Glycopeptide export from mammalian microsomes is independent of calcium and is distinct from oligosaccharide export

Glycopeptides are exported from the endoplasmic reticulum to the cytosol of eukaryotic membranes in an ATP- and cytosol-requiring process (Romisch and Ali, 1997, Proc. Natl. Acad. Sci. USA,94, 6730-6734). Oligosaccharides of the polymannose-type are also exported from the endoplasmic reticulum of mammalian cells to the cytosol in an ATP-dependent fashion. These findings raise the strong possibility that the two substrate classes are transported by the same mechanism but the precise identity of the trans-location machinery for each substrate class has not been fully defined. Here we have investigated the mechanism by which a glycopeptide is exported from rat liver microsomes, and compare this to the export of free polymannose oligosaccharides. Using EGTA and the endoplasmic reticulum calcium mobilizing agents thapsigargicin and calcium ionophores A23187 and ionomycin, we show that glycopeptides, in contrast to oligosaccharides, are exported by a calcium-independent mechanism. On the other hand, Mg(2+)is required in the assay for the transport of glycopeptide from mammalian microsomes which is in common with oligosaccharide export. Deoxynojirimycin and castanospermine, inhibitors of ER glucosidases, when added to rat liver microsomes prior to loading with peptide that bears an N -glycosylation sequon, had no effect on the release of glucosylated glycopeptides from membranes, indicating that removal of the alpha-glucose units from the oligomannose glycan structure of the glycopeptide is not required for export. In contrast to oligosaccharides, where transport is efficiently inhibited, mannosides were without effect or only weak inhibitors of glycopeptide export. Taken together, these data suggest that glycopeptides are exported by a distinct mechanism from oligosaccharides of the polymannose-type and that the peptide moiety is an important structural determinant for glycopeptide export and capable of directing translocation of substrates to a specific transport pathway.     

Cytosol-to-lysosome transport of free polymannose-type oligosaccharides. Kinetic and specificity studies using rat liver lysosomes

In hepatocellular carcinoma HepG2 cells, free polymannose-type oligosaccharides appearing in the cytosol during the biosynthesis and quality control of glycoproteins are rapidly translocated into lysosomes by an as yet poorly defined process (Saint-Pol, A., Bauvy, C., Codogno, P., and Moore, S. E. H. (1997) J. Cell Biol. 136, 45-59). Here, we demonstrate an ATP-dependent association of [2-3H]mannose-labeled Man5GlcNAc with isolated rat liver lysosomes. This association was only observed in the presence of swainsonine, a mannosidase inhibitor, which was required for the protection of sedimentable, but not nonsedimentable, Man5GlcNAc from degradation, indicating that oligosaccharides were transported into lysosomes. Saturable high affinity transport (Kuptake, 22.3 microM, Vmax, 7.1 fmol/min/unit of beta-hexosaminidase) was dependent upon the hydrolysis of ATP but independent of vacuolar H+/ATPase activity. Transport was inhibited strongly by NEM and weakly by vanadate but not by sodium azide, and, in addition, the sugar transport inhibitors phloretin, phloridzin, and cytochalasin B were without effect on transport. Oligosaccharide import did not show absolute specificity but was selective toward partially demannosylated and dephosphorylated oligosaccharides, and, furthermore, inhibition studies revealed that the free reducing GlcNAc residue of the oligosaccharide was of critical importance for its interaction with the transporter. These results demonstrate the presence of a novel lysosomal free oligosaccharide transporter that must work in concert with cytosolic hydrolases in order to clear the cytosol of endoplasmic reticulum-generated free oligosaccharides.     

Oligosaccharide transport: pumping waste from the ER into lysosomes.

N-glycans play important roles during the folding and secretion of glycoproteins. Surprisingly, during the N-glycosylation of glycoproteins, considerable amounts of unconjugated polymannose-type oligosaccharides ('free OS') are generated. Although free oligosaccharides have no known function in mammalian cells, a sophisticated cellular machinery enables them to be cleared from the endoplasmic reticulum (ER) into the cytosol and then re-enter the endomembrane system at the level of the lysosome. One possible function of this pathway is to stop free OS from interfering with the carbohydrate-dependent aspects of glycoprotein folding and transport along the secretory pathway.     

