Laboratoire de Chimie Biologique, CNRS-UMR 8576, Université des Sciences et Technologies de Lille, 59655 Villeneuve dAscq Cedex, France
Accepted on February 25, 2000;
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Abstract |
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Key words: N-glycosylation/oligomannoside/endoplasmic reticulum/transport
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Introduction |
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Besides this main flow toward the Golgi apparatus, several different lines of research have demonstrated that free oligomannosides, glycopeptides, and misfolded glycoproteins do not follow the secretory pathway but are transported across the membrane from the ER lumen to the cytosol.
This review will focus on what is known about the export mechanisms of these three different molecular species. We will also discuss the physiological significance of this efflux of glycosylated molecules produced during the N-glycosylation process.
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Efflux of free oligomannosides |
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Regarding its specificity, this carrier recognizes the terminal nonreducing mannosyl part of the free oligomannosides. Indeed, competitive inhibition of transport is observed during in vitro assays in the presence of mannose derivatives modified at the carbon 1 position (Moore, 1998). Moreover, it has been shown by inhibiting glucosidases with castanospermine in vivo, that oligomannosides which have retained the glucose residues are not transported (Moore et al., 1995
). It is interesting to note that the transport process is suspected to be unidirectional since the presence of 1mM OS-Gn2 outside the vesicles did not inhibit the exit transport (Moore, 1998
). Assuming that the lumenal concentration of OS-Gn2 does not exceed 1 mM, it can be postulated that this transport process can operate against a concentration gradient.
As proposed (Verbert and Cacan, 1999), this efflux of oligomannosides provides an ER lumen clearing mechanism. It is indeed suspected that free oligomannosides would otherwise compete for oligomannoside recognizing proteins such as glycosidases, chaperones, and glycosyltransferases. In addition, free oligomannosides would follow the secretory pathway and interfere with the Golgi processing machinery.
Another possible function for this oligomannoside trafficking would be to provide the cytosol with potential oligomannoside ligands for lectins which have been described in other subcellular locations such as the nucleus. However this possibility still needs to be explored experimentally.
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Efflux of glycopeptides |
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This same transmembrane export mechanism has been described in mammalian cells (dog pancreas, rat liver), but in this case the glycopeptides are not recovered in the cytosol due to their rapid degradation by a cytosolic PNGase (Römish and Ali, 1997). Experiments performed with ER and cytosol from heterologous sources have indicated that the process is conserved throughout evolution and thus appears to be of importance.
This transport mechanism has been studied by Ali and Field (2000). It was shown to be distinct from the oligomannoside carrier previously described by Moore (1998)
, through a number of criteria. First, glycopeptide export requires Mg++ but not Ca++; second, thapsigargicin and calcium ionophore stimulates the export; and third, the presence of glucosyl residues does not impair the export. Furthermore it has been recently shown in yeast that export of glycopeptides is not affected by the structure of their oligosaccharide chains (Suzuki and Lennarz, 2000
).
Neither the physiological significance of this phenomenon nor the origin of the glycopeptides formed in the lumen of the ER are yet understood. They could originate from glycosylation of peptides which have entered the ER lumen. Indeed such peptide carriers have been described (Kleijmeer et al., 1992). On the other hand, they could be generated through proteolytic cleavage of glycoproteins in the ER. The latter process could be a pathway parallel to the proteasome dependent degradation of newly synthesized glycoproteins.
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Retrotranslocation of glycoproteins |
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The retrotranslocation involves the Sec 61 complex in what appears to be a reversal of the reaction by which nascent peptide chains are translocated into the endoplasmic reticulum (Bonifacino, 1996; Wiertz, 1996b
; Pilon, 1997
; Plemper et al., 1997
). This reverse transport through the translocon machinery strongly suggests that the glycoproteins have to be unfolded to reengage the Sec 61 complex for translocation thus leading to cytosolic destruction.
So far, the putative driving forces would be the association either with calnexin (McCracken and Brodsky, 1996; Qu et al., 1996
; Liu et al., 1999
), Kar2p/Bip (Plemper et al., 1997
; Skowronek et al., 1998
; Brodsky et al., 1999
), or PDI (Gillece et al., 1999
) in the lumenal side, and the association with heat-shock protein (hsp 70) in the cytosolic compartment (Fischer et al., 1997
). The ATPase activity associated to proteasome could also be involved as suggested by Mayer et al. (1998)
.
However, cytosolic deglycosylation of retrotranslocated glycoprotein has to occur before ubiquitination and degradation. This observation raises the question of the occurrence of cytosolic endoglycanases. Two endoglycanase activities have been already pointed out in the cytosol: a ß-endo N-acetylglucosaminidase described by Pierce et al., 1979, 1980), and more recently a PNGase characterized and isolated by Suzuki et al., 1994
, 1998). By metabolic labeling of the glycan moieties we have correlated the presence of OS-Gn1 in the cytosol with the degradation of newly synthesized glycoproteins (Villers et al., 1994
; Duvet et al., 1998
). Thus, two deglycosylation pathways are possible: the complete deglycosylation by PNGase releasing OS-Gn2 which is immediately degraded to OS-Gn1 by a cytosolic chitobiase (Cacan et al., 1996
) or the cleavage of the glycoprotein by the ß-endo N-acetylglucosaminidase releasing directly OS-Gn1. The latter process produces glycoproteins with a single GlcNAc attached to their N-glycosylation sites. Such intermediates have been recently reported in recombinant human interferon-
produced in CHO and insect cells (Hooker et al., 1999
).
This whole process appears to be correlated with the expression of misfolded or misassembled glycoproteins. This suggests that this glycoprotein efflux is used to clear the ER from such denatured glycoproteins as a result of quality control.
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Conclusion |
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On the other hand, glycopeptides can originate from proteolytic cleavage of glycoproteins. However, this raises the question of a subcompartment of ER allowing the segregation of the biosynthetic and degradation pathways. It can be equally postulated that they originate from glycosylation of cytosolic peptides which have entered the ER. The possibility of such a posttranslational glycosylation reaction has already been demonstrated by numerous experiments involving oligosaccharyl transferase assays performed with synthetic peptides. It is interesting to note that whatever is the origin of the glycosylated species transported to the cytosol (free oligomannosides, glycopeptides, retrotranslocated glycoproteins), the end product is a unique oligosaccharide Man5GlcNAc1 specifically recognized by a lysosomal carrier for entry and degradation. The different transports of free and N-linked oligomannoside species across the rough endoplasmic reticulum membranes are summarized in the Figure 1.
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Footnotes |
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References |
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