Differential role of mannose and glucose trimming in the ER degradation of asialoglycoprotein receptor subunits

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1 Journal of Cell Science 112, (1999) Printed in Great Britain The Company of Biologists Limited 1999 JCS Differential role of mannose and glucose trimming in the ER degradation of asialoglycoprotein receptor subunits Michal Ayalon-Soffer, Marina Shenkman and Gerardo Z. Lederkremer* Department of Cell Research and Immunology, George Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel, *Author for correspondence ( Accepted 26 July; published on WWW 22 September 1999 SUMMARY To gain insight into how sugar chain processing events modulate endoplasmic reticulum (ER)/proteasomal degradation we looked at human asialoglycoprotein receptor polypeptides H2a and H2b, variants which differ only by an extra pentapeptide (EGHRG) present in H2a. Membrane-bound H2a is a precursor of a soluble secreted form while H2b reaches the plasma membrane. Uncleaved precursor H2a molecules are completely retained in the ER and degraded as well as a portion of H2b. Inhibition of N- linked sugar chain mannose trimming stabilized both variants. In contrast, inhibition of glucose trimming with castanospermine greatly enhanced the degradation rate of H2a but not that of H2b. We studied a possible involvement of the ER chaperone calnexin, as inhibitors of glucose trimming are known to prevent calnexin binding. Incubation of cells with low concentrations of castanospermine (30 µg/ml) did not interfere with calnexin binding to H2a while causing the same accelerated degradation as high concentrations (>100 µg/ml) which did inhibit the association. Castanospermine treatment after calnexin binding blocked the dissociation of the chaperone but still caused accelerated degradation. The increased degradation could be blocked by a specific proteasome inhibitor, ZL 3 VS. Our results suggest that extensive mannose trimming or retention of glucose residues due to lack of glucose trimming are signals for ER/proteasomal degradation independent of interaction with calnexin. Key words: Calnexin, Chaperone, Endoplasmic reticulum degradation, Proteasome, Glycosylation, Asialoglycoprotein receptor INTRODUCTION The degradation of proteins from the endoplasmic reticulum (ER) has recently been found to involve the cytosolic ubiquitin/proteasomal pathway (reviewed by Bonifacino and Weissman, 1998; Sommer and Wolf, 1997). However, it is not clear yet what early events target the proteins to the proteasome. A postranslational modification that could possibly be involved in this targeting could be a differential processing of glycoprotein sugar chains. Removal of glucose (Glc) residues from N-linked Glc 3 Man 9 GlcNAc 2, in a process called Glc trimming, is accomplished in the endoplasmic reticulum (ER) by the sequential action of glucosidase I and glucosidase II, which remove the outermost and two innermost Glc residues, respectively (Kornfeld and Kornfeld, 1985). Another sugar chain processing event that occurs in the ER is the removal of 1 to 3 mannose (Man) residues and the function of this Man trimming is unknown as the process can also occur in the cis-golgi by the action of Golgi α-mannosidase I. Retention of a glycoprotein in the ER leads to extensive Man trimming of its sugar chains to yield Man 6 GlcNAc 2 (Atkinson and Lee, 1984; Hebert et al., 1995). We have studied the role of Man and Glc trimming in relation to the ER degradation of the H2 subunit of the human asialoglycoprotein receptor (ASGPR). The ASGPR H2 subunit has two alternatively spliced variants, H2a and H2b, that differ only by the presence in H2a of an extra five amino acid miniexon, EGHRG, immediately carboxy-terminal to the transmembrane domain. H2a can be cleaved yielding a soluble secreted ectodomain but the membrane-bound form is completely retained and degraded at the ER by the proteasome (Kamhi-Nesher and G. Z. Lederkremer). In contrast, a significant portion of H2b is processed through the Golgi and reaches the cell surface (Lederkremer and Lodish, 1991; Tolchinsky et al., 1996). We have recently shown that the extra pentapeptide in H2a functions as a signal for ER retention and induces a prolonged association to calnexin (Shenkman et al., 1997). Calnexin, an ER resident protein, is a non-glycosylated, type I