Glycoprotein Quality Control in the Endoplasmic Reticulum: Mannose Trimming by ER

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1 JBC Papers in Press. Published on January 24, 2001 as Manuscript M Glycoprotein Quality Control in the Endoplasmic Reticulum: Mannose Trimming by ER Mannosidase I Times the Proteasomal Degradation of Unassembled Immunoglobulin Subunits. Claudio Fagioli and Roberto Sitia * # Department of Molecular Pathology and Medicine DIBIT-San Raffaele Scientific Institute, Milano, Italy * Università Vita-Salute San Raffaele, Milano, Italy # Address correspondence to: Via Olgettina, Milano, Italy tel fax r.sitia@hsr.it Running Title: Key words: Assembly; dislocation; folding; glycan processing; IgM; polymerization; proteasomes. Abbreviations. C, castanospermine; dm, deoxymannojirimicin; dn, deoxynojirimycin; ER, endoplasmic reticulum; H, heavy chain; Ig, immunoglobulin; K, kifunensine; L, light chain; P, phenylarsine oxide; S, swainsonine; TM, tunicamycin; Z, ZL3H. Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

2 Summary Quality control in the endoplasmic reticulum (ER) must discriminate nascent proteins in their folding process from terminally unfolded molecules, selectively degrading the latter. Unassembled Ig-µ and J chains, two glycoproteins with five and one N-linked glycans respectively, are degraded by cytosolic proteasomes after a lag from synthesis, during which glycan trimming occurs. Inhibitors of mannosidase I (kifunensine), but not of mannosidase II (swainsonine), prevent the degradation of µ chains. Kifunensine inhibits also J chain dislocation and degradation, without inhibiting secretion of IgM polymers. In contrast, glucosidase inhibitors do not significantly affect the kinetics of µ and J degradation. These results suggest that removal of the terminal mannose from the central branch acts as a timer in dictating the degradation of transport-incompetent, glycosylated Ig subunits in a calnexin independent way. Kifunensine does not inhibit the degradation of an unglycosylated substrate (λ Ig light chains) or of chimeric µ chains extended with the transmembrane region of the α TCR chain, implying the existence of additional pathways for extracting proteins from the ER lumen for proteasomal degradation. 2

3 Introduction The endoplasmic reticulum (ER) is the port of entry and the main folding compartment for proteins destined to the exocytic pathway. A stringent quality control system is coupled to the folding machinery, ensuring that only structurally mature proteins reach the Golgi (1). Proteins that fail to attain their correct three-dimensional structure are retained in the ER and eventually degraded after a lag that varies amongst individual substrates (2, 3). Cytosolic proteasomes are responsible for the degradation of many membrane and soluble ERsynthesised proteins (4-14), implying that proteins targeted for degradation must be delivered to the cytosol (15). Sec61, a component of the translocon complex that mediates the entry of proteins into the ER (16), seems to be involved also in the retrograde translocation, or dislocation, to the cytosol (17,18). This observation raises intriguing questions on the mechanisms that gate Sec61 for dislocation and determine the directionality of transport across the ER membrane (19-22). ER quality control prevents the deployment of potentially harmful molecules to their final destinations and maintains homeostasis within the ER. Somehow, the system must be capable of discriminating terminally unfolded and/or unassembled molecules from newly translocated polypeptides, which have not had the time to complete folding. Therefore, the processes of retention and degradation must be not only specific, but also precisely timed. The oligosaccharide moieties present on many ER-synthesised proteins have been shown to play a crucial role in the quality control (see 1 for a comprehensive review). A branched oligosaccharide consisting of three glucoses, nine mannoses and two N-acetyl-glucosammines (Glc 3 Man 9 GlcNAc 2 ) is added to the nascent polypeptide when an appropriate sequence is recognised by oligosaccharyltransferase ER (Figure1). Immediately after synthesis, the terminal glucoses are removed by two glucosidases (I and II) localised in the ER. Another ER-resident enzyme, UDP-glucose: glycoprotein glucosyltransferase (UGT), is capable of adding a glucose residue to the A branch of the glycans present on unfolded molecules, favouring their binding to calnexin or calreticulin. These two ER chaperones bind mono-glucosylated proteins and retain them in the folding-promoting ER environment. ER mannosidases I and II remove the terminal mannoses from the B and C branches, respectively. It is 3