Endoplasmic reticulum-to-cytosol transport of free polymannose oligosaccharides in permeabilized HepG2 cells.

Free polymannose oligosaccharides have recently been localized to both the vesicular and cytosolic compartments of HepG2 cells. Here we investigated the possibility that free oligosaccharides originating in the lumen of the endoplasmic reticulum (ER) are transported directly into the cystosol. Incubation of permeabilized cells in the absence of ATP at 37 degrees C led to the intravesicular accumulation of free Man9GlcNAc2 which was generated from dolichol-linked oligosaccharide in the ER. This oligosaccharide remained stable within the permeabilized cells unless ATP was added to the incubations at which time the Man9GlcNac2 was partially converted to Man8GlcNAc2, and both these components were released from an intravesicular compartment into the cytosolic compartment of permeabilized cells. In contrast, when permeabilized cells, primed with either free triglucosyl-oligosaccharide or a glycotripeptide, were incubated with ATP both these structures remained associated with the intravesicular compartment. As the conditions in which free oligosaccharides were transported out of the intravesicular compartment into the cytosolic compartment did not permit vesicular transport of glycoproteins from the ER to the Golgi apparatus our data demonstrate the presence of a transport process for the delivery of free polymannose oligosaccharides from the ER to the cytosol.     

Structural investigation of glycan recognition by the ERAD quality control lectin Yos9

Yos9 is an essential component of the endoplasmic reticulum associated protein degradation (ERAD) system that is responsible for removing terminally misfolded proteins from the ER lumen and mediating proteasomal degradation in the cytosol. Glycoproteins that fail to attain their native conformation in the ER expose a distinct oligosaccharide structure, a terminal α1,6-linked mannose residue, that is specifically recognized by the mannose 6-phoshate receptor homology (MRH) domain of Yos9. We have determined the structure of the MRH domain of Yos9 in its free form and complexed with 3α, 6α-mannopentaose. We show that binding is achieved by loops between β-strands performing an inward movement and that this movement also affects the entire β-barrel leading to a twist. These rearrangements may facilitate the processing of client proteins by downstream acting factors. In contrast, other oligosaccharides such as 2α-mannobiose bind weakly with only locally occurring chemical shift changes underscoring the specificity of this substrate selection process within ERAD.     

Man2C1, an alpha-mannosidase, is involved in the trimming of free oligosaccharides in the cytosol

The endoplasmic-reticulum-associated degradation of misfolded (glyco)proteins ensures that only functional, correctly folded proteins exit from the endoplasmic reticulum and that misfolded ones are degraded by the ubiquitin-proteasome system. During the degradation of misfolded glycoproteins, they are deglycosylated by the PNGase (peptide:N-glycanase). The free oligosaccharides released by PNGase are known to be further catabolized by a cytosolic alpha-mannosidase, although the gene encoding this enzyme has not been identified unequivocally. The findings in the present study demonstrate that an alpha-mannosidase, Man2C1, is involved in the processing of free oligosaccharides that are formed in the cytosol. When the human Man2C1 orthologue was expressed in HEK-293 cells, most of the enzyme was localized in the cytosol. Its activity was enhanced by Co2+, typical of other known cytosolic alpha-mannosidases so far characterized from animal cells. The down-regulation of Man2C1 activity by a small interfering RNA drastically changed the amount and structure of oligosaccharides accumulating in the cytosol, demonstrating that Man2C1 indeed is involved in free oligosaccharide processing in the cytosol. The oligosaccharide processing in the cytosol by PNGase, endo-beta-N-acetylglucosaminidase and alpha-mannosidase may represent the common 'non-lysosomal' catabolic pathway for N-glycans in animal cells, although the molecular mechanism as well as the functional importance of such processes remains to be determined.     

N-glycan structure dictates extension of protein folding or onset of disposal

The endoplasmic reticulum (ER) is the site of folding for proteins that are resident in the ER or that are destined for the Golgi, endosomes, lysosomes, the plasma membrane, or secretion. Cotranslational addition of preassembled glucose(3)-mannose(9)-N-acetylglucosamine(2) core oligosaccharides (N-glycosylation) is a common event for polypeptides synthesized in this compartment. Protein-bound oligosaccharides are exposed to several ER glycanases that sequentially remove terminal glucose or mannose residues. Their activity must be tightly regulated because the N-glycan composition determines whether the associated protein is subjected to folding attempts in the ER lumen or whether it is retrotranslocated into the cytosol and degraded.     