membrane protein that binds transiently to a wide array of newly synthesized membrane and secretory proteins. It also exhibits prolonged association with misfolded proteins or unassembled subunits and prevents their exit from the ER (Jackson et al., 1994; Rajagopalan et al., 1994). However, calnexin does not seem to be essential for the ER/proteasomal degradation to occur (Schubert et al., 1998). Calnexin has been shown to interact most preferentially with N-linked glycoproteins (Ou et al., 1993) and removal of two Glc residues from nascent oligosaccharide chains is crucial for its initial association to glycoproteins (Hammond et al., 1994; Hebert et al., 1995). In fact calnexin is a lectin with specificity for the

2 3310 M. Ayalon-Soffer, M. Shenkman and G. Z. Lederkremer monoglucosylated N-linked oligosaccharide Glc 1 Man 9 GlcNAc 2 (Trombetta and Helenius, 1998). Treatment of cells with inhibitors of the two ER glucosidases, castanospermine (CST) or 1-deoxynojirimycin (dnm), prevents the association of glycoproteins with calnexin (Hammond et al., 1994; Hebert et al., 1995; Kearse et al., 1994). Treatment with these inhibitors also accelerates degradation of several glycoproteins through an unknown mechanism (Kearse et al., 1994; Keller et al., 1998; Moore and Spiro, 1993). In the present report we show that inhibition of Man trimming stabilizes ASGPR H2a and H2b. On the other hand inhibition of Glc trimming results in an accelerated proteasomal degradation of ASGPR H2a whereas H2b is not affected. Most importantly, the increased degradation of H2a is independent of its interaction with calnexin. MATERIALS AND METHODS Materials Rainbow [ 14 C]-labeled methylated protein standards were obtain from Amersham (Buckinghamshire, UK). Pro-mix cell labeling mix ([ 35 S]methionine plus [ 35 S]cysteine) was from Amersham; >1000 Ci/mmol. Protein A-Sepharose was from Repligen (Cambridge, MA). Endo-N-acetylglucosaminidase H (endo H) was obtain from New England Biolabs (Beverly, MA). CST was from Genzyme Corp. (Boston, MA). 1-deoxymannojirimycin (dmm) was from Biomol (Plymouth Meeting, PA). N-acetyl-leucyl-leucyl-norleucinal (ALLN) was obtained from Boehringer Mannheim Biochemicals (Indianapolis, IN). Carboxybenzyl-leucyl-leucyl-leucyl-vinylsulphone (ZL 3VS) was a kind gift from Hidde Ploegh. Other inhibitors and common reagents were from Sigma Chem. Co. (St Louis, MO). Cell lines and culture Mouse NIH 3T3 fibroblasts expressing H2a (2-18 cells) or H2b (2C cells) were grown in Dulbecco s modified Eagles s medium (DMEM) plus 10% calf serum under 5% CO 2 as described (Lederkremer and Lodish, 1991). Antibodies A polyclonal antibody specific for a peptide corresponding to the carboxy terminus of H2 was the one used in earlier studies (Lederkremer and Lodish, 1991; Tolchinsky et al., 1996). A polyclonal antibody specific for a carboxy-terminal peptide of calnexin used in early experiments was a gift from Ari Helenius and in most experiments was SPA-860 from Stressgen Biotechnologies Corp. (Victoria, BC, Canada). Metabolic labeling and immunoprecipitation Subconfluent (90%) monolayers of cells in 60-mm tissue culture dishes were rinsed and preincubated for 30 minutes at 37 C with cysteine-free DMEM plus 10% dialyzed calf serum. They were then pulse labeled for 10 to 40 minutes in the same medium containing 0.5 mci/ml of Pro-Mix mixture of [ 35 S]cysteine and [ 35 S]methionine with addition of 5 mm unlabeled methionine. The monolayers were then rinsed and chased for different periods of time with normal DMEM plus 10% calf serum as described (Lederkremer and Lodish, 1991; Tolchinsky et al., 1996). Immunoprecipitations of H2a and H2b from cell lysates and endo H treatments were performed as described before (Tolchinsky et al., 1996). For the experiments of association with calnexin, cells were lysed in 2% sodium cholate as in (Ou et al., 1993). Sequential immunoprecipitation was done first by immunoprecipitation with anti-calnexin antibody. The pellets were then washed 3 times with 50 mm HEPES, ph 7.4, and boiled with 1% SDS and 2 mm DTT for 5 minutes. The supernatants were then diluted with 10 volumes of 1% Triton X-100 and 0.5% sodium deoxycholate plus 2 mm oxidized glutathione and subjected to a second immunoprecipitation with anti-h2 antibody. Gel electrophoresis, fluorography and quantitation Reducing SDS-PAGE was