition, chaperones have a role
in transferring misfolded proteins for degradation by the ER-associated degradation (ERAD)
pathway. It seems reasonable to assume that the chaperone system in the ER can shift the
equilibrium away from the native conformation if it is not sufficiently stable. So, high
conformational stability is likely to protect proteins from recapture by the ER quality control
system and to favour ER export. Modified with permission from REF. 26. © the American Society
for Biochemistry and Molecular Biology (1998).
hydrophobic peptide sequences to chaperones might
depend not only on exposure, but also on the dynamic
properties of the target sequence22,39.
LECTIN
A protein that binds
carbohydrates.
F-BOX PROTEIN
A protein component of a
ubiquitin-ligase complex that
contains an F-box domain,
which is responsible for the
interaction with a specific
substrate protein.
UBIQUITIN LIGASE
A protein or protein complex
that mediates the ubiquitylation
of a substrate protein through
interactions with other
components of the
ubiquitylation machinery.
CFTR
(Cystic fibrosis transmembrane
conductance regulator). A
plasma membrane Cl– channel.
CFTR∆F508
A folding-defective and
principal disease-causing allele
of the cystic fibrosis
transmembrane conductance
regulator (CFTR).
184
The ‘stamp of disapproval’. An example of a particularly
well-characterized primary QC system is the so-called
calnexin/calreticulin cycle (FIG. 2). This system is responsible for promoting the folding of glycoproteins, retaining non-native glycoproteins in the ER until they are
correctly folded and, in some cases, targeting misfolded
glycoproteins for degradation8,41–45.
Calnexin and calreticulin are homologous proteins that are resident in the ER, and both are LECTINS
that interact with monoglucosylated, trimmed
intermediates of the N-linked core glycans on newly
synthesized glycoproteins7,44,46,47. Calnexin is a transmembrane protein and calreticulin is a soluble
lumenal protein. Their interaction with glycans
occurs through a binding site in their globular lectin
domain, which is structurally related to legume
lectins48. The specificity of calnexin and calreticulin
for binding monoglucosylated glycan (Glc 1Man 7GlcNAc 2) (where Glc is glucose, Man is mannose,
9
and GlcNAc is N-acetylglucosamine) leads to the
transient association of one or both of these chaperones with almost all of the glycoproteins that are
synthesized in the ER 21. Whether there are further
contacts, beyond the glycan, between substrate glycoproteins and the lectin chaperones is a matter of
ongoing investigation49–51.
Both calnexin and calreticulin form complexes with
ERp57 (REFS 52,53) — a thiol-disulphide oxidoreductase
that is known to form transient disulphide bonds with
calnexin- and calreticulin-bound glycoproteins54.
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NMR and biochemical studies have shown that
ERp57 binds to the tip of an arm-like domain that
extends ~110 and ~140 Å from the lectin domains of
calreticulin and calnexin, respectively55–57. A protected
space is formed for the bound substrate between
ERp57, the arm-like domain and the lectin domain
(FIG. 2). Other ER chaperones and folding enzymes,
such as BiP and PDI, are also often involved in the
folding of glycoproteins.
Two functionally independent ER enzymes mediate
the on- and off-cycle in this chaperone system (FIG. 2).
Glucosidase II is responsible for dissociating the substrate glycoprotein from calnexin or calreticulin by
hydrolysing the glucose from the monoglucosylated
core glycan. UDP-glucose:glycoprotein glucosyltransferase (GT), on the other hand, is reponsible for reglucosylating the substrate so that it can reassociate with
calnexin or calreticulin. It is well known from in vivo
and in vitro studies that re-glucosylation by GT happens
only if the glycoprotein is incompletely folded8. In this
way, GT works as a folding sensor. A protein can only
exit the cycle when GT fails to re-glucosylate it. The glucose acts as a selective tag for incompletely folded proteins — it is, in a sense, a ‘stamp of disapproval’ that tells
the system that the protein is not yet ready to be
deployed.