4 thought that glycans lacking the terminal mannoses are poorer substrates of glucosidase II and UGT, thereby bringing the calnexin cycle to an end (1, 23). Hence, it is possible that the different kinetics of the ER sugar processing enzymes provide a molecular mechanism to time retention and dislocation/degradation. Evidence for a role of mannose trimming in diverting misfolded glycoproteins to proteasomal degradation has been produced in yeast (24) and in mammalian cells (25-29). To investigate how ER-synthesised proteins are diverted from retention into the degradative pathway, we have compared the fate of Ig µ, J and λ chains in assembly-deficient myeloma transfectants. These Ig-subunits fold primarily under the assistance of BiP (30-34). While µ and J carry five and one N- linked glycans, respectively, λ are not glycosylated. Evidence is presented indicating that the trimming of the B-branch terminal mannose by ER mannosidase I times the dislocation of µ and J chains from the ER lumen to the cytosol for proteasomal degradation, without the involvement of calnexin and/or calreticulin. 4

5 Experimental procedures Cell lines. NSO, a myeloma cell line that produces endogenous J chains but no heavy (H) or light (L) chains, and the transfectants expressing wild type secretory (µs) or membrane (µm) µ chains (N[µ1] and N[µm] respectively), or a mutant λ chain (N[λGly214]), were described previously (34-36). N[µ-TCR αtm], N[γ2b-TCR αtm] were generated by stably transfecting NSO with chimeric µ or γ2b heavy chains extended at their C-termini with the transmembrane region of the T cell receptor α chain (37). Antibodies and reagents. A rabbit anti mouse J chain antiserum (38) was kindly provided by Dr. R.M.E. Parkhouse (Pirbright Laboratory, Surrey, UK). Horseradish peroxidase conjugated goat anti mouse-µ, goat anti mouse-ig or goat anti rabbit-ig were from Southern Biotechnology Associates Inc. (Birmingham, AL). Purified rabbit anti-µ and anti-λ antibodies were obtained from Zymed (San Francisco, CA). Dithiothreitol (DTT), leupeptin, N-ethyl maleimide (NEM), thapsigargin (Tg) and TritonX100 were purchased from Sigma (St. Louis, MO); the protease inhibitors Cocktail Complete and endoglycosidase H from Roche Diagnostic (Monza, Italy). The proteasome inhibitor carboxybenzyl-leucyl-leucyl-leucinal (ZL3H) (39, 40), was a kind gift of Drs. M. Bogyo and H. Ploegh (Harvard University, Cambridge, MA). Phenylarsine oxide (Sigma) was used at µm (26). Castanospermine (C) and deoxynojirimycin (dn) were purchased from Sigma and used at the final concentration of 1mM to inhibit glucosidases I and II. Kifunensine and swainsonine were purchased from Toronto Research Chemicals (Toronto, Canada), dissolved in water and used at a final concentration of 2 µg/ml and 100 µm, to selectively block mannosidase I and mannosidase II, respectively (41-44). 1-deoxymannojirimicin (dm, Sigma) was used at 1 mm as an inhibitor of ER mannosidases I and II. The effects of kifunensine and swainsonine on the degradation and electrophoretic mobility of µ and J chains were similar either when the drugs were added before, or immediately after the pulse (CF and RS, unpublished observations). Therefore, to simplify the experimental procedures and to 5

6 reduce variability in incorporation, cells were pulse-labelled in bulk, and then aliquoted and chased with the different drugs. Pulse chase assays, immunoprecipitation and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Pulse chase assays were performed as previously described (21). In the experiment shown in Figure 7, cells were chased in Krebs Ringer Hepes medium (125mM NaCl, 5mM KCl, 1mM Na 2 HPO 4, 5.5mM glucose, 25mM Hepes, 0,5mM MgCl 2, FCS 2%, ph7) supplemented with different concentrations of CaCl 2 (3µM-1mM). Ionomycin was added at the final concentration of 1 µm. Films were scanned and relevant bands quantitated by the ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Glucosylation assay. Following extensive washings, immunoprecipitates on protein A beads were incubated overnight at 37 C with jack bean α-mannosidase (α-man) (Sigma) in 20µl of 50µM sodium citrate, ph4.4 before boiling in sample buffer and electrophoresis. 6