performed on 10% Laemmli gels except where stated otherwise. For non-reducing conditions β- mercaptoethanol was not included in the buffer. The gels were analyzed by fluorography using 20% 2,5-diphenyloxazole and exposing to BioMax MR film from Eastman-Kodak (Rochester, NY). Quantitation was performed in a Fuji BAS 1000 phosphorimager (Japan). In the cases where quantitations are shown for the gels that are illustrated in the figures the values are representative of those obtained in several repeat experiments. Immunofluorescence microscopy The procedures employed were as described previously (Lederkremer and Lodish, 1991; Shenkman et al., 1997). As secondary antibodies were used indocarbocyanine (Cy3)- or fluoresceine isothiocyanate (FITC)- conjugated goat anti-rabbit IgG, from Jackson Labs (West Grove, PA). For treatment with CST, cells on coverslips were incubated with medium containing (100 µg/ml) of the drug at 37 C in a CO 2 incubator for 4 hours. Cycloheximide was then added to the medium at 300 µm for 1 hour. Digital photography on a Leica DMRBE fluorescence microscope was done at identical exposure times between samples to be able to compare signal intensities. RESULTS Inhibition of Glc trimming by CST results in accelerated ER/proteasomal degradation of ASGPR H2a but not of the H2b subunit In order to study the relation between Glc trimming and the ER/proteasomal degradation of ASGPR polypeptides, singly transfected NIH 3T3 cells expressing H2a or H2b were cultured for several hours in the absence or presence of CST. The cells were then metabolically labeled, and the survival of H2a and H2b assessed. As shown in Fig. 1A, in the presence of CST the electrophoretic mobility of H2a and H2b was decreased, which is consistent with the retention of nine Glc residues distributed among the three N-linked oligosaccharides of H2. CST caused a dramatic reduction in the stability of H2a even after short periods of chase. In Fig. 1A, after 1 hour chase H2a was just emerging from a lag before the degradation starts as was seen before (Lederkremer and Lodish, 1991; Wikstrom and Lodish, 1991) and nevertheless CST strongly accelerated the degradation (Fig. 1A, lanes 4 and 5). In contrast to H2a the stability of H2b was somehow increased by CST after 1 hour chase (Fig. 1A, lanes 8 and 9). A similar experiment was performed with a longer chase time (2 hours), not long enough for complete degradation so H2a could be detected in the absence and also in the presence of CST. After 2 hours chase whereas the stability of H2b was unaffected by CST that of H2a was much decreased (Fig. 1B). As described previously (Lederkremer and Lodish, 1991), when H2a is expressed alone in 3T3 cells, it is inserted normally into the ER and acquires three N-linked oligosaccharides but fails to be processed to its mature form. Instead it is retained and degraded in the ER. To find out if the CST induced degradation is similar in nature to the previously described pre- Golgi degradation, we used a series of inhibitors utilized before to study the degradation of H2a. As shown in Fig. 2A, agents

3 Man and Glc trimming in ER degradation 3311 affecting lysosomal degradation such as leupeptin, NH 4 Cl or chloroquine, did not affect the increased degradation by CST. However, it was inhibited by ALLN, a cysteine protease and proteasome inhibitor, and even more strongly by incubation of the cells at 18 C, characteristic of degradation from the ER (Amara et al., 1989; Neumann et al., 1996). As we have recently shown that the degradation of H2a involves the ubiquitin-proteasome pathway we also tested a specific inhibitor of the proteasome, ZL 3 VS (Wiertz et al., 1996), which inhibited significantly, although not completely the increased degradation (Fig. 2B). Thus, inhibition of Glc trimming by CST results in a specific accelerated degradation of H2a. This degradation is non-lysosomal, similar to the described ER degradation of H2a and it involves the proteasome. CST does not prevent the folding of H2a or H2b One explanation for the increased degradation of H2a in the presence of CST could be that CST prevents the folding or A B % remaining relative to pulse CST chase (min) H2a H2b Fig. 1. CST causes accelerated degradation of ASGPR H2a but not of H2b. (A) Singly transfected NIH 3T3 cells expressing H2a (2-18 cell line, lanes 2-5) or H2b (2C