The cycles of glucosylation and de-glucosylation
continue until the glycoprotein has either reached its
native conformation or is targeted for degradation. For
the degradation of glycoproteins, trimming of a single
mannose by the ER α1,2-mannosidase I in the middle
branch of the oligosaccharide leads to an association
with ER degradation-enhancing 1,2-mannosidase-like
protein (EDEM) — a newly discovered lectin58,59. This
interaction probably diverts the glycoprotein from the
calnexin/calreticulin cycle and promotes its degradation. The recent discovery that the F-BOX PROTEIN Fbx2
of the SCFFbx2 ubiquitin ligase complex functions as
a UBIQUITIN LIGASE specifically for proteins that carry
N-linked high-mannose oligosaccharides provides
another link between the processes of ER QC and
ERAD of glycoproteins60.
So, how does GT distinguish native from incompletely folded proteins? In vitro studies have shown
that it preferentially re-glucosylates glycoproteins in
partially folded, molten globule-like conformations,
and that an important feature for recognition is the
exposure of hydrophobic amino-acid clusters 61. It
ignores glycoproteins in native and random-coil
conformations, as well as short glycopeptides and
isolated glycans8,62. GT is able to recognize limited,
highly localized folding defects and, in doing so, it
only re-glucosylates those glycan chains that are present in the misfolded regions63 (C. Ritter, K. Quirin
and A.H., unpublished observations). This means
that the enzyme focuses specifically on regions of
the substrate glycoproteins that are partially misfolded and contain glycans, and highlights the fact
that GT is a sophisticated and sensitive detector of
misfolding in the ER lumen and in ER exit sites. The
placement of N-linked glycans in the sequence of
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glycoproteins might have evolved to direct the
attention of the QC system to particular regions of
the folding protein. Conversely, N-linked glycans
might be missing from other regions where an
increased mobility is needed.
Ribosome
Cytosol
Translocon complex
ER
Nascent protein chain
M GG
N-linked glycan
G
Glucosidases
I and II
G
M
UDP
Calreticulin
UDP-glucose:glycoprotein
glucosyltransferase
M
UDP– G
–S
–S
–
G
M
Glucosidase II
ERp57
α1,2-mannosidase I
M
EDEM
Translocon
complex
ERAD
ER exit site
Figure 2 | The calnexin/calreticulin cycle. Calnexin and calreticulin assist the folding of
glycoproteins in the endoplasmic reticulum (ER) (for simplicity, only calreticulin is depicted). After
transfer of the core oligosaccharide (Glc3Man9GlcNAc2, where Glc is glucose (red circles), Man is
mannose (blue circles) and GlcNAc is N-acetylglucosamine) to the nascent chain of the protein,
two glucoses are removed by glucosidases I and II. This generates a monoglucosylated
(Glc1Man9GlcNAc2) glycoprotein that can interact with calnexin and calreticulin. Both chaperones
associate with the thiol-disulphide oxidoreductase ERp57 through an extended arm-like domain.
During the catalysis of disulphide-bond formation, ERp57 forms interchain disulphide bonds
(S–S) with bound glycoproteins. Cleavage of the remaining glucose by glucosidase II terminates
the interaction with calnexin and calreticulin. On their release, correctly folded glycoproteins can
exit the ER. By contrast, non-native glycoproteins are substrates for the
UDP-glucose:glycoprotein glucosyltransferase, which places a single glucose back on the
glycan and thereby promotes a renewed association with calnexin and calreticulin. If the protein
is permanently misfolded, the mannose residue in the middle branch of the oligosaccharide is
removed by ER α1,2-mannosidase I. This leads to recognition by the ER degradation-enhancing
1,2-mannosidase-like protein (EDEM), which probably targets glycoproteins for ER-associated
degradation (ERAD). Please note that, for simplicity, only some of the sugar moieties of the
oligosaccharide core structure are depicted fully in the figure.
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Chemical and pharmacological chaperones. Given a system in which retention is based on conformational criteria and on differences in the stability of native and
non-native conformations, it should be possible to
improve maturation and secretion by stabilizing proteins in the early secretory pathway. This approach
could potentially pave the road for therapeutic intervention in ER-storage diseases in which folding-defective
proteins fail to be secreted.
