Glycobiology vol. 10 no. 7 pp. 645–648, 2000
MINI REVIEW
Transport of free and N-linked oligomannoside species across the rough endoplasmic
reticulum membranes
René Cacan1 and André Verbert
Laboratoire de Chimie Biologique, CNRS-UMR 8576, Université des
Sciences et Technologies de Lille, 59655 Villeneuve d’Ascq Cedex, France
Accepted on February 25, 2000
The N-glycosylation process occurs in the rough endoplasmic reticulum. It requires the transport of glycosyl
donors into the lumen and the exit of the glycosylated products toward the secretory pathway. Besides this main flow,
the formation of free oligomannosides, glycopeptides, and
misfolded glycoproteins which do not enter the secretory
pathway and are cleared out of the endoplasmic reticulum
by specific transports has been demonstrated. This review
focuses on the export mechanisms of these three side products of the N-glycosylation process and discusses their
physiological significance.
Key words: N-glycosylation/oligomannoside/endoplasmic
reticulum/transport
Introduction
In eukaryotic cells, the rough endoplasmic reticulum (ER)
plays a key role in the biosynthesis and the quality control of
secretory and membrane glycoproteins. This requires an input
of glycosyl donors into the system and an output of glycosylated products. With respect to the dolichol cycle, this input
involves the transmembrane transport of mannose-phosphodolichol (Man-P-Dol) and glucose-phospho-dolichol (Glc-PDol) as well as the Man5GlcNAc2-PP-Dol intermediate. The
final Glc3Man9GlcNAc2-pyrophospho-dolichol is used as
donor in an “en bloc” transfer reaction to asparagine residues
of the acceptor protein. In the ER, the input also concerns the
entry of UDP-Glc (Vanstapel and Blanckaert, 1988; Castro et
al., 1999) as a substrate for unfolded protein reglucosylation, a
prerequisite for retention by calnexin or calreticulin. Once core
glycosylation and folding are completed successfully,
membrane and secretory glycoproteins are packaged into ERto-Golgi transport vesicles. This constitutes the major pathway
for the sorting of glycosylated proteins synthesized in the ER.
Besides this main flow toward the Golgi apparatus, several
different lines of research have demonstrated that free oligomannosides, glycopeptides, and misfolded glycoproteins do
1To
whom correspondence should be addressed
© 2000 Oxford University Press
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.
Efflux of free oligomannosides
It has been demonstrated in several biological models that the
synthesis of glycoconjugates generates substantial quantities
of free oligomannosides in the lumen of the rough endoplasmic
reticulum (for review see Cacan and Verbert, 1999; Moore,
1999). This material is in part constituted of oligomannosides
possessing di-N-acetyl-chitobiosyl moieties at the reducing
end (mainly OS-Gn2), derived from the precursor
Glc3Man9GlcNAc2 structure. These originate from the hydrolysis of oligosaccharide-PP-dolichol, presumably as a result of
transfer to water. Several lines of evidence, such as the effect
of EDTA (Anumula and Spiro, 1983) or cycloheximide
(Villers et al., 1994), indicate that this phenomenon was
enhanced when the level of newly synthesized proteins to be
glycosylated was low. This material does not follow the secretory pathway since it is not recovered in the cell culture
medium. However, pulse chase experiments have shown that
this oligosaccharide material is recovered in the cytosol as
oligomannosides possessing a single GlcNAc residue (OSGn1) at the reducing end. Moore and Spiro (1994) have clearly
shown that oligomannosides are transported into the cytosol as
OS-Gn2. We demonstrated (Cacan et al., 1996) that it is further
cleaved by a cytosolic chitobiase. This cleavage is a prerequisite to the action of cytosolic mannosidase, which is known to
act specifically on OS-Gn1 species (Grard et al., 1996).
Sequential action of cytosolic chitobiase and α-mannosidase
leads to the formation of a specific Man5Gn1 isomer: Manα1,2
Manα1,2 Manα1,3 (Manα1,6) Manβ1,4 GlcNAc, (Kmiécik et
al., 1995; Saint-Pol et al., 1997). This compound is finally
targeted to the lysosomes where it is further degraded into
monosaccharides. The key step of this oligomannoside trafficking is the transport from the ER lumen to the cytosol by a
specific carrier which has been thoroughly studied (Moore et
al., 1995; Moore, 1998). In vitro experiments have shown that
the carrier is effective only if ATP is available for hydrolysis.
