Download Signals and mechanisms for protein retention in the endoplasmic

Journal of Experimental Botany, Vol. 50, No. 331, pp. 157–164, February 1999
Signals and mechanisms for protein retention in the
endoplasmic reticulum
Sophie Pagny, Patrice Lerouge, Loı̈c Faye and Véronique Gomord1
Laboratoire des Transports Intracellulaires, CNRS–ESA 6037, IFRMP 23, Université de Rouen, 76821 Mont
Saint Aignan, France
Received 3 February 1998; Accepted 19 March 1998
Abstract
After their co-translational insertion into the ER lumen
or the ER membrane, most proteins are transported
via the Golgi apparatus downstream on the secretory
pathway while a few protein species are retained in
the ER. Polypeptide retention in the ER is either signalindependent or depends on specific retention signals
encoded by the primary sequence of the polypeptide.
A first category, i.e. the newly synthesized polypeptides that are unable to reach their final conformation,
are retained in the ER where this quality control generally results in their degradation. A second category,
namely the ER-resident proteins escape the bulk flow
of secretion due to the presence of a specific N- or
C-terminal signal that interacts with integral membrane or soluble receptors. ER retention of soluble
proteins mediated by either KDEL, HDEL or related
sequences and membrane receptors has been relatively well characterized in plants. Recent efforts have
aimed at a characterization of the retention signal(s)
of type I membrane proteins in the plant ER.
Key words: Endoplasmic reticulum, plant, retention signal,
soluble proteins, membrane proteins.
ER before being further transported downstream in the
secretory system. Polypeptides that fail to fold properly
or never become part of a multisubunit complex are
retained within this compartment. It seems that multiple
proteolytic pathways exist for purging the ER from these
misfolded products. The cell then performs a ‘quality
control’ of neosynthesized peptides, allowing elimination
of non-functional proteins. The process whereby polypeptides attain their native structure is directly influenced by
ER-resident molecular chaperones. The latter bind either
nascent proteins, next releasing them so that they can
exit from the ER, or persistently malfolded and incompletely assembled proteins, then retaining them within the
ER until they are further proteolysed (Hammond and
Helenius, 1995). Several soluble or membrane ER chaperones have been identified in plants including the binding
protein BiP, calreticulin, endoplasmin, protein disulphide
isomerase, and calnexin (Denecke et al., 1991, 1993, 1995;
Chen et al., 1994; Dresselhaus et al., 1996, Huang et al.,
1993, Hasenfratz et al., 1997; Ou et al., 1993; Shorrosh
and Dixon, 1991; Walther-Larsen et al., 1993). These
ER-resident molecular chaperones harbour specific signals that are responsible for residency in the ER. In this
short review, current data will be presented and discussed
that clarify which signals and mechanisms are responsible
for protein retention in the plant ER.
Introduction
In plant cells, the secretory system delivers many different
proteins to four principal locations: the cell wall, the
vacuole, the plasma membrane, and the tonoplast. All
proteins directed to these compartments are
co-translationally inserted into the endoplasmic reticulum
( ER) and are then selectively targeted to and/or retained
in various intracellular compartments. A newly synthesized protein must acquire a specific conformation in the
Signal-independent retention in ER
Most luminal, resident proteins are retained in the ER
due to the presence of a specific C-terminal signal, namely
KDEL, HDEL or related sequences. However, for a few
resident proteins, the retention is signal-independent. This
is the case for some cereal prolamin storage proteins that
directly accumulate in the ER lumen where they are
1 To whom correspondence should be addressed. Fax: +33 2 35 14 67 87. E-mail: [email protected]
© Oxford University Press 1999
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Pagny et al.
assembled into protein bodies. It seems that these prolamins remain in the ER of maize, rice or sorghum endosperm
cells because they aggregate or assemble within the ER
lumen to give large insoluble deposits that are physically
impossible to transport (for a detailed review on
ER-derived protein bodies see Galili et al., 1998). As
proposed for rice prolamins (Li et al., 1993) another
possibility for a protein to reside in a signal-independent
manner in the ER would be through prolonged interaction
with molecular chaperones, such as BiP, having specific
sequences for ER retention. This mechanism of ER
retention has been illustrated for newly synthesized and
assembly-defective secretory proteins in yeast, mammals
and plants. For instance, in a recent study, Pedrazzini
et al. (1997) have shown that the ER chaperone BiP
remains associated with an assembly-defective form of
the bean vacuolar storage protein phaseolin (D363
phaseolin) for much longer than with the assembly competent form when these reporter proteins are expressed
in transgenic tobacco. While the assembly-competent
phaseolin is transported to the vacuole, D363 phaseolin
is retained and slowly degraded in the ER or in a related,
pre-Golgi compartment (Pedrazzini et al., 1997).