Indeed, several studies using tissue-culture cells
have shown that such an effect can be obtained, for
example, by lowering the temperature or by using small
cell-permeable molecules such as glycerol, dimethylsulphoxide and trimethylamine N-oxide. These ‘chemical chaperones’ exert a nonspecific, folding-promoting
effect presumably by stabilizing native or native-like
conformers or by reducing aggregation. A well-known
case involves the primary disease-causing allele of the
cystic fibrosis transmembrane conductance regulator
(CFTR) — the CFTR ∆508 folding-defective mutant. In this
case, the use of different chemical chaperones leads to
increased cell-surface expression of the structurally
destabilized, but functional, protein64.
Similar, but more specific, effects can be achieved with
‘pharmacological chaperones’. This term refers to ligands
that target a specific protein (for a review, see REF. 65).
Recent elegant work, on G-protein-coupled receptors
(such as the δ opioid receptor66 and V2 vasopressin receptor mutants67), tyrosinase68, P-glycoprotein mutants69 and
antibodies that carry mutations in the heavy chain complementarity-determining region70, has demonstrated the
potential of these molecules. The specificity of the
approach is shown by a correlation between ligand-binding affinity and ligand-mediated rescue66,70. The beneficial
effects of pharmacological chaperones probably stem
from the structural stabilization of native-like conformers
that are able to interact with ligands. Therefore, they
might help to shift the equilibrium from the incompletely
folded (I) forms of the protein to the native (N) form
(FIG. 1) enough to increase secretory efficiency.
Pharmacological chaperones mimic the effect that
certain physiological ligands have on protein secretion.
For example, the retinol binding protein (RBP) is
retained in the ER in the unliganded form. Only on
binding retinol does export of RBP take place18. As
retinol binding results in only a minor conformational
change in RBP, as detected by X-ray crystallography71,
this effect is probably best explained by ligand-induced
stabilization of the RBP structure.
Secondary quality control
To be secreted, many proteins must fulfil criteria beyond
those that are imposed by the primary QC system. The
term secondary QC refers to various selective mechanisms that regulate the export of individual protein
species or protein families19. Each of the factors involved
has its own specific recognition mechanism and many
of these factors interact with the folded cargo proteins
or late folding intermediates19,24,72. Among the colourful
mixture of secondary QC proteins, Herrmann and colleagues72 have classified those proteins that are needed
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to fold and assemble specific proteins as ‘outfitters’, those
needed to accompany proteins out of the ER as ‘escorts’
and those needed to provide signals for intracellular
transport as ‘guides’. The group of outfitters includes
specialized chaperones and enzymes such as Nina A — a
peptidyl-prolyl cis/trans isomerase that ensures the
transport competence of specific rhodopsins in
Drosophila melanogaster73. A well-known escort is the
receptor-associated protein (RAP), which binds to members of the low density lipoprotein-receptor (LDLR)
family in the ER and escorts them to the Golgi complex
to protect them from premature ligand-binding in the
early secretory pathway74. Among the guides, the lectin
that is known as ER–Golgi intermediate compartment
(ERGIC)-53 cycles between the ER and the Golgi
complex and seems to act as a transport receptor for
certain proteins that carry high-mannose N-linked
glycans75,76. For a more complete listing of secondary
QC factors, see REF. 19.
Secondary QC processes are often cell-type specific.
In addition, they are frequently involved in the regulation of ER retention and export. For example, cellular
cholesterol levels are controlled by the regulated ER
export of the sterol regulatory element binding protein
(SREBP) through its cholesterol-dependent interaction
with the escort factor SCAP (SREBP cleavage-activating
protein)77. In this case, cholesterol binding by SCAP
brings about a conformational change in the molecule
that leads to ER retention of the SCAP–SREBP
complex16. This retention is facilitated through the interaction of the sterol-sensing domain of SCAP with either
of two homologous transmembrane ER proteins (insig-1
and insig-2)78,79. At present, the ER-retention mechanism for the ternary SCAP–SREBP–insig complex is not
known.