7 Results Glycan trimming determines diversion of µ and J chains into the proteasomal degradation pathway. In the absence of λ chains, secretory µ (µs) and J chains cannot assemble into transport competent IgM polymers and are degraded by cytosolic proteasomes (21). When cells were pulse-labelled for a short period (5min), a lag was evident before degradation ensued (Figure 2, panels A and C). During this period, both subunits undergo some folding, as determined by the formation of intrachain disulfide bonds (A. Mezghrani, CF and RS, unpublished results) and, in the case of µ, by acquisition of partial protease resistance (21). Changes in the electrophoretic mobility of µ chains were evident during the lag period (panel A). These mobility shifts likely corresponded to glycan processing, as they were no longer detectable after treatment with endoglycosidase H (data not shown). In contrast, if N[µ1] cells were pulse labelled for a long period (20h) so as to label the whole intracellular pool, µ and J chain degradation could be observed immediately (Figure 2, panels B and C) consistent with the fact that a fraction of the molecules has reached a degradation competent state. Inhibiting binding to calnexin and calreticulin does not alter significantly the kinetics of µ or J chain degradation. As some newly synthesised µ can be co-precipitated with calnexin and calreticulin (21) the effects described above could reflect a role of the two chaperones in µ chain quality control, as described for certain α1-antitrypsin mutants (23). To verify this, the glucosidase inhibitor castanospermine was added before the pulse, so as to inhibit the rapid removal of glucose residues from newly made proteins. Consistent with the presence of additional glucoses, µ and J chains synthesised in the presence of CST migrated more slowly (Figure 3A). Under these conditions, µ chains were not co-immunoprecipitated by anti-calnexin antibodies (data not shown). Preventing the binding to calnexin and calreticulin, however, did not affect the kinetics of µ chain degradation: after the characteristic lag, clearance was efficient (Figure 3B, lanes 1-4). Castanospermine had little if any effect also on the degradation of J chains (Figure 3C, lanes 2-5). Pre-treatment with castanospermine did not affect the dislocation of J chains, which can be monitored by the appearance of the two faster migrating bands (>Æ and >>Æ,) when cells 7

8 are chased in the presence of proteasome inhibitors (21). Similar amounts of cytosolic J chains accumulated in the presence or absence of castanospermine (panel C, compare lanes 1 and 9 and the histograms below). These results indicated that calnexin and calreticulin do not play a major role in timing the degradation of the two Ig subunits. We therefore investigated other glycan processing steps known to occur in the ER lumen, in particular mannose trimming which has been implicated in the quality control of mutated α1 antitrypsin (23, 25-27) and thyroglobulin (29). Inhibitors of ER mannosidase I prevent µ chain degradation. Inhibiting the activity of ER mannosidase I with kifunensine prevented the degradation of µ chains synthesised in the presence of castanospermine as efficiently as proteasome inhibitors (Figure 3B, compare lanes 6 and 9). The simultaneous presence of kifunensine and castanospermine did not have synergistic effects (lanes 8B). As shown in panel C, kifunensine partially inhibited J chain degradation as well (see below). These results suggested that the trimming of the B branch mannose is important in timing the degradation of unassembled Ig subunits. To further confirm this, specific inhibitors of the main ER glycosidases were added after a 5 min pulse without castanospermine preincubation (Figure 4). As observed above in castanospermine pre-treated cells, kifunensine was as effective as ZL3H in preventing the degradation of µ chains (panel A, compare lanes 2, 3, 6 and 9). 1-deoxymannojirimicin (dm) an inhibitor of both ER mannosidases I and II, also inhibited µ degradation (lane 17). In contrast, the ER mannosidase II inhibitor swainsonine was not effective (lane 12). Consistent with the presence of additional mannoses, µ chains accumulating in the presence of dm, kifunensine or swainsonine migrated more slowly than those present in untreated cells (lane 2) or in cells incubated with proteasome inhibitors (lane 3). When both swainsonine and kifunensine were present during the chase, the effects of the ER mannosidase I inhibitor prevailed, and abundant µ accumulated intracellularly (lane 22). The mobility shift was more pronounced than that induced by kifunensine or swainsonine alone. Densitometric quantitations (see bars below each lane) confirmed that the removal of the terminal mannose from the central branch is crucial for the degradation of µ chains. As the removal of mannoses may alter the substrate recognition by glucosidases and UGT, it was possible that the drugs influenced the binding of µ chains to calnexin. To exclude this possibility, 8