cell line, lanes 6-9) or untransfected (lane 1) were metabolically labeled for 20 minutes with [ 35 S]cysteine and chased for 0 or 60 minutes with complete medium in the absence (lanes 1, 2, 4, 6, 8) or presence of CST (100 µg/ml) (lanes 3, 5, 7, 9). Cells treated with CST were preincubated with the drug for 2.5 hours before the labeling and it was also present during the starvation and labeling periods. At the end of the pulse or chase periods, the cells were lysed, immunoprecipitated with anti-h2 carboxy-terminal antibody and subjected to 12% SDS-PAGE followed by fluorography. The bands were quantitated in a phosphorimager and the values relative to the pulse are indicated at the bottom of the panel. The values were normalized to the amounts of label present after the pulse in the presence or absence of CST to compensate for a slight decrease of labeling in the presence of the drug. The shift caused by the presence of Glc residues on the 3 N-linked chains is indicated. (B) An experiment similar to the one in A but with a chase period of 120 minutes was performed several times and phosphorimager quantitations of the average amounts of H2 remaining were plotted relative to the pulse. White bars show H2 remaining in the absence and black bars in the presence of 100 µg/ml CST. Fig. 2. The increased degradation of H2a caused by CST displays characteristics of proteasome mediated ER degradation. (A) NIH 3T3 cells (lane 1) or the same cells stably transfected with expression vectors encoding H2a (2-18 cell line, lanes 2-8) were metabolically labeled for 20 minutes with [ 35 S]cysteine and chased for 120 minutes with complete medium (lane 2) or with complete medium containing CST (100 µg/ml) (lanes 3, 5) or with complete medium containing CST in the presence of ALLN (100 µm, lane 4), leupeptin (400 µg/ml, lane 6), NH 4Cl (20 mm, lane 7) or chloroquine (800 µm, lane 8). In lane 5, the cells were incubated at 18 C during the chase period. Cells treated with CST were preincubated as in Fig. 1. Cell lysates were immunoprecipitated with anti-h2 antibody and analyzed by SDS-PAGE followed by fluorography. The lower panel shows a phosphorimager quantitation plotted in relation to untreated (=100, white bar). The black bar shows treatment with CST only and in gray with CST and addition of the inhibitors indicated above. (B) An experiment similar to that in A was performed but with addition (lane 3) of the specific proteasome inhibitor ZL 3VS. Cells treated with ZL 3VS were incubated with the drug at 50 µm during the starvation, labeling and chase periods.

4 3312 M. Ayalon-Soffer, M. Shenkman and G. Z. Lederkremer causes misfolding of H2a, increasing its ability to serve as a substrate for the quality control mechanism of the ER. In order to study the effect of CST on folding we metabolically labeled cells expressing H2a or H2b in the absence or presence of the drug, immunoprecipitated the cell lysates and ran the immunoprecipitated proteins on non-reducing SDS-PAGE, in which compact folded forms run faster than unfolded molecules. Before lysis cells were treated with 0.1 M iodoacetamide to prevent formation of any disulfide bonds that did not already exist in vivo. As shown in Fig. 3A, after two hours chase both H2a and H2b bands shifted similarly in the presence or absence of CST to a higher mobility. To analyze the folding in more detail cells were treated with CST and DTT was added to the cells in vivo for 5 minutes at the end of the pulse or chase periods to analyze the acquisition of resistance to disulfide bond reduction upon folding (Braakman et al., 1992). Cells were then incubated with iodoacetamide to block free sulfhydryl groups. The cell lysates were immunoprecipitated and the proteins were treated with endo H (to eliminate heterogeneity due to the sugar chains) before nonreducing SDS-PAGE. As seen in Fig. 3B, after chase both H2a and H2b bands shifted similarly to a higher mobility. H2a became mostly resistant to in vivo reduction by DTT and H2b partially resistant. The similar folding pattern of H2a and H2b in the presence of CST suggests that the increased degradation of H2a by CST is not due to a general misfolding of the protein. Fig. 3. Folding of H2a and H2b in the presence of CST. (A) Singly transfected NIH 3T3 cells expressing H2a (2-18 cell line, lanes 1-6) or H2b (2C cell line, lanes 7-12) were