Protein sorting and ER quality control
TYPE I MEMBRANE PROTEIN
A transmembrane protein that is
orientated with its carboxyl
terminus in the cytosol.
14-3-3 PROTEINS
A family of abundant proteins
that bind to phosphoserine- and
phosphothreonine-containing
motifs in a sequence-specific
manner.
INVARIANT CHAIN
A specific chaperone and escort
protein for MHC class II
molecules.
TYPE II MEMBRANE PROTEIN
A transmembrane protein that is
orientated with its amino
terminus in the cytosol.
186
ER-retention signals. The ER is continuously feeding
cargo-containing vesicles into the secretory pathway
and receiving retrograde membrane traffic from the
Golgi complex in return (BOX 1). A cohort of folded,
assembled, native secretory and membrane proteins,
as well as membrane lipids, are exported continuously from the ER. To avoid this fate, most resident
ER proteins, including chaperones and folding
enzymes, contain ER-retention and -retrieval signals
(for reviews, see REFS 80,81). Of these signals, the wellcharacterized tetrapeptide KDEL sequence (where K
is lysine, D is aspartate, E is glutamate and L is
leucine) is present at the carboxyl terminus of many
soluble ER proteins. This motif interacts with the
KDEL receptor and thereby ensures that the protein
is retrieved from the Golgi complex by coatomer
protein (COP)I-coated vesicles (BOX 1)82–84. Similar to
the KDEL sequence, KKXX or KXKXX signals
(where X is any amino acid) that are present at the
carboxyl terminus of TYPE I MEMBRANE PROTEINS are
known to mediate retrieval by direct interaction with
the COPI coat 85–88. In addition, exposed cysteine
residues can also function as retention signals in the
lumen of the ER20.
| MARCH 2003 | VOLUME 4
Retention and retrieval signals that are present in
proteins destined for export are also important in QC,
as they help to retain proteins in the ER until correct
folding or assembly has taken place. The best characterized of these signals is the RKR motif (where R is
arginine), which was first described in subunits of
ATP-sensitive potassium channels14. In most cases, this
signal helps to retain individual subunits and incomplete oligomers in the ER until they are masked by the
correct oligomeric assembly89.
Another intriguing example of transport regulation
involves the recently described role of a 14-3-3 PROTEIN in
promoting ER export of proteins that contain dibasic
COPI-interacting retention signals90. The binding of
14-3-3 to short sequence motifs — which contain a
phosphorylated serine residue and are present, for
example, in different potassium channels and in the
INVARIANT CHAIN — was shown to exclude simultaneous
binding to the COPI-protein β-COP and to result in ER
export. So, mutually exclusive binding of 14-3-3 and
β-COP seems to underlie this phosphorylation-dependent
regulation of ER export and retention.
In the case of the TYPE II TRANSMEMBRANE PROTEIN ATF6
(activating transcription factor 6), transport from the
ER is regulated by ER stress. Under stress conditions that
lead to the accumulation of misfolded proteins in the
ER, the transcription factor ATF6 is transported to the
Golgi complex where intramembrane proteolysis
releases the transcription-factor domain, which is
exposed to the cytosol91. The transcription-factor
domain then traffics to the nucleus in which it activates
ER-chaperone-gene transcription. The ER export of
ATF6 is controlled by BiP92. Using a mechanism that
probably involves the masking of signals for forward
transport to the Golgi, binding of BiP retains ATF6 in
the ER under normal cellular conditions. On the accumulation of misfolded protein in the ER, it is conceivable that such conformers compete for binding of BiP.
This then results in BiP release from the lumenal
domain of ATF6 and, as a consequence, the protein
becomes transport competent.
ER exit sites. Exit of proteins from the ER occurs at socalled transitional elements or ER exit sites93,94 (FIG. 3).