9 castanospermine or deoxynojirimycin (dn) were added after the pulse so as to block glycans in a monoglucosylated state. The effects of castanospermine and dn were confirmed by the mobility shift of µ chains accumulating in treated cells (compare lanes 18A-20A), and by mannosidase sensitivity assays (see Figure 6 below). Under these experimental conditions, which should favour the binding of unfolded glycoproteins to calnexin and/or calreticulin (1), castanospermine and dn had minor effects on µ chain degradation. Analysis of the anti-j immunoprecipitates confirmed the role of mannose trimming in targeting unassembled Ig subunits to degradation. Castanospermine and dn had minor effects (lanes 10, 18, 19). Kifunensine and dm inhibited the degradation of J, although to a lesser extent than µ chains. A single J chain band was precipitated from treated cells, with mobility slightly slower than the upper band accumulating in the presence of proteasome inhibitors (compare lanes 3-4 in panel B). Inhibitors of ER mannosidase I prevented only in part the dislocation of J chains, as indicated by the appearance of the two bands with faster mobility, corresponding to deglycosylated, cytosolic J chains (21), when proteasome inhibitors were added during the chase (compare lanes 4-9B). Therefore, glycan trimming is not essential for the dislocation of J chains. The differences between µ and J chains may reflect the number of N-glycans present on the two substrates, and may correlate with our previous observation that some J but no µ chains can dislocate to the cytosol when proteasomal degradation is inhibited (21). Neither kifunensine nor castanospermine inhibit the degradation of unglycosylated λ chains. To exclude the possibility that the different drugs acted on general cellular functions, we investigated their effects on λ, a non-glycosylated Ig subunit. Neither kifunensine nor castanospermine had any effect on the degradation of λg213, a mutant that is also degraded by proteasomes (Figure 5), albeit with slower kinetics than µ or J (36, 45), A. Sparvoli and RS, unpublished results). µ chains accumulating in the presence of kifunensine are not glucosylated. The presence of a terminal glucose on the A branch of a N-glycan can be monitored by a mannosidase sensitivity assay (46). When the glucose is present (G1), the mannosidase cannot remove the underlying mannoses, yielding a small mobility shift. In contrast, glycans lacking the glucose (G0) are digested to a greater extent (see Figure 1). As expected, µ chains present in castanospermine treated cells were only 9

10 marginally affected by mannosidase (Figure 6A, compare lanes 9-10). In contrast, ZL3H (lanes 5-6) and kifunensine (lanes 7-8) caused the accumulation of µ chains that were sensitive to the enzyme. Similarly, J chains appeared to be sensitive to mannosidase except after chase in the presence of castanospermine (Figure 6B). µ and J chains accumulating in the presence of swainsonine (lanes 11A-12A and 6B) were largely resistant to mannosidase, suggesting that the presence of a terminal mannose on the C branch inhibits glucosidase activity on the two Ig subunits Calcium is required for the degradation of unassembled µ chains. The recent solution of the structure of yeast class I α1,2-mannosidase I revealed a crucial role for calcium ions in enzymatic activity (28). Therefore, we investigated whether perturbing calcium homeostasis in the ER lumen affected µ chain degradation. As shown in Figure 7, the ER Ca ++ ATPase inhibitor thapsigargin caused the intracellular accumulation of µ chains, which migrated with a mobility slower than those present in untreated cells (compare lanes 2-3). Similarly to what reported above for kifunensine, also thapsigargin inhibited partially J chain degradation (not shown). The requirements for calcium were further confirmed by chasing pulse-labelled N[µ1] cells with ionomycin and different Ca ++ concentrations (lanes 4-8). In the presence of the ionophore, the Ca ++ levels in the ER are expected to be similar to those in the extracellular medium. Based on this assumption, it would seem that 100 µm Ca ++ is required to sustain the processes underlying µ chain degradation. The reduced degradation of µs in this experiment (lane 2) could be due to the low extracellular Ca ++ concentration. Mannose trimming is important for the degradation of membrane µ (µm) chains as well. Both substrates analysed so far, secretory µ (µs) and J chains, are soluble proteins retained in the ER lumen. It was of interest to analyse the role of glycan processing on an integral membrane protein. Alternate RNA processing generates two forms of µ chains, µs and µm, which differ only in their C- terminal ends (47) and are degraded with similar kinetics (35). Clearly, kifunensine prevented the degradation of membrane bound µ chains (Figure 8). Cytosolic proteasomes are responsible for the 10