Ordered assembly of the asymmetrically branched lipid-linked oligosaccharide in the endoplasmic reticulum is ensured by the substrate specificity of the individual glycosyltransferases.

The assembly of the lipid-linked core oligosaccharide Glc3Man9GlcNAc2, the substrate for N-linked glycosylation of proteins in the endoplasmic reticulum (ER), is catalyzed by different glycosyltransferases located at the membrane of the ER. We report on the identification and characterization of the ALG12 locus encoding a novel mannosyltransferase responsible for the addition of the alpha-1,6 mannose to dolichol-linked Man7GlcNAc2. The biosynthesis of the highly branched oligosaccharide follows an ordered pathway which ensures that only completely assembled oligosaccharide is transferred from the lipid anchor to proteins. Using the combination of mutant strains affected in the assembly pathway of lipid-linked oligosaccharides and overexpression of distinct glycosyltransferases, we were able to define the substrate specificities of the transferases that are critical for branching. Our results demonstrate that branched oligosaccharide structures can be specifically recognized by the ER glycosyltransferases. This substrate specificity of the different transferases explains the ordered assembly of the complex structure of lipid-linked Glc3Man9GlcNAc2 in the endoplasmic reticulum.     

Analysis of the glycosylation and phosphorylation of the alpha-subunit of the lysosomal enzyme, beta-hexosaminidase A, by site-directed mutagenesis.

The glycosylation and subsequent phosphorylation of mannose residues is a pivotal modification during the biosynthesis of lysosomal enzymes. We have identified the sites of N-linked glycosylation and oligosaccharide phosphorylation on the alpha-subunit of beta-hexosaminidase and have determined the influence of the oligosaccharides on the folding and transport of the enzyme. The potential glycosylation sequences, either singly or in combination, were eliminated through site-directed mutagenesis of the cDNA. By expression of the mutant cDNAs in COS-1 cells, each of the three glycosylation sites on the alpha-subunit was found to be modified by an oligosaccharide. One of the three oligosaccharides was the preferred site of phosphorylation. The absence of any individual oligosaccharide did not diminish the expression of the catalytic activity associated with the alpha-chain, implying proper folding and assembly of subunits. A profound effect was observed, however, when all three oligosaccharides were absent. The unglycosylated alpha-subunit, resulting from genetic alteration of all three glycosylation sites or synthesis of the wild-type protein in the presence of tunicamycin, was catalytically inactive. It was found to be improperly folded into an insoluble aggregate, linked through inappropriate disulfide bonds. The unglycosylated protein was trapped in the lumen of the endoplasmic reticulum and was found in a complex with the Ig heavy chain-binding protein, BiP. The properties of the nonglycosylated, misfolded alpha-subunit were similar to some mutant alpha-subunits in Tay-Sachs disease patients. The results indicate that the oligosaccharides are essential, although not in a site-specific manner, for proper folding and cellular transport of the alpha-subunit.     

Evidence for an alpha-mannosidase in endoplasmic reticulum of rat liver

An alpha-mannosidase activity has been identified in a preparation of rat liver endoplasmic reticulum and shown to be distinct from the previously described Golgi alpha-mannosidases I and II and the lysosomal alpha-mannosidase. The enzyme was solubilized with deoxycholate and separated from other alpha-mannosidases by passage over concanavalin A-Sepharose to which it does not bind. The endoplasmic reticulum alpha-mannosidase cleaves alpha-1,2-linked mannoses from high mannose oligosaccharides and, unlike Golgi alpha-mannosidase I, is active against p-nitrophenyl-alpha-D-mannoside (Km = 0.17 mM). It has no activity toward GlcNAc-Man5GlcNAc2 peptide, the specific substrate of the Golgi alpha-mannosidase II. The endoplasmic reticulum alpha-mannosidase activity toward p-nitrophenyl-alpha-D-mannoside is relatively insensitive to swainsonine, an inhibitor of both the lysosomal alpha-mannosidase and Golgi alpha-mannosidase II. We propose that the endoplasmic reticulum alpha-mannosidase is responsible for the removal of mannose residues from asparagine-linked high mannose type oligosaccharides prior to their entry into the Golgi.     