These form buds or small membrane clusters that are
contiguous with the ER membrane and are coated with
the COPII coat95. Importantly, incompletely folded
cargo proteins and ER chaperones are generally
excluded from exit sites13,96,97. If the tsO45 VSV G-protein
is rendered misfolded by a temperature shift when present in exit sites, it is re-glucosylated by GT and, instead
of being transported to the Golgi complex, is returned
to the ER13. Therefore, the exit sites function as an
intrinsic part of the ER QC system.
The reasons why misfolded proteins are not included
in export vesicles could be: that they are retained by
interactions with molecules elsewhere in the ER; that
they are somehow prevented from entering the exit sites
by restrictions imposed by the exit sites themselves; or
that ‘cargo receptors’ that are present in the exit sites fail
to recognize them. Here, the underlying models are
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a
b
c
ER
ER exit sites
Golgi complex
Figure 3 | Trafficking of a cargo glycoprotein from the endoplasmic reticulum through exit sites to the Golgi complex.
a | A light-microscopy image of the endoplasmic reticulum (ER). Tissue-culture cells were transfected with a green fluorescent
protein-labelled version of the temperature sensitive tsO45 variant of the vesicular stomatitis virus (VSV) G-protein and were
incubated at 39.5 °C. At this temperature, the VSV G-protein is partially unfolded and is therefore retained in the ER (white
meshwork) by the quality-control system. b | A light-microscopy image of the ER and ER exit sites. On shifting the cells from 39.5 °C
to 10 °C for 1 h, the VSV G-protein that is discussed in part a refolds, but it remains in the ER and in ER exit sites, which are visible
as small ‘dots’ (the labels highlight two examples of ER exit sites)13. c | A light-microscopy image of the Golgi complex. On shifting
the temperature from 39.5 °C to 20 °C, followed by incubation for 2 h, the VSV G-protein refolds and traffics to the Golgi complex,
which is visible as an intensely stained, bright area (see label). A weak reticular ER stain is visible from VSV G-protein that is still
present in the ER. This figure was kindly provided by A. Mezzacasa, ETH Zürich, Switzerland.
those of ‘bulk flow’, in which selectivity is based on
retaining interactions98, and of ‘cargo capture’, in which
folded cargo is selectively recognized in the exit sites by
receptors that are thought to be transmembrane proteins, which associate directly or indirectly with the
COPII coat99. Although evidence has been presented in
favour of both models, neither adequately explains the
full range of sorting phenomena that are observed during the ER-to-Golgi trafficking of proteins. As is often
the case in biology, elements taken from numerous
models might together provide the full picture.
Protein mobility in the ER. Some misfolded proteins fail
to be transported to the Golgi complex because they
aggregate in the ER2. In addition to non-native proteins,
such aggregates typically contain molecular chaperones
and thiol-disulphide oxidoreductases, and they are often
stabilized by non-native, interchain disulphide
bonds100,101. Protein aggregation in the ER is most often
irreversible and in many cases connected directly to a
disease state. However, recent work showed that aggregates of a genetically engineered variant of the FKBP12
protein (FK506 binding protein), which was recombinantly expressed in the ER, could be dissolved on the
addition of a small synthetic ligand102. When fusion proteins — of FKBP12 linked to therapeutically important
hormones (such as insulin) through a cleavage site for
furin (which is a Golgi-resident protease) — were
expressed in tissue-culture cells or mice, regulated ligandinduced secretion of the hormone could be achieved102.
This approach shows that the modulation of the
processes that are involved in protein sorting and ER
QC can potentially lead to the development of new
therapeutic strategies.
Sheer size might be enough to explain why aggregates in the ER fail to be exported. However, the upper
size limit for particles that can be exported is quite high;
for example, viruses and virus-like particles that are 50 nm
NATURE REVIEWS | MOLECUL AR CELL BIOLOGY
in diameter can bud into the ER, and procollagen fibres
with a length of 300 nm can be transported to the Golgi
complex103–105. Nevertheless, it is possible that some of
the aggregates that are formed by misfolded proteins
exceed these limits.