11 degradation of µm, as ZL3H caused their accumulation (lane 11). Castanospermine had little if any effects on the clearance of membrane bound µm chains (lane 10). Kifunensine does not inhibit IgM secretion. In the presence of λ chains, most µ chains synthesised by plasma cells are assembled into IgM polymers and secreted (34). Only part of them, corresponding to molecules that fail to assemble with λ or to polymerise, are degraded (37). We therefore tested the effects of kifunensine and ZL3H on J[µ1] cells, which express µ, λ and J chains. As shown in Figure 9, kifunensine did not inhibit IgM secretion (compare lanes 5-6). The proteasome inhibitor ZL3H had only a marginal effect (lane 7). Both kifunensine and ZL3H caused a small accumulation of intracellular µ chains (lanes 2-3), suggesting that secretory µ chains that fail to polymerise were degraded by proteasomes. These results imply that the molecular mechanisms that recognise µ chains lacking the central mannose(s) and divert them to proteasomal degradation, are specific for structurally immature molecules (either unassembled or unpolymerized). The degradation of µ-tcr αtm chains does not require mannose trimming. We have previously shown that appending the transmembrane region of the TCR α chain to the C- terminus of Ig µ and γ2b chains induces brefeldin A insensitive degradation of chimeric immunoglobulins (37). When expressed in the absence of L chains in NSO, degradation of µ-tcr αtm was rapid. Unlike in the case of µs and µm chains, however, kifunensine did not alter the kinetics of µ- TCR αtm degradation (Figure 10A). Thapsigargin failed to inhibit the proteasomal disposal of µ-tcr αtm as well (panel B), in agreement with the notion that Ca++ is not required for the degradation of T cell receptor subunits (48). Also the proteasomal degradation of γ2b -TCR αtm chains, which occurs with kinetics slower than µ-tcr αtm (37), was insensitive to glycosidase inhibitors and thapsigargin (panel C), suggesting that the extraction of these chimeric molecules follows a different pathway, independent from the activity of ER mannosidase I. It has been recently reported that inhibitors of tyrosine phosphatases, such as phenylarsine oxide (P) 11

12 prevent the degradation of certain α1 anti-trypsin mutants (26). Phenylarsine oxide clearly inhibited the degradation of both µs and µ-tcr αtm chains (Figure 10 D). Unlike kifunensine, phenylarsine oxide inhibited the dislocation of J chains, as the simultaneous presence of phenylarsine oxide and proteasome inhibitors during the chase did not yield the characteristic cytosolic J isoforms (see arrows) in either N[µ- TCR αtm] or N[µ1] cells (lanes 5D and 9D). Pervanadate (not shown) and the oxidant diamide (21) gave similar results. 12

13 Discussion To mediate folding efficiency and homeostasis, the processes of retention and degradation that underlie quality control within the ER must be precisely coordinated. Sufficient time must be allocated to allow the folding of newly made polypeptides in a milieu that provides optimal assistance by specialised chaperones and enzymes. On the other hand, molecules that fail to attain the correct three-dimensional structure after some time should be degraded to prevent their accumulation or aggregation in the ER lumen (49). The data presented in this paper demonstrate that the trimming of N-linked glycans provides a molecular mechanism for co-ordinating the quality control of two glycosylated unassembled Ig subunits. This timer function seems to be based on the sequential activity of the ER sugar processing enzymes. While glucosidases act rapidly as substrates emerge from the translocon, ER mannosidases I and II come into the scene at later (1). The absence of the terminal mannose on the B branch of asparagine-linked glycans enables to target terminally unfolded glycoproteins for dislocation to the cytosol and proteasomal destruction. The distinct patterns of µ and J chain electrophoretic mobility and in vitro sensitivity to jack bean α- mannosidase, confirmed the specificity of the pharmacological inhibitors. Interestingly, µ chains isolated from cells chased in the presence of swainsonine are largely resistant to mannosidase, while kifunensine leads to the accumulation of sensitive molecules. This suggests that -at least in myeloma cells- removal of the B branch mannose does not impair glucosidase II to a greater extent than UGT. On the other hand, on glycans lacking a C branch terminal mannose, the activity of UGT prevails and glucosylated molecules accumulate, which are nonetheless dislocated and degraded by proteasomes. Thus, it appears that calnexin has a minor role in the clearance of µ chains. In contrast, kifunensine and dm were as effective as proteasome inhibitors in preventing µ chain degradation. Under both conditions, µ chain dislocation across the ER membrane was blocked, and glycosylated heavy chains accumulated. Kifunensine (figures 3-4) and proteasome inhibitors (21) blocked only partially the dislocation of J chains, perhaps reflecting the different number of glycans or folding state of the two subunits. The involvement of mannose trimming in targeting glycoproteins for proteasomal destruction has been demonstrated in several experimental models. In yeast, biochemical and genetic evidence indicates that 13