Maturation of lipoprotein lipase. Expression of full catalytic activity requires glucose trimming but not translocation to the cis-Golgi compartment.

The relationship between maturation of lipoprotein lipase (LPL) and its translocation from the endoplasmic reticulum (ER) to the Golgi complex was determined by measuring lipolytic activity under conditions preventing transport of the enzyme from the ER to the Golgi compartment. In the presence of brefeldin A, a reagent that inhibits movement of proteins from the ER and causes the disassembly of the Golgi complex, pro-5 Chinese hamster ovary cells accumulated catalytically active LPL, while secretion of the enzyme was effectively blocked. LPL retained intracellularly by brefeldin A treatment possessed oligosaccharide chains that were processed to the complex form by the Golgi enzymes redistributed into the ER. At 16 degrees C, a condition disrupting protein transport to the cis-Golgi, the retained enzyme again remained catalytically active although the oligosaccharides remained in the high mannose form. Lastly, attachment of the specific ER retention signal KDEL (Lys-Asp-Glu-Leu) to the carboxyl terminus of LPL also resulted in intracellularly retained enzyme that was fully active. The importance of oligosaccharide processing for attainment of LPL catalytic activity in vitro was also determined. LPL was active and secreted when trimming of the mannose residues was inhibited by deoxymannojirimycin and when addition of complex sugars was blocked using Chinese hamster ovary mutants (lec1 and lec2), indicating that these processing events are not necessary for the expression of a functional enzyme. However, blocking glucose removal by glucosidase inhibitors (castanospermine and N-methyl-deoxynojirimycin) resulted in a significant reduction in LPL specific activity and secretion. Thus, glucose trimming of LPL oligosaccharides is essential for enzyme activation; however, further oligosaccharide processing or translocation of the enzyme to the cis-Golgi is not required for full expression of lipolytic activity in vitro.     

Chemical modification of the glucosidase inhibitor 1-deoxynojirimycin. Structure-activity relationships.

The ability of the glucosidase inhibitor 1-deoxynojirimycin (dNM) and a series of N-alkylated dNM derivatives to interfere with biosynthesis, transport, and maturation of the glycoprotein alpha 1-antitrypsin in HepG2 cells was investigated. Inhibition of endoplasmic reticulum glucosidase I and II by dNM and its derivatives resulted in an intracellular accumulation of alpha 1-antitrypsin with glucose-containing high mannose type oligosaccharides (precursor). N-alkylation of dNM increased its potency in inhibiting endoplasmic reticulum glucosidases, as determined from the concentration required for half maximal inhibition. N-Alkylated derivatives of dNM were better able to inhibit glucosidase I than glucosidase II (deduced from the number of glucose residues retained in Endo H-releasable oligosaccharides). The inhibition of glucosidase activity imposed by alkylated dNM derivatives was less easily reversed than that by dNM, an effect most pronounced for N-methyl-dNM. Branching of the alkyl group of dNM derivatives decreased the inhibitory potency. Although dNM and its derivatives interfered strongly with intracellular oligosaccharide processing, they did not completely block N-glycan maturation of alpha 1-antitrypsin even at the highest concentrations tested.     

The soluble form of rat liver alpha-mannosidase is immunologically related to the endoplasmic reticulum membrane alpha-mannosidase