Fluorescence recovery after photobleaching (FRAP)
analysis of green fluoresent protein (GFP)-labelled
cargo molecules has shown that certain aggregated,
non-native proteins are effectively immobilized in the
rough ER106–108. So, another reason why aggregates of
misfolded proteins fail to be transported to the Golgi is
probably a lack of mobility in the ER. They might not
be able to move to ER exit sites.
It has been proposed that molecular chaperones
and other resident proteins that are present in the ER
lumen at high concentrations form a viscous, interconnected protein network that might restrict mobility,
not only of large aggregates but also of smaller complexes that contain incompletely folded proteins109–112.
However, recent reports, also based on FRAP analysis,
show that several transport-incompetent cargo proteins
are actually quite mobile in the ER. These transportincompetent cargo proteins include GFP-labelled
tsO45 VSV G-protein106, the CFTR ∆508 mutant113, a
mutant of aquaporin 2 (REF. 107) and the heavy chain of
unassembled major histocompatibility complex
(MHC) class I molecules108. When compared to the
folded forms of the same proteins, little difference in
lateral mobility was observed. The estimated diffusion
constants indicate that the proteins retained in the ER
are in the form of monomers or small to mid-size
aggregates. So, although aggregation can be a factor
that might help to exclude certain misfolded proteins
from exit sites, the FRAP data imply that many misfolded proteins are mobile in the ER. More work is
needed to address the important question of why such
proteins are not exported through exit sites like their
folded counterparts.
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Quality control in the Golgi complex. Although most of
the reported QC events occur in the ER, the Golgi complex can also participate32,33. In certain cases, retrieval of
misfolded proteins from the Golgi complex to the ER
occurs. For unassembled T-cell antigen receptor α-chains,
this retrieval pathway involves COPI-coated vesicles and
is dependent on BiP and the KDEL-receptor114.
Similarly, when the misfolded tsO45 variant of the VSV
G-protein was overexpressed in Chinese hamster ovary
(CHO) cells, the protein was seen to cycle between the
ER and the cis-Golgi in a complex with BiP115. However,
the KDEL-receptor has the ability to retrieve KDELcontaining proteins and complexes not only from the
cis-Golgi, but from the entire Golgi complex116. The
retrieval pathway from the Golgi complex probably represents a back-up system for when the ER-retention
capacity for a protein is saturated. In other cases, the
recycling of misfolded proteins might be required for
ERAD33,117–119. The Golgi complex of mammalian cells
seems to lack resident chaperones and folding enzymes,
but the localization of calreticulin, GT and glucosidase II
to the ERGIC might indicate a potential for glycoprotein
folding in this compartment120,121.
Particularly in S. cerevisiae, the Golgi complex seems
to be an important site for QC.Vps10 — a Golgi protein
that is responsible for sorting vacuolar hydrolases — has
been implicated as a folding sensor for the vacuolar
transport of misfolded proteins122,123. Recently, a multispanning membrane protein of the Golgi — Tul1, which
is a putative ubiquitin ligase — was identified as an
enzyme that recognizes polar residues in the transmembrane region of membrane cargo proteins124. It is possible that Tul1 singles out membrane proteins that are
either not folded correctly in the transmembrane region
or are not completely oligomerized for vacuolar targeting.
The efficiency of protein maturation
Although some proteins fold quite efficiently, many fold
inefficiently. One possible explanation for the poor success rate of protein maturation might be that, during
evolution, proteins have been optimized for function,
but not necessarily for folding and assembly. Therefore,
this would mean that these latter processes are intrinsically error-prone. Often polypeptides get caught in nonnative conformations from which they cannot escape
within a reasonable time, even in the presence of chaperones. For certain ER proteins, such as CFTR and the
δ opioid receptor, 40% or more of newly synthesized
wild-type protein molecules fail to mature and are
degraded125,126. Although the physiological temperature
might be optimal for protein function, it might be too
high for efficient folding. Consequently, an increase in
folding efficiency is often found when cells are grown at
reduced temperatures (for a review, see REF. 2).