14 the degradation of CPY* requires the activity of a Class I α1,2 mannosidase (50, 51). Trimming of the central mannose is important also for the degradation of yeast pre-pro-α-factor ectopically expressed in mammalian cells (52) and of certain α1 anti-trypsin mutants associated with familial emphysema (23, 25-27). The degradation of a truncated α1 anti-trypsin mutant was shown to depend on a prolonged interaction with calnexin (23). In this respect the degradation of µ and J chains seems to be mechanistically different, as it is only marginally affected by castanospermine. This indicates that more than one pathway can co-ordinate the dislocation/degradation of glycoproteins, a notion that is not surprising since at least two parallel systems exist in the ER to assist glycoprotein folding. Whether a glycoprotein selects calnexin/calreticulin or BiP seems to depend on the location of the glycan(s). The closer the oligosaccharide moieties are to the N-terminus, the higher are the chances that the protein engages with calnexin (53). This may explain why µ chains, in which the first of the five N-linked glycans is located at position 171 (see 54 and references therein) fold primarily on BiP. However, a fraction of newly made µ chains can be co-precipitated with calnexin (21). It is not known whether these have detached from a first round of interaction with BiP or represent direct binding to the lectin, possibly facilitated by the numerous glycan moieties present on µ chains. Although the single glycan of J chains is bound to asparagine 48, we failed to detect interactions of this small Ig subunit with calnexin or calreticulin (21). In contrast, J chains can be easily co-precipitated with BiP, especially in tunicamycin treated cells (CF and RS, unpublished observations). Since unassembled µ and J chains preferentially associate with BiP, it is not surprising that pre-incubation with castanospermine (which prevents binding to calnexin/calreticulin) fails to affect their degradation. The minor effects observed when the glucosidase inhibitor is administered after the pulse could be due to a reduced activity or accessibility of ER mannosidase I on mono-glucosylated substrates. Therefore, our results suggest the involvement of glycan trimming in timing glycoprotein degradation independently from interactions with calnexin or calreticulin. Many ER-resident proteins bind Ca ++, and perturbing the homeostasis of this ion is bound to have pleiotropic effects. However, since ER mannosidase I contains Ca ++ (28), thapsigargin could inhibit µ 14

15 chain degradation by interfering with the function of this enzyme, as also suggested by the similar electrophoretic mobility of µ chains accumulating in thapsigargin or kifunensine treated cells. How can removal of the B-branch terminal mannose activate dislocation? Terminally unfolded substrates, in particular soluble molecules like µs and J chains, must be recruited in the vicinities of a functional dislocon, which must eventually be opened and activated. Dislocation across the ER membrane, which in the case of µ chains requires active proteasomes (21) is likely to be facilitated by unfolding and reduction of the substrate. It is tempting to speculate that lectin molecules endowed with specificity for man8 glycans play an important role in this chain of events (24). In principle, if such lectins had a binding specificity similar to ER mannosidase I, kifunensine, thapsigargin and other drugs may compete with or inhibit the lectin itself. The observation that kifunensine does not affect IgM secretion indicates that, if a lectin plays a role in timing dislocation, it must be specific for unfolded molecules. It also suggests that the inhibition of degradation caused by kifunensine is not due to prolonged retention in the ER lumen. Taken together, these results are consistent with an active role of man8 binding lectin molecules in targeting misfolded or unassembled glycoproteins to dislocation/degradation. The degradation of the PiZ and null variants of α1-antitrypsin seems to follow different pathways (23, 26, 27). The results obtained with µ-tcr αtm indicate that indeed more than one pathway can be utilised to dislocate a protein from the ER to the cytosol. In µ-tcr αtm, the last residues of µs including the carboxy terminal N-glycan (54) and Cys575, had been replaced with the transmembrane and cytosolic domains of the α TCR chain, known to contain a dominant ERAD targeting signal (11,14,55,56). Like µm, µ-tcr αtm contains 4 of the 5 glycans present in wild type µs and is an integral membrane protein with a short cytosolic tail. Therefore, neither the number of sugars nor the different topology can explain the distinct requirements of the three ERAD substrates for dislocation and degradation. Also γ2b-tcr αtm, whose slower kinetics of degradation allow glycan processing by ER mannosidase I, is efficiently degraded in the presence of kifunensine. These observations suggest that information contained in the transmembrane and cytosolic tail of the α TCR chain can induce 15

16 mannosidase I-independent degradation of a polypeptide that normally requires removal of the B terminal mannoses. Also this pathway seems to utilise cytosolic proteasomes, since it can be inhibited by ZL3H (Figure 10), and in this respect differs from the one described for the PiZ α1-antitrypsin variant (26). In view of the importance of maintaining ER homeostasis and fidelity of quality control, it is not surprising that multiple pathways are operative in mammalian cells to eliminate misfolded proteins from the ER. While differing in the sensitivity to kifunensine and thapsigargin, µs and µ TCR αtm share the property of being stabilised by inhibitors of tyrosine phosphatases, such as phenylarsine oxide (Figure 10D) and pervanadate (data not shown). Also the degradation of endogenous J chains is blocked by phenylarsine oxide. The absence of deglycosylated J isoforms suggests that this drug blocks dislocation from the ER lumen to the cytosol, a molecular phenotype similar to that induced by the reversible oxidant diamide (21). The involvement of tyrosine phosphatases could be important for integrating ERAD with other cellular signalling pathways, including the responses to unfolded proteins and ER overload (57, 58 and references therein). However, as many steps in the complex process of ERAD are redox sensitive, the precise targets of phenylarsine oxide, pervanadate and diamide remain to be identified. Acknowledgements We thank Drs. M. Aebi, M. Alessio, A. Cabibbo, A. Fassio, A.M. Fra, A. Helenius, C. Jakob, R. Mancini, A. Mezghrani and T. Simmen for helpful discussions and suggestions, M. Bogyo, H. Ploegh and R.M.E. Parkhouse for generously providing reagents and Ms. S. Trinca for impeccable secretarial assistance. This work was supported through grants from Associazione Italiana per la Ricerca sul Cancro (AIRC), Consiglio Nazionale delle Ricerche (Target Project on Biotechnology, PF49 and 5% PF31), and Ministero della Sanità (ISS, Special AIDS Project and Ricerca Finalizzata RF 98.53). 16

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20 Figure Legends Figure 1 Processing of N-glycans in vivo and in vitro. A branched glycan consisting of two N-acetyl-glucosammines (squares) nine mannoses (open circles) and three glucoses (striped or dotted circles) can be added to the nascent polypeptide when an asparagine (Asn) is followed by the sequence Aaa / Ser or Thr, where Aaa is any aminoacid but proline (59). Soon after translation, glucosidases (Glu) I and II remove the glucoses, while UGT can add a single glucose (striped circle) to the terminal mannose of the A branch, yielding a monoglucosylated glycan. ER mannosidases (ER Man) I and II remove the terminal mannoses from the B and C branches, respectively. Castanospermine and deoxynojirimycin (dn) can be used to inhibit glucosidases, while kifunensine and swainsonine block the activity of ER Man I and ER Man II, respectively. 1-deoxymannojirimicin (dm) inhibits both ER mannosidases (1, 43 and references therein). Jack bean α-mannosidase (α Man) can be used in vitro to discriminate between glycans carrying or not a glucose on the A branch (46). Only in the absence of the glucose (G0) αman cleaves the mannoses of the A branch. Figure 2 Newly made µ and J chains are degraded after a lag. N[µ1] cells were pulse-labelled for 5 min (panel A) or 20 hours (panel B) and chased for the indicated times before lysis and sequential immunoprecipitation with anti-µ and anti-j antibodies. Immunoprecipitates were resolved by SDS-PAGE (12 % acrylamide for anti-j, 10% or 9% for the anti-µ immunoprecipitates shown in panels A and B respectively). Note the mobility shifts in µ chains during chase after a short pulse (panel A). Autoradiograms were scanned and the percentage of µ and J chains present at each time point determined relative to the intensity at time 0 of chase (panel C). After a 20h pulse, the degradation of µ (closed squares) and J (closed circles) chains clearly begins without the lag which is evident after a short pulse. Figure 3 Preventing the binding to calnexin and calreticulin by continuous exposure to castanospermine does not alter the kinetics of µ and J chain degradation. 20

21 N[µ1] cells were pre-incubated for 30 min in the presence of castanospermine, and then pulsed for 5 min and chased for the indicated times in the presence or absence of the glucosidase inhibitor, alone or in combination with other drugs (C= castanospermine, K= kifunensine, Z= ZL3H). As indicated by the mobility shifts in both µ and J (Panel A) chains, pre-treatment with castanospermine was effective in preventing glucose removal from newly synthesised glycoproteins (see also Figure 6). After chase in the presence of the indicated drugs, cells were lysed and sequentially immunoprecipitated with anti-µ (panel B) and anti-j (panel C) and resolved by SDS-PAGE. The two arrows on the left-hand margin of panel C (>Æ and >>Æ) point to deglycosylated, cytosolic J chains (21). The densitometric quantitation of the relevant bands is shown below the corresponding autoradiograms. Bars represent the percentage of µ and J chains present at each time point relative to the intensity at time 0 of chase. In panel C, the shaded area indicates the percentage of deglycosylated J isoforms relative to total J chains accumulating at individual time points. Figure 4 Mannosidase I is involved in timing the degradation of glycosylated Ig subunits. N[µ1] cells were pulse-labelled for 5 min and chased for 1, 2 or 4 hours in the presence of the indicated combinations of inhibitors (Z= ZL3H; K= kifunensine; C= castanospermine; S= swainsonine, dm, 1- deoxymannojirimicin, dn= deoxynojirimycin). Lanes 1-13 and 14-22, derive from two independent experiments. Lysates were sequentially immunoprecipitated with anti-µ (panel A) and anti-j (panel B) antibodies and resolved by SDS-PAGE. The densitometric quantitation of the relevant bands is shown below the corresponding autoradiograms. Arrows and bars as in legend to Figure 3. Figure 5 Kifunensine does not affect the proteasomal degradation of λ chains. N[λG213] cells were pulse-labelled for 5 min and chased for 4 hours in the presence of the indicated inhibitors (C = castanospermine, K = kifunensine, Z = ZL3H). Lysates were immunoprecipitated with anti-λ antibodies, and resolved by SDS-PAGE. As anti- λ antibodies react poorly with unfolded λ chains, they fail to bring down all radioactive λ present at the end of the pulse. 21

22 Figure 6 µ and J chains accumulating in kifunensine treated cells are not glucosylated. N[µ1] cells were pulse-labelled for 5 min and chased for 4 h in the presence of the indicated inhibitors (Z= ZL3H; K= kifunensine; C= castanospermine; S= swainsonine). Aliquots of the anti-µ (panel A) and anti-j (panel B) immunoprecipitates were digested in vitro with jack bean α-mannosidase before electrophoresis. The arrow at the right-hand margin indicates the mobility of untreated J chains. Figure 7 Calcium is required for µ chain degradation. N[µ1] cells were pulse-labelled for 5 min and chased for 4 hours in the presence of thapsigargin (Tg) or ionomycin in calcium-free Krebs Ringer Hepes medium before lysis and immunoprecipitation with anti- µ antibodies. Ionomycin treated cells were chased in the presence of the indicated concentrations of CaCl 2. Figure 8 Kifunensine prevents the proteasomal degradation of membrane µ chains. N[µm] cells were pulse-labelled for 5 min and chased for the indicated times with kifunensine (K), castanospermine (C) or ZL3H (Z) before lysis and immunoprecipitation with anti-µ antibodies. Figure 9 Neither kifunensine nor proteasome inhibitors significantly perturb IgM secretion. J[µ1] cells, which produce µ, λ and J chains and secrete IgM polymers (34), were pulse-labelled for 5 min and chased for 4 hours in the presence or absence (-) of the indicated inhibitors (K= kifunensine; Z= ZL3H). The anti-µ immunoprecipitates from the cell lysates and supernatants were resolved by SDS- PAGE under reducing conditions. The diagonal arrow on lane 3 highlights the altered migration of intracellular µ chains present in kifunensine-treated cells. Figure 10 Proteasomal degradation of µ-tcr αtm is not inhibited by kifunensine or thapsigargin. A) Kifunensine does not inhibit the degradation of µ-tcr αtm. N[µ-TCR αtm] cells were pulse-labelled for 5 min and chased for the indicated times in the presence (+) or absence (-) of kifunensine. Anti-µ immunoprecipitates were resolved by SDS-PAGE, 22

23 and the intensity of the µ-tcr αtm chain bands quantitated by densitometry. The graph shows the percentage of µ-tcr αtm present at individual chase points relative to time 0. B) Unlike µs, µ-tcr αtm chains are degraded in the presence of thapsigargin. N[µ-TCR αtm] and N[µ1] cells were pulse-labelled for 5 min and chased for the indicated times in the presence or absence (-) of thapsigargin (Tg) or ZL3H (Z). Bars represent the densitometric quantitation, performed as described in legend to Figure 3. C) Also the more slowly degraded chimeric γ2b-tcr αtm chains are insensitive to kifunensine. N[γ2b-TCR αtm] cells were pulse-labelled for 5 min and chased for the indicated times in the presence or absence (-) of kifunensine (K), thapsigargin (Tg) or ZL3H (Z). D) Inhibitors of tyrosine phosphatases block the degradation of both µs and µ-tcr αtm chains. N[µ-TCR αtm] and N[µ1] cells were pulse-labelled for 5 min and chased for 4 h with phenylarsine oxide (P), ZL3H (Z), or both drugs (PZ). Bars represent the densitometric quantitation, performed as described in Figure 3. The absence of deglycosylated J chains (>Æand >>Æ) in cells treated with both P and Z suggests that P inhibits J chain dislocation. Chase in the presence of pervanadate (not shown) or diamide (21) gave similar results 23

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34 Glycoprotein quality control in the endoplasmic reticulum: Mannose trimming by ER mannosidase I times the proteasomal degradation of unassembled immunoglobulin subunits Claudio Fagioli and Roberto Sitia J. Biol. Chem. published online January 24, 2001 Access the most updated version of this article at doi: /jbc.M Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts