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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 158 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 160 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- 162 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). 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