This process whereby misfolded proteins are retained
and subsequently degraded in the ER was termed quality
control (Hammond and Helenius, 1995). This mechanism
which prevents secretion and clear the secretory pathway
of misassembled and misfolded proteins is highly conserved in eukaryotic cells.
Localization signals for soluble, ER-resident
proteins
In order to remain in the ER, and therefore to be diverted
from the bulk flow of secretory proteins, ER-resident
proteins and particularly molecular chaperones generally
require specific signals for retention or retrieval. By
comparing the polypeptide sequences of many soluble
proteins that reside in the ER lumen, a consensus tetrapeptide H/KDEL has been identified at their C-terminal
end. Also, a carboxy-terminal tetrapeptide KDEL has
been shown to function as an ER retention signal in
animal cells, and a mutated form of BiP, which lacked
the KDEL C-terminal extension, was slowly secreted
instead of being retained in the ER when expressed in
COS cells (Munro and Pelham, 1987). Likewise ER
retention in yeast is mediated by an HDEL sequence
(Pelham et al., 1988). However, the fact that the
animal/yeast BiP (with its original retention signal ) is
poorly retained in the ER of yeast/animal cells, respectively, indicates that the receptors used for ER retention
in yeast and animals differ in their specificity.
In plants, both HDEL and KDEL C-terminal extensions have been identified in the sequences of soluble,
ER-resident proteins that are collectively referred to as
reticuloplasmins (Gomord and Faye, 1996). For example,
the alfalfa protein disulphide isomerase (Shorrosh and
Dixon, 1991) harbours a KDEL sequence, although most
plant reticuloplasmins carry an HDEL signal (for review
see Vitale et al., 1993). The bulk of proteins containing
either HDEL or KDEL signals at their C-terminal end
fractionate with the plant ER on a density gradient,
whereas their respective intracellular distribution is markedly different when visualized by immunofluorescence.
Indeed, HDEL proteins show an ER-characteristic distribution whereas KDEL proteins are immunodetected on
a discrete part of the ER network that might be the
cortical ER (Napier et al., 1992).
Many laboratories have shown that the addition of
KDEL to the C-terminal end of various secreted proteins
leads to the retention of these proteins in the ER of
animal cells or at least to a significant retardation of their
transport downstream on the secretory pathway (for
illustration see Rose and Doms, 1988). In a similar
approach, reporter proteins fused with an H/KDEL tetrapeptide are at least partly retained in the ER of plant
cells (Hermann et al., 1990; Wandelt et al., 1992; Denecke
et al., 1993; Boevink et al., 1996; Gomord et al., 1997).
In a recent study it was shown that an HDEL sequence
fused to vacuolar or extracellular forms of sweet potato
sporamin relocalizes these reporter proteins within the
ER of tobacco cells. In addition, probably because of the
very high stability of recombinant sporamin in the vacuole
of transgenic tobacco cells, it has been observed that a
significant fraction of these fusion proteins that escape
the ER retention machinery, is transported to the vacuole.
On such grounds it may be proposed that, in addition to
its function as an ER retention signal, HDEL could also
participate in quality control by targeting chaperonemisfolded protein complexes that escape the ER to the
plant lytic vacuole for degradation (Gomord et al., 1997).
This putative mechanism and a recently identified retrotransport to the cytosol would involve molecular chaperones in the delivery of ER proteins that fail to properly
fold or oligomerize either to vacuolar proteases or to
cytosolic proteasomes for a final degradation (Gomord
et al., 1997; Lord, 1996; Brodsky and McCracken, 1997).