The soluble alpha-mannosidase of rat liver, originally described as a cytoplasmic alpha-mannosidase, has been purified to homogeneity by conventional techniques. The purified enzyme has an apparent molecular weight of 350,000 and is composed of 107-kDa subunits. The soluble alpha-mannosidase has the same enzymatic properties as the endoplasmic reticulum (ER) membrane alpha-mannosidase of rat liver (Bischoff, J., and Kornfeld, R. (1983) J. Biol. Chem. 258, 7909-7910) which is believed to play a role in oligosaccharide processing in the rough ER. Like the membrane-bound ER alpha-mannosidase, the soluble alpha-mannosidase can hydrolyze alpha-linked mannose from both p-nitrophenyl alpha-mannoside (Km = 0.14 mM) and high mannose oligosaccharides, is not inhibited by the mannose analogues swainsonine and 1-deoxymannojirimycin, is stabilized by MnCl2 or CoCl2, and does not bind to concanavalin A-Sepharose. A goat polyclonal antibody raised against the purified soluble alpha-mannosidase specifically recognizes the rat liver membrane-bound ER alpha-mannosidase, leading us to propose that they are two forms of the same enzyme and that the soluble form is derived from the ER membrane alpha-mannosidase by proteolysis. The antibody also cross-reacts with both the soluble and membrane-bound forms of ER alpha-mannosidase activity in cultured Chinese hamster ovary cells and rat H35 hepatoma cells. Since the ER alpha-mannosidase is presumed to be involved in the early steps of oligosaccharide processing, the action of the purified soluble form of the enzyme on high mannose oligosaccharides was examined. Surprisingly, the enzyme released free mannose from oligosaccharides ranging in size from Glc1Man9GlcNAc to Man5GlcNAc with almost equal efficiency. However, a long term incubation of the enzyme with Man9GlcNAc led to the accumulation of Man7GlcNAc and produced only small amounts of Man6GlcNAc and Man5GlcNAc. Structural analysis of these reaction products indicated that the purified soluble form of ER alpha-mannosidase shows little specificity for which mannose residues it removes from Man9GlcNAc. In contrast, as shown in the accompanying paper, the intracellular action of ER alpha-mannosidase on glycoprotein-bound Man9GlcNAc2 is highly specific.     

Discrimination between lumenal and cytosolic sites of deglycosylation in endoplasmic reticulum-associated degradation of glycoproteins by using benzyl mannose in CHO cell lines

Recent studies demonstrated that deglycosylation step is a prerequisite for endoplasmic reticulum (ER)-associated degradation of misfolded glycoproteins. Here, we report the advantages of using benzyl mannose during pulse-chase experiments to study the subcellular location of the deglycosylation step in Chinese hamster ovary (CHO) cell lines. Benzyl mannose inhibited both the ER-to-cytosol transport of oligomannosides and the trimming of cytosolic-labeled oligomannosides by the cytosolic mannosidase in vivo. We pointed out the occurrence of two subcellular sites of deglycosylation. The first one is located in the ER lumen, and led to the formation of Man8GlcNAc2 (isomer B) in wild-type CHO cell line and Man4GlcNAc2 in Man-P-Dol-deficient cell line. The second one was revealed in CHO mutant cell lines for which a high rate of glycoprotein degradation was required. It occurred in the cytosol and led to the liberation of oligosaccharides species with one GlcNAc residue and with a pattern similar to the one bound onto glycoproteins. The cytosolic deglycosylation site was not specific for CHO mutant cell lines, since we demonstrated the occurrence of cytosolic pathway when the formation of truncated glycans was induced in wild-type cells.     

Trafficking of oligomannosides released during N-glycosylation: a clearing mechanism of the rough endoplasmic reticulum

The main reaction of N-glycosylation of proteins is the transfer 'en bloc' of the oligosaccharide moieties of lipid intermediates to an asparagine residue of the nascent protein. For the past 15 years, a few laboratories including ours have shown that the process was accompanied by the release of oligosaccharide-phosphates and of neutral oligosaccharides possessing one GlcNAc (OS-Gn(1)) or two GlcNAc (OS-Gn(2)) at the reducing end. The aim of this review is to gather the evidence for the different origins of these soluble oligomannosides, to examine their subcellular location and intracellular trafficking. Furthermore, using Brefeldin A we demonstrated that this released oligomannoside material could be the substrate for the Golgi glycosidases and glycosyltransferases. Indeed, released oligomannoside never reach the Golgi vesicles either because they are directly produced in the cytosol as has been demonstrated for oligosaccharide-phosphates and for neutral oligosaccharides possessing one GlcNAc at the reducing end or because they are actively transported out of the rough endoplasmic reticulum to the cytosol. One of the functions of oligomannoside trafficking between rough endoplasmic reticulum, cytosol and lysosomes could be to prevent these oligosaccharides for competing with glycosylation in the Golgi.     