metabolically labeled for 10 minutes with [ 35 S]cysteine and chased for the indicated times with complete medium in the absence or presence of 100 µg/ml CST as in Fig. 1. Cells were then incubated with 0.1 M iodoacetamide for 5 minutes at 4 C, lysed and immunoprecipitated with anti-h2 antibody. The immunoprecipitates were run on SDS-PAGE under non-reducing conditions followed by fluorography. Besides the depicted monomer bands, disulfide bonded dimers, trimers and higher oligomers of H2a and especially of H2b were also present in the nonreducing gel and are not shown. (B) Cells expressing H2a (lanes 1-5) or H2b (lanes 6-10) were pulsed and chased as in A except that all samples were treated with CST. In lanes 2, 5, 7, 10 DTT (5 mm) was added to the cell medium for the last 5 minutes of the pulse or chase periods. Cells were then incubated with 0.1 M iodoacetamide for 5 minutes at 4 C, lysed and immunoprecipitated with anti-h2 antibody. The immunoprecipitates were treated with endo H and subjected to non-reducing SDS-PAGE followed by fluorography. Folded S-S bonded and unfolded molecules are indicated. Note the slightly faster migration of all H2b bands compared to H2a because of the absence of the EGHRG pentapeptide. CST causes ER retention of H2b but nevertheless does not increase its degradation In the presence of CST H2b does not mature to its Golgiprocessed form (Fig. 4a, lanes 5 and 6). Two possibilities can explain this finding. The first is that H2b cannot exit the ER in the presence of CST, therefore it is not available for the processing enzymes of the Golgi. The second is that H2b exits the ER, but as a result of the untrimmed Glc residues, it cannot be processed to the complex form. The latter alternative could explain the differential degradation of H2a and H2b in the presence of CST, as H2b by leaving the ER, could escape from the place of degradation and therefore survive. If this were the case one would expect that if the transport from the ER is prevented, H2b would be degraded in the presence of CST, the same as H2a. We have thus blocked the ER to Golgi transport by incubation with brefeldin A (BFA) and analyzed the effect of CST. As shown in Fig. 4a, incubating the cells with CST caused an increased degradation of H2a while H2b remained stable (compare lanes 1,2 with lanes 5,6). Treatment of the cells with BFA alone did not affect the survival of the molecules. Upon incubation of the cells with CST plus BFA, H2a was degraded while H2b remained stable, the same effect as seen with CST alone (compare lanes 3,4 with lanes 7,8). Thus, when exit from the ER is prevented H2b still remains stable upon inhibition of Glc trimming. To analyze the subcellular localization of H2b upon treatment with CST we performed immunofluorescence after incubation of cells expressing H2b with CST for 5 hours. To minimize detection of newly synthesized molecules cycloheximide was added for the last hour of the incubations. Before treatment H2b appeared in a mixed ER/Golgi pattern, much of it being in a perinuclear location, colocalizing with a Golgi marker, Cab45 (Scherer et al., 1996) (Fig. 4b, A and B). After treatment with CST, H2b was no longer found in the Golgi and appeared only in an ER-like distribution (Fig. 4b, C). Cab45 was also partially redistributed to the ER (it also bears an N-linked sugar chain), but to a much lesser degree than H2b probably because of its much slower turnover rate (Fig. 4b, D). From these results we conclude that the differential degradation of H2a and H2b in the presence of CST cannot be explained by a mechanism of escape from the ER of H2b. Upon inhibition of Glc trimming both H2a and H2b are in the ER although only H2a shows increased degradation. The increased degradation of H2a in the presence of CST is not due to a prevention of its association to calnexin Since inhibition of Glc trimming is known to prevent the association of glycoproteins to calnexin, a differential involvement of calnexin in the maturation of H2a and H2b could account for the effect of CST on their degradation. To study the interaction of H2a and H2b with calnexin and the