It was demonstrated that the transport is inhibited if the transport assay medium is depleted of calcium ions; if thapsigargin,
an inhibitor of [Ca++-Mg ++]ATPase, is added; or if calcium
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ionophore is present. These observations suggest that the transport process requires the presence of calcium sequestered in
the lumen of ER.
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.
Efflux of glycopeptides
Another source of OS-Gn2 in the cytosol could be the cleavage
by a cytosolic peptide N-glycanase (PNGase) of glycopeptides
which are exported from the ER lumen as described by Römish
and Ali (1997). The first observation was made by GeethaHabib et al. (1990), who investigated the fate of peptides
injected in the cytosol of Xenopus oocytes. They observed that
the peptides are glycosylated but are neither recovered in the
Golgi nor secreted. In fact, they are degraded and their degradation is inhibited by chloroquine thereby indicating it is
achieved by lysosomal enzymes. This result implies that glycopeptides have first to enter the ER to be glycosylated and then
are re-exported to the cytosol to be finally degraded in lysosomes. This export of glycopeptides has been further studied in
Saccharomyces cerevisiae and compared to the Golgi-directed
vesicular transport of glycoproteins (Römisch and Schekman,
1992). Both processes are cytosol-, temperature-, and ATPdependent, but the traffic of glycopeptides is not inhibited by
antibodies against two proteins essential for the budding of
transport vesicles (Sec23p and p105). Thus, the released
glycopeptides, in contrast to glycoproteins, are not entrapped
into vesicles since they do not acquire yeast Golgi specific α1,6 linked mannose residues.
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 (Römisch and
Ali, 1997). Experiments performed with ER and cytosol from
heterologous sources have indicated that the process is
646
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.
Retrotranslocation of glycoproteins
Wiertz (1996a) first demonstrated that infection with human
cytomegalovirus induces the degradation of the N-glycosylated major histocompatibility complex (MHC) class I molecules of host cells. They showed that this degradation process
requires the retrotranslocation of the glycoprotein from the ER
lumen to the cytosol to be further degraded by the proteasome.
They have shown the presence in the cytosol of a deglycosylated MHC intermediate suggesting the action of a putative
peptide N-glycanase, indicating that the protein has to be retrotranslocated in its glycosylated form. A similar retrotranslocation of the glycosylated form of glycoproteins has also been
demonstrated for misfolded MHC class I heavy chain in nonvirally infected cells (Hughes et al., 1997) for T-cell receptor αchain (Yu et al., 1997) and for truncated ribophorin I (de
Virgilio et al., 1998).
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 heatshock 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 β-endo N-acetylglu-
Export mechanisms for side products of N-glycosylation
Fig. 1. Schematic representation of the ER lumen illustrating the transport of free and N-linked oligomannosides. On left, import of glycosyl donors, peptides and
nascent proteins for the formation of free oligomannosides (1), glycopeptides (2), and glycoproteins (3). On right, besides the vesicular secretory pathway to Golgi,
export of free oligomannosides A, glycopeptides B and retrotranslocation of glycoproteins C. OS, Oligosaccharide; G, glucose residue; M, mannose residue; Gn,
N-acetyl-glucosamine residue; TAP, transporter associated with antigen processing. Dashed lines indicate putative pathways.
cosaminidase 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 β-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-γ 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.
Conclusion
The N-glycosylation process can produce four different types
of glycosylated species: correctly folded glycoproteins,
misfolded or misassembled glycoproteins, glycopeptides, and
free oligomannosides. Among these four end products, only
the correctly folded glycoproteins are transported to the Golgi
apparatus for plasma membrane insertion or for secretion.
Thus, the three other ones have to be eliminated from the ER
lumen. Presumably, if they would follow the secretion
pathway they would interfere and compete with the glycosylation machinery within the Golgi. This review has discussed
three different export processes for each of these N-glycosylation side products. Indeed, this appears to answer why a more
strict control of the N-glycosylation early steps (i.e., the dolichol cycle) does not seem to be exerted. So far, few reports
have described the regulation of the dolichol cycle and these
mainly concern the inhibition of the formation of GlcNAc-PPDol by Man-P-Dol (Kean, 1982) and the control of Man-P-Dol
synthase activity by phosphorylation (Banerjee et al., 1987).
This could suggest that regulation might occur at other downstream steps such as the degradation of unsuitable products
formed in excess. This would explain the degradation of
oligosaccharide-PP-Dol into soluble OS-Gn2 and the degradation of misfolded glycoproteins.
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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R.Cacan and A.Verbert
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