For hetero-oligomeric proteins, a poor success rate of
maturation is often observed. Orphan subunits and partially assembled oligomers are retained in the ER and
eventually degraded. For example, this has been
observed in cultured muscle cells, in which only ~30%
of acetylcholine receptor α-subunits were incorporated
into the native hetero-pentameric receptor molecules127.
188
| MARCH 2003 | VOLUME 4
However, non-stoichiometric production of protein
subunits can also have a distinct purpose. In the case of
immunoglobulin assembly in plasma cells, the light
chains are expressed in excess of the heavy chains to
ensure high rates of heavy-chain incorporation into the
oligomeric complex, because the heavy chain is aggregation prone and potentially toxic in the free form128.
Likewise, the level of protein expression often exceeds
the availability of specific ligands that are essential for
secretion. This is the case for MHC class I molecules —
only the fraction that is loaded with high affinity peptides is transported to the cell surface129 — and for RBP,
which needs to associate with retinol18.
Recent reports, which used pulse-labelling and proteasome inhibitors to block the degradation of newly
synthesized proteins, indicated that 30–75% of the proteins that are synthesized are degraded within 20 min,
and that some of the proteins that are being degraded
are growing nascent chains130–133. How can the cell
afford this large-scale degradation of newly synthesized
proteins? Yewdell and co-workers130 have argued that
the apparent wastefulness actually has an important
function, because the ongoing degradation of newly
synthesized and nascent proteins provides a continuous
supply of peptides that are presented on the cell surface
bound to MHC class I molecules. It is known that most
of the peptides that are presented in this way are, in fact,
from proteins that have just been synthesized134.
Therefore, when a cell is infected by a virus and starts to
produce viral protein, this infection can be rapidly
detected by T-cells.
Although the formation of misfolded proteins might
have a useful role in immune defence, the downside is
the various diseases in which aggregated proteins accumulate in the cells and tissues in non-native conformations. For example, the formation of amyloid plaques is
clearly due to a failure in QC and/or in the proteindegradation system135. It is apparent that these conformations are either not recognized efficiently as being
misfolded or, once they are formed, they cannot be
degraded.
Conclusions and perspectives
The ER is unique among cellular compartments in the
sense that most newly synthesized proteins are exported
from the ER and find their final location in places that
are devoid of chaperones or other factors that could
help them refold if they were to become damaged.
Therefore, the requirements for protein stability might
be set higher in the ER than in the cytosol. The stability
of proteins that are produced in the ER is enhanced by
the formation of disulphide bonds and by the addition
of protein-bound glycans. Although these modifications
make proteins more stable, they impose more complexities on the folding process. The number of enzymes that
are involved solely in the oxidation and isomerization of
disulphide bonds is large, and so is the number of
enzymes that are needed to synthesize and trim N-linked
glycans. It is also probable that the ER exposes folded
proteins to further ‘prodding’ by chaperones to ensure
their stability.
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REVIEWS
As we learn more about the folding and maturation of
individual proteins in the ER, and about the mechanisms
that are used by individual chaperones, it will be important to consider the overall principles of these systems.
How are degradation decisions made in this compartment? How do chaperones and folding enzymes work
together? Which are the crucial folding sensors, and how
do they recognize their substrates? What is the mechanism for selective cargo loading in ER exit sites? Whether
at the level of structural fluctuations in the folded and
unfolded proteins or the mobility of the proteins and
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Acknowledgements
We thank A. Smith, A. Mezzacasa and E. Frickel for their critical
reading of the manuscript. This work was supported by the
Swiss National Science Foundation.
Online links
DATABASES
The following terms in this article are linked online to:
European Bioinformatics Institute:
http://srs.ebi.ac.uk/
BiP | calnexin | calreticulin | CFTR | ERp57 | PDI |
UDP-glucose:glycoprotein glucosyltransferase
FURTHER INFORMATION
Ari Helenius’ laboratory:
http://www.bc.biol.ethz.ch/professors/helenius/helenius.html
Lars Ellgaard’s laboratory:
http://www.bc.biol.ethz.ch/groups/ellgaard/ellgaard.html
Access to this interactive links box is free online.
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