The ER retention machinery for soluble
reticuloplasmins
Soluble reticuloplasmins are thought to be retained in the
ER by membrane receptors that recognize H/KDEL
C-terminal extensions. In yeast, two HDEL-specific receptors have been identified: ERD1 (Hardwick et al., 1990)
and ERD2 (Semenza et al., 1990). ERD2 is a 26 kDa
membrane protein whose sequence is 50% similar to its
human equivalent (Hsu et al., 1992; Lewis and Pelham,
1992) and exhibits 52% similarity with Arabidopsis thaliana ERD2 (Lee et al., 1993). The A. thaliana erd2 gene is
Protein retention in the endoplasmic reticulum 159
able to complement a yeast erd2 deletion mutant, while
the human erd2 gene is not.
It was first thought that the ER-resident ERD receptors
would bind the K/HDEL retention signal to divert reticuloplasmins from the bulk flow of secreted proteins. There
is now some evidence, at least in mammals and yeast,
that these receptors do not stricto sensu retain reticuloplasmins in the ER, but rather are responsible for their
transport back to the ER when they escape this compartment (Pelham et al., 1988). Consistent with this recycling
mechanism, it has been shown that the human KDEL
receptor concentrates mainly in a post-ER intermediate
compartment and throughout the Golgi apparatus, but
with a concentration that markedly decreases towards the
trans-side (Griffith et al., 1994). Also arguing for a
recycling of ER proteins is the fact that reporter glycoproteins fused with H/KDEL motifs undergo structural
modifications in their N-glycans that are believed to be
post-ER events. For example, in animal cells, the lysosomal glycoprotein, cathepsin D, when modified by addition
of a KDEL C-terminal extension is retained in the ER,
but also carries N-acetylglucosamine-1-phosphate residues which are added in the cis-Golgi compartment
(Pelham et al., 1988). Some post-ER glycan maturations
have also been found on reporter glycoproteins retained
in yeast ER after fusion with the HDEL sequence (Pelham
et al.,1988; Dean and Pelham, 1990). Although these
results should be considered with caution as reporter
proteins fused with H/KDEL are generally retained in
the ER to a limited extent, they imply that these ‘artificial’
reticuloplasmins indeed pass through a post-ER compartment and are subsequently retrieved back to the ER from
locations that can be as distal as the trans-Golgi network
(Miesenböck and Rothman, 1995). Data obtained by
several groups also indicate that some natural reticuloplasmins exhibit N-glycan modifications such as a terminal galactose addition that are known to occur in the
Golgi, or even in the trans-Golgi (Nguyen Van et al.,
1989). As illustrated in Fig. 1, all this information
obtained from the glycosylation state of ER-resident
glycoproteins is consistent with the existence of a continual retrieving of H/KDEL-containing proteins from
an early or a late Golgi compartment back to the ER in
mammals and yeast. This mechanism implies a bidirectional transport between ER and Golgi mediated by
COPI coated vesicles moving retrogradely while COPII
coated vesicles move anterogradely. However, while
retrieval of mammalian KDEL containing proteins may
occur from several distinct Golgi regions (Lewis and
Pelham, 1992), yeast cells appear to recycle soluble ER
proteins only from an early Golgi compartment (Dean
and Pelham, 1990). Consistent with the hypothesis that
anterograde and retrograde pathways are linked to form
a cycle are the effects induced by a fungal drug, namely
brefeldin A (BFA) that blocks the anterograde traffic by
preventing the COPII coat assembly. This BFA-induced
exaggeration of the Golgi to ER retrograde pathway
results in the loss of Golgi stacks and their mixing with
the ER (Lipincott-Schwartz et al., 1989, 1990). However,
in contrast with KDEL receptors and ‘artificial’ reticuloplasmins, natural ER-resident proteins have never been
clearly identified in a post-ER compartment in physiological conditions except in developmentaly immature mammalian cells ( Wiest et al., 1995) and in protein bodies
directly derived from the ER in maize, wheat and rice
( Zhang and Boston, 1992; Levanony et al., 1992; Muench
et al., 1997).
The secretory systems in animal, yeast or plant cells
share many similarities. However, there is no evidence
that plant reticuloplasmins are retained in the ER by
cognate receptors that would constantly retrieve reticuloplasmins escaped from an intermediate, early or late
Golgi compartment back to the ER, in contrast with
what is shown in Fig. 1 for animals cells. Indeed, the
glycan structures of natural, resident, soluble glycoproteins of the plant ER such as calreticulins are not consistent with an active recycling of these proteins between the
ER and the Golgi apparatus. For instance, using immunoand affinodetection on blots (Navazio et al., 1998), chromatography, and NMR data (Navazio et al., 1996;
Lerouge et al., unpublished results), it was shown that all
plant calreticulins analysed so far have N-linked glycans
that are devoid of Golgi modifications. These results
strongly contrast with the typical Golgi modifications
observed on mammalian calreticulins as illustrated in
Fig. 2. In a similar approach, where N-glycan structures
are used as markers of a putative recycling, immunolabelling of the ER in BY2 tobacco cells with antibodies
directed against N-glycans that have been matured in the
Golgi apparatus (Rayon et al., 1998) has not been
observed. This result was confirmed with antibodies
specific for glycan structures corresponding to early or
late Golgi modifications in tobacco cells, whether or not
the latter were treated with high or low amounts of BFA
(Denmat et al., unpublished results).
Of course, the lack of complex N-linked glycans in the
plant reticuloplasmins analysed so far does not exclude a
recycling from post-ER compartments back to the ER.
Indeed, recent data showing that (i) the Arabidopsis
ERD2 receptor fused to green fluorescent protein (GFP)
expressed in transgenic tobacco plants is targeted to both
Golgi apparatus and ER and (ii) a retrograde transport
of both ERD2 and a Golgi resident enzyme from the
Golgi membrane into the ER in response to BFA, can be
considered as preliminary evidence for a retrograde transport pathway in plants (Boevink et al., 1998). However,
structural analyses of ER-resident glycoproteins strongly
suggest that either the recycling efficiency is so high in
plants that reticuloplasmins do not travel very far downstream from the Golgi apparatus prior to their transport
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Pagny et al.
Fig. 1. Possible mechanisms of ER protein retention. ER retention may involve two different and non-exclusive mechanisms. Either proteins are
retained in the ER due to their inability to enter transport vesicles destined to the next compartment (1) or they enter these vesicles (2) but they
are subsequently transported back to the ER from early or late Golgi compartments after binding to the ERD2 receptor (3).
Fig. 2. Structures of the N-linked carbohydrate chains of calreticulins in plants or mammals. Rat liver (a) and bovine brain (b) calreticulins have
complex-type or highly trimmed (Man-5) N-glycans, respectively. These oligosaccharide structures are typical of processing in the Golgi.
Calreticulins from spinach (c) and maize (d) contain Man-8 and/or Man-9 oligomannose structures that cannot be accounted for by a post-ER
processing.
back to the ER (hence, no complex glycans as marker)
or that this travel is just for a minority of them (i.e.
complex glycans are not abundant enough to be detectable). Besides the C-terminal tetrapeptide-mediated recycling, a further recently proposed ER retention mechanism
involves ill-defined sequences that limit the leakage of
resident proteins and is in keeping with the second
hypothesis (Murakami et al., 1994). The basis for such
retention is still unclear, but it may involve associations
with other ER proteins, similar to those described for
resident Golgi proteins in animal cells.
Several pieces of evidence favour a mechanism whereby
the diffusion of reticuloplasmins is limited to those ER
domains where proteins are concentrated before their
Protein retention in the endoplasmic reticulum 161
transport downstream in the secretory pathway. This
limited access would very efficiently exclude the reticuloplasmins from transport vesicles (Balch et al., 1994;
Mizuno and Singer, 1993). For instance, when the
C-terminal retention signal of the best characterized
ER-resident protein BiP is removed, only a poor secretion
of the truncated molecule is observed. Also, a stable
complex formation between several ER chaperones and
particularily between BiP and calreticulin, as recently
reported in plant and mammalian cells, is consistent with
the hypothesis of a network of resident proteins in the
ER ( Tatu and Helenius, 1997; Crofts et al., 1998).
The existence of two mechanisms including one that
keeps the reticuloplasmins in the ER and another that
recycles them from intermediate or post-ER compartments could explain the highly efficient retention of
natural reticuloplasmins in plant ER. This contrasts with
the partial retention usually observed with ‘artificial’
reticuloplasmins that are made of extracellular or vacuolar proteins fused with H/KDEL and whose location in
the ER would exclusively rely upon the efficiency of the
recycling machinery.
Retention of integral membrane ER proteins
Membrane proteins move along the secretory pathway
with the bulk of membrane flow. While the default
pathway for the vectorial flow of membrane proteins
leads from the ER to the plasma membrane (in animal
cells) or to the vacuole membrane (in yeast and possibly
in plant cells) specific signals and mechanisms are necessary to retain resident proteins within the ER or the
Golgi membranes.
A cytosolic di-lysine motif has been identified at the
C-terminal end of many type I integral membrane proteins
(amino terminus in the lumen) that reside in the ER of
yeast and animal cells. This sequence is both necessary
and sufficient for protein retention in the ER membrane.
The two critical lysine residues that are constitutive of
this retention signal must be in a −3 and a −4/−5
position relative to the C-terminus (Jackson et al., 1990,
1993; Townsley et al., 1993; Letourneur et al., 1994;
Nilsson et al., 1993; Gaynor et al., 1994; Rajagopalan
et al., 1994). As for H/KDEL-containing, soluble ER
proteins, the C-terminal di-lysine motif has been shown
to mediate retrieval of type I membrane proteins from
the Golgi back to the ER both in mammalian (Jackson
et al., 1993) and yeast cells (Gaynor et al., 1994). The
mechanism involved in this recycling process is not completely identified. While the ERD receptors that bind
K/HDEL sequences have been identified, the receptor(s)
that retain(s) type I integral membrane proteins in the
ER remains elusive. It was recently shown that the
di-lysine consensus motif (i.e. KKXX or KXKXX ) interacts specifically with the coatomer, a polypeptide complex
COPI implicated in vesicle-mediated transport between
Golgi cisternae as well as in retrograde transport from
the Golgi to the ER (Cosson and Letourneur, 1994;
Letourneur et al., 1994).
For the type II membrane proteins (C-terminus in the
lumen) a double arginine motif at the cytoplasmically
oriented N-terminus has also been identified as a retention/retrieval signal in the ER (Schutze et al., 1994).
These two critical arginine residues have to be within the
first five amino-terminal residues of the proteins.
However, as suggested above for soluble reticuloplasmins,
the correct location of ER membrane proteins may involve
more than one mechanism. Some evidences suggest that
retention and retrieval signals can also coexist in ER-resident
membrane proteins. For instance, the removal of the double
lysine motif from a mammalian, endogenous ER enzyme
( UDP-glucurosyl transferase) does not result in the loss of
ER retention (Jackson et al., 1993). This is also illustrated
with yeast Sec12p, a type II transmembrane glycoprotein of
the ER membrane. It was recently shown by Sato et al.
(1996) that the Sec12p ER retention is mediated by its
cytoplasmic portion while the retrieval is only dependent on
the transmembrane domain.
The recent cloning of plant ER type I membrane
proteins such as omega-3 desaturase from Arabidopsis
(Arondel et al., 1992) or calnexin from different plant
species (Huang et al., 1993; Coughlan et al., 1997;
Denecke et al., 1995; Hasenfratz et al., 1997) has suggested that similar motifs in the cytosolic tail of these
proteins could also be involved in their retention in the
ER membrane ( Table 1).
Calnexin is an abundant type I membrane protein first
described in mammalian ER ( Wada et al., 1991). In
plants, a calnexin-like protein presenting 63% similarity
with human calnexin was first cloned in A. thaliana
(Huang et al., 1993). Calnexin is a well-known ER
chaperone that transiently binds nascent glycoproteins
(Helenius et al., 1997; Coughlan et al., 1997). In recent
studies, calnexin was used as a model to define the signals
and mechanisms whereby a type I membrane protein is
retained in the plant ER. In both plants and mammals
calnexin has no di-lysine motif in its C-terminal cytosolic
tail which is surprising for a type I integral ER membrane
protein. Instead it harbours a consensus di-arginine motif
previously described as a retention/retrieval motif for type
II membrane ER proteins (Schutze et al., 1994).
Preliminary results have shown that, when fused with
GFP or phosphinothricin acetyl transferase (PAT ) used
as reporter proteins, the transmembrane domain and
cytosolic tail of tobacco or castor bean calnexins are
sufficient for retention of the transiently expressed fusion
proteins in the ER of tobacco protoplasts. When the
cytosolic tail or the last 10 amino acids including the
di-arginine motif of the C-terminal domain of these
calnexins are deleted from the fusion proteins the trun-
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Pagny et al.
Table 1. Alignment of the C-terminal sequences of two sets of type I membrane proteins, namely omega-3 desaturase from A. thaliana
and plant/mammalian calnexins.
cDNA accession numbers and species are given on the left.
Source
Sequence
D26508 A. thaliana
Omega-3 fatty acid
desaturase
.......... D L Y V Y A S D K S K I
L18888 Mus musculus
Calnexin mRNA
L18889 Rattus rattus
Calnexin mRNa
M94859 Homo sapiens
Calnexin mRNA
D78590 Rana rugosa
Calnexin mRNA
Z18242 A. thaliana
Calnexin mRNA
Z35108 H. tuberosus
Calnexin mRNA
U20502 Glycine max
Calnexin mRNA
X99488 D. melanogaster
Calnexin mRNA
X77569 Zea mays
Calnexin mRNA
N *
.......... L N R S P R N R K P R R E *
.......... L N R S P R N R K P R R E *
.......... L N R S P R N R K P R R E *
.......... L N R S P R N R K P R R D *
.......... E T A A P R K R Q P R R D N *
.......... E V A A A P R R R P R R D T *
.......... E A S N A A R R R P R R E T *
.......... S N T K T R K R Q A R K E *
........P R P R R R
.......... R K R M K R T R Q A H V G G P E E E T*
cated proteins are, at least in part, exported out of the
ER (Coughlan et al., 1997; Phillipson and Denecke,
1997). When human calnexin was expressed in COS cells
it was also shown that a truncated form lacking the
C-terminal six amino acids (RKPRRE ) was only partly
retained in the ER while a form deleted from the entire
cytoplasmic tail completely escapes the ER retention
machinery (Rajagopalan et al., 1994). These results could
indicate that the cytosolic tail of calnexin and more
particularly the highly conserved amino acid sequence
with a di-arginine motif at its C-terminal end is involved
in the retention in the ER membrane in both plant and
mammalian cells.
Recent studies suggest that several signals and mechanisms may concurrently play a role in retention of membrane
proteins both in the ER and the Golgi apparatus. The
relatively low stability of reporter proteins such as PAT or
GFP when they are transported downstream of the ER in
the plant secretory pathway does not allow one to exclude
the fact that several signals are necessary for an optimal
retention of membrane proteins in the plant ER.
Particularly, the participation of both soluble and transmembrane domains in the retention of membrane proteins
has been very well illustrated in mammalian cells for
several Golgi glycosyltransferases (Colley, 1997) and for
cytochrome p450 in the ER (Szezesna-Skorupa et al., 1995).
From pathways to freeways or what do we need to
speed up the description of the plant secretory
system?
The rapidly growing amount of information on tobacco
BY2 cell suspension cultures will probably provide plant
cell biology with a counterpart of what CHO and COS
cells are in mammalian studies. This will provide standard
immunostaining patterns for the different organelles in
plant cells, and such standards will result in a more
efficient description of the plant secretory system.
To address novel questions on protein transport and
targeting in the secretory pathway, such as retention of
proteins in the plant Golgi apparatus, further cellular
markers are needed. Antibodies to marker proteins that
are not only specific for the different organelles but also
for the vesicles that shuttle between these compartments
are still to be identified.
The analysis of glycan structures in reporter glycoproteins
is a major approach in mammalian and yeast cell biology.
A similar approach in plants is also needed which should
provide new information about the plant secretory pathway
(for a recent review, Lerouge et al., 1998). Getting new
probes for these oligosaccharide structures is probably one
of the major limitations that may be overcome in the not
too distant future and this will eventually result from an
unambiguous description of plant oligosaccharides and a
subcellular localization of their processing events.
Acknowledgements
This work was supported by a grant from CNRS, INSERM,
University of Rouen and Region Haute-Normandie. SP is a
recipient of a doctoral fellowship from MESRT. We are grateful
to JP Salier for critical reading of this review.
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