5 a b Fig. 4. The fact that CST does not increase the degradation of H2b is not due to its escape from the ER. (a) The NIH 3T3 cells expressing H2a or H2b were metabolically labeled for 20 minutes with [ 35 S]cysteine and chased for 120 minutes with complete medium in the absence (lanes 1,5) or in the presence of CST (100 µg/ml) (lanes 2, 6), BFA (5 µg/ml, to block ER-Golgi traffic) (lanes 3,7) or both CST and BFA (lanes 4, 8). Cells treated with CST were preincubated as in Fig. 1, while BFA was present only during the chase. Cell lysates were immunoprecipitated with anti-h2 antibody and analyzed by SDS-PAGE followed by fluorography. The full arrowhead points to Golgi-processed H2b. The asterisk indicates a non-specific band. Migration of a molecular mass marker is indicated on the right side in kilodaltons. (b) The cells expressing H2b (2C cell line) were incubated for 5 hours in the absence (A and B) or presence (C and D) of 100 µg/ml CST. 300 µm cycloheximide were added during the last hour of the incubation. Double labeling immunofluorescence was performed on permeabilized cells with anti-h2 carboxy-terminal antibody (secondary goat anti-rabbit IgG-Cy3; A and C), a block with unlabeled goat anti-rabbit IgG and an incubation with anti- Cab45 antibody (as Golgi marker) (secondary goat anti-rabbit IgG- FITC; B and D). Bar, 10 µm. effect of CST, cells expressing H2a or H2b were metabolically labeled in the absence or in the presence of a relatively low concentration, 30 µg/ml or a higher concentration, 100 µg/ml of CST. Immunoprecipitation of cell lysates was done first with anti-calnexin antibody followed by elution and reimmunoprecipitation (recapture) with anti-h2 carboxyterminal antibody. As shown in Fig. 5A both H2a and H2b interact with calnexin although to a relatively small extent (~5% of total H2 coprecipitates with calnexin). It would be Man and Glc trimming in ER degradation 3313 unlikely that this small extent of association could account for the large increase in the degradation caused by CST. Nevertheless, as calnexin functions by cycles of binding and release a small extent of association at any time could have a much more profound overall effect. When we inhibited Glc trimming by using CST at a high concentration (100 µg/ml), we could abolish completely the interaction of H2a and H2b with calnexin as expected (Fig. 5A, lanes 4, 8). However, when we used 30 µg/ml of CST, H2a and H2b were associated with calnexin to the same extent as without CST. It is interesting to point out the shift to a lower mobility in the presence of 30 µg/ml of CST (compare Fig. 5A, lanes 2 to 3 and 6 to 7), which indicates that at least one of the three sugar chains in H2a and H2b remains with additional untrimmed Glc residues and nevertheless this does not affect calnexin binding. We then analyzed if the lower concentration of CST (30 µg/ml) while not abrogating the association to calnexin could still cause accelerated degradation of H2a. Cells expressing H2a were metabolically labeled in the absence or in the presence of 30 µg/ml or 100 µg/ml CST and the survival of H2a assessed (Fig. 5B). Treatment of the cells with 30 µg/ml CST resulted in partial inhibition of Glc trimming, as can be seen by a smaller shift in the molecular mass compared to that obtained with a concentration of CST of 100 µg/ml (compare Fig. 5B, lane 5 to lane 6). However, the degradation of H2a was accelerated to a similar extent following partial or complete inhibition of Glc trimming. Thus, partial inhibition of Glc trimming results in accelerated degradation of H2a, but does not prevent its association to calnexin. CST causes increased degradation of H2a even in conditions that inhibit the dissociation from calnexin We analyzed the fate of H2a and H2b when their dissociation from calnexin is prevented. In contrast to previous experiments where cells were pre-incubated for several hours with CST leading to inhibition of glucosidases I and II, in this experiment we pulse-labeled cells expressing H2a or H2b and then only chased them in the presence of CST, inhibiting only the removal of the last Glc by glucosidase II (Hebert et al., 1995). Inhibition of the removal of this last Glc leads to an inhibition in calnexin dissociation. As shown in Fig. 6A and C after two hours of chase without any treatment (lanes 2 and 5) only 14% of H2a and 25% of H2b were still bound to calnexin compared to H2a or H2b bound to calnexin after the pulse. CST inhibited the dissociation of H2a and H2b from calnexin, because after two hours of chase in the presence of the drug (lanes 3 and 6), 61% of H2a and 77% of H2b were bound to calnexin compared to the pulse. Nevertheless the presence of CST only during the chase still caused an accelerated degradation of H2a and not of H2b. As shown in Fig. 6B and D after two hours of chase in the presence of CST, only 40% of H2a molecules remained (lane 9) compared to 78% for untreated cells (lane 8). Altogether our results suggest that the accelerated ER degradation caused by inhibition of Glc trimming is independent of interaction of the molecules with calnexin. Inhibition of glycosylation or of mannose trimming result in an increased stability of H2a, opposite to the effect of inhibition of Glc trimming We decided to dissociate calnexin from H2a and H2b in a

6 3314 M. Ayalon-Soffer, M. Shenkman and G. Z. Lederkremer Fig. 5. Partial inhibition of Glc trimming, although does not prevent the association of H2a to calnexin still results in its accelerated degradation. (A) The cell lines expressing H2a or H2b were metabolically labeled for 40 minutes with [ 35 S]cysteine in the absence (lanes 1, 2, 5, 6) or in the presence of CST at a concentration of 30 µg/ml (lanes 3, 7) or 100 µg/ml (lanes 4, 8). Cells treated with CST were preincubated as in Fig. 1. Cell lysates were immunoprecipitated with anti-h2 antibody (lanes 1, 5) or with anticalnexin antibody (Cx, lanes 2-4, 6-8). The immunoprecipitates were then boiled in SDS, the supernatants were then diluted with excess of Triton X-100 and re-immunoprecipitated with anti-h2 antibody, as described in materials and methods. The co-precipitated proteins were analyzed by SDS-PAGE and fluorography. (B) The same as in A except that cells were metabolically labeled for 20 minutes with [ 35 S]cysteine, chased in the indicated cases for 120 minutes with complete medium in the absence or in the presence of CST at a concentration of 30 µg/ml or 100 µg/ml and cell lysates were immunoprecipitated with anti-h2 antibody and directly analyzed by SDS-PAGE followed by fluorography. On the left are indicated the migrations of precursor H2a containing Glc residues or nonglucosylated. On the right the migration of a molecular mass marker in kilodaltons. The bottom panel shows an average of phosphorimager quantitations of several similar experiments where H2a remaining after the chase has been plotted relative to pulse (=100, white bar). Gray bar: in the presence of 30 µg/ml CST. Black bar: in the presence of 100 µg/ml CST. As in Fig. 1 the values were normalized to the amounts of label present after the pulse in the presence or absence of CST. Fig. 6. Incubation with CST only during a chase period inhibits calnexin dissociation but still causes an accelerated degradation of H2a and not of H2b. (A) Cells expressing H2a (lanes 1-3) or H2b (lanes 4-6) were metabolically labeled for 40 minutes with [ 35 S]cysteine and chased for 0 or 120 minutes with complete medium in the absence (lanes 1, 2, 4, 5) or in the presence of CST (100 µg/ml) (lanes 3,6). In this experiment, cells treated with CST were not preincubated with the drug; it was present only during the chase period. Cell lysates were immunoprecipitated with anti-calnexin (αcx) antibody. The immunoprecipitates were then boiled in SDS and re-immunoprecipitated with anti-h2 antibody as in Fig. 5A. The co-precipitated proteins were analyzed by SDS-PAGE and fluorography. The asterisk indicates a non-specific band. H2 indicates H2a or H2b precursors. (B) The same as in A, except that cell lysates were immunoprecipitated with anti-h2 antibody and directly analyzed by SDS-PAGE followed by fluorography. The gel was exposed to film for a much shorter time than in A. (C and D) Phosphorimager quantitations of the gels in A and B, respectively. In C values are plotted relative to the label recovered (H2 associated to calnexin) after the pulse labeling (=100, white bars). Gray bars show amounts remaining after chase in the absence and black bars in the presence of CST. In D values are plotted for total H2 remaining after the chase period relative to that present after the pulse (=100, white bars). Gray bars show amounts remaining after chase in the absence and black bars in the presence of CST.

7 Man and Glc trimming in ER degradation 3315 different manner and analyze the effect on the stability of the proteins. For this purpose we used tunicamycin (TM), an inhibitor of N