Lipid carriers in the synthesis of high-mannose glycoproteins in algae.

Particulate preparations from the chlorophyta Prototheca zopfii catalyze the incorporation of mannose and N-acetylglucosamine into glycolipids. These had been characterized as lipid monophosphate mannose, lipid pyrophosphate N,N'-diacetylchitobiose and various lipid-linked oligosaccharides containing two N-acetylglucosamine residues plus a variable number of mannose residues. The lipid moiety has the properties expected for dolichyl phosphate. The oligosacchride-linked lipids serve as precursors for the formation of a polymer sensible to pronase digestion. The oligosaccharide is linked by N-glycosidic linkage to an asparagine residue. In longer incubation periods, a polymer insensitive to pronase hydrolysis, but precipitable by copper salts such as cell wall mannans is formed. Polymer formation is inhibited by 1 mM bacitracin. The reactions leading to the formation of the mannoprotein were found associated to the rough endoplasmic reticulum. The synthesis of mannans was found to occur in the Golgi vesicles.     

Glucose removal from N-linked oligosaccharides is required for efficient maturation of certain secretory glycoproteins from the rough endoplasmic reticulum to the Golgi complex

1- Deoxynojirimycin is a specific inhibitor of glucosidases I and II, the first enzymes that process N-linked oligosaccharides after their transfer to polypeptides in the rough endoplasmic reticulum. In a pulse- chase experiment, 1- deoxynojirimycin greatly reduced the rate of secretion of alpha 1-antitrypsin and alpha 1-antichymotrypsin by human hepatoma HepG2 cells, but had marginal effects on secretion of the glycoproteins C3 and transferrin, or of albumin. As judged by equilibrium gradient centrifugation, 1- deoxynojirimycin caused alpha 1- antitrypsin and alpha 1-antichymotrypsin to accumulate in the rough endoplasmic reticulum. The oligosaccharides on cell-associated alpha 1- antitrypsin and alpha 1-antichymotrypsin synthesized in the presence of 1- deoxynojirimycin , remained sensitive to Endoglycosidase H and most likely had the structure Glu1- 3Man9GlcNAc2 . Tunicamycin, an antibiotic that inhibits addition of N-linked oligosaccharide units to glycoproteins, had a similar differential effect on secretion of these proteins. Swainsonine , an inhibitor of the Golgi enzyme alpha- mannosidase II, had no effect on the rates of protein secretion, although the proteins were in this case secreted with an abnormal N- linked, partially complex, oligosaccharide. We conclude that the movement of alpha 1-antitrypsin and alpha 1-antichymotrypsin from the rough endoplasmic reticulum to the Golgi requires that the N-linked oligosaccharides be processed to at least the Man9GlcNAc2 form; possibly this oligosaccharide forms part of the recognition site of a transport receptor for certain secretory proteins.     

Mannose processing is an important determinant in the assembly of phosphorylated high mannose-type oligosaccharides

Phosphorylation of the high mannose-type oligosaccharides attached to newly synthesized acid hydrolases occurs in two sequential steps within the endoplasmic reticulum and the Golgi apparatus, and the products generated at the two sites differ with respect to the location of the phosphorylated mannose residue. To investigate the mechanism of this two-step phosphorylation, biosynthesis of the Man-6-P recognition marker was studied in class E Thy-1- and J774 cells metabolically labeled with [2-3H]mannose. Class E Thy-1- cells produce truncated high mannose oligosaccharides that lack 4 mannose residues from the alpha 1,6-branch of the core beta-linked mannose residue; three of the missing residues are potential phosphorylation sites. Acid hydrolases produced by these mutant cells were phosphorylated on the alpha 1,3-branch of the truncated oligosaccharide even when transport to the Golgi apparatus was inhibited. J774 cells produce normal high mannose oligosaccharides, but they secrete a large percentage of their newly synthesized acid hydrolases. The secreted enzymes contained primarily diphosphorylated units in which a phosphate was positioned to both the alpha 1,3- and alpha 1,6-branches of the core beta-linked mannose. J774 cells treated with deoxymannojirimycin continued to phosphorylate and to secrete acid hydrolases. The secreted hydrolases, however, contained only monophosphorylated oligosaccharides in which the phosphate was restricted to the alpha 1,6-branch. These results indicate that mannose residues within high mannose oligosaccharides impose constraints on the phosphorylation of their composite structures. We conclude that the two-step phosphorylation occurs as a result of a common phosphotransferase at both the pre-Golgi and Golgi locations and a change in the conformation of the oligosaccharides attached to the acid hydrolases through the action of Golgi-associated alpha-mannosidase I.     

Free Ricin A Chain Reaches an Early Compartment of the Secretory Pathway before It Enters the Cytosol

During the intoxication of mammalian cells by ricin, the catalytically active A chain must cross the membrane of an intracellular compartment in order to reach its ribosomal substrates in the cytosol. The actual site of ricin A chain translocation is unclear, and conflicting views hold that it enters the cytosol from endosomes or from an early compartment of the secretory pathway, possibly the lumen of the endoplasmic reticulum. Here we show that treating cells with brefeldin A, or transiently overexpressing mutant GTPases known to inhibit biochemical complexes mediating anterograde and retrograde transport between the endoplasmic reticulum and the Golgi complex, protected cells from intoxication by free ricin A chain. These data indicate that ricin A chain, either free or as part of intact ricin, reaches an early compartment of the secretory pathway before translocation into the cytosol occurs.     

Structural diversity of cytosolic free oligosaccharides in the human hepatoma cell line, HepG2

It is thought that free oligosaccharides in the cytosol are an outcome of quality control of glycoproteins by endoplasmic reticulum-associated degradation (ERAD). Although considerable amounts of free oligosaccharides accumulate in the cytosol, where they presumably have some function, detailed analyses of their structures have not yet been carried out. We isolated 21 oligosaccharides from the cytosolic fraction of HepG2 cells and analyzed their structures by the two-dimensional high-performance liquid chromatography (HPLC) sugar-mapping method. Sixteen novel oligosaccharides were identified in the cytosol in this study. All had a single N-acetylglucosamine at their reducing-end cores and could be expressed as (Man)n (GlcNAc)1. No free oligosaccharide with N,N'-diacetylchitobiose was detected in the cytosolic fraction of HepG2 cells. This suggested that endo-beta-N-acetylglucosaminidase was a key enzyme in the production of cytosolic free oligosaccharides. The 21 oligosaccharides were classified into three series--series 1: oligosaccharides processed from Manalpha1-2Manalpha1-6 (Manalpha1-2Manalpha1-3)Manalpha1-6(Manalpha1-2Manalpha1-2Manalpha1-3) Manbeta1-4GlcNAc (M9A') and Manalpha1-2Manalpha1-6(Manalpha1-3) Manalpha1-6(Manalpha1-2Manalpha1-2Manalpha1-3)Manbeta1-4GlcNAc (M8A') by digestion with cytosolic alpha-mannosidase; series 2: oligosaccharides processed with Golgi alpha-mannosidases in addition to endoplasmic reticulum (ER) and cytosolic alpha-mannosidases; and series 3: glucosylated oligosaccharides produced from Glc1Man9GlcNAc1 by hydrolysis with cytosolic alpha-mannosidase. The presence of the series "2" oligosaccharides suggests that some of the misfolded glycoproteins had been processed in pre-cis-Golgi vesicles and/or the Golgi apparatus. When the cells were treated with swainsonine to inhibit cytosolic alpha-mannosidase, the amounts of M9A' and M8A' increased remarkably, suggesting that these oligosaccharides were translocated into the cytosol. Four oligosaccharides of series "2" also increased. In contrast, there were obvious reductions in Manalpha1-6(Manalpha1-2Manalpha1-2Manalpha1-3)Manbeta1-4GlcNAc (M5B'), the end product from M9A' by digestion with cytosolic alpha-mannosidase, and Manalpha1-6(Manalpha1- 2Manalpha1-3)Manbeta1-4GlcNAc, derived from series "2" oligosaccharides by digestion with cytosolic alpha-mannosidase. Our data suggest that (1) some of the cytosolic oligosaccharides had been processed with Golgi alpha-mannosidases, (2) the major oligosaccharides translocated from the ER were M9A' and M8A', and (3) M5B' and Glc1M5B' were maintained at relatively high concentrations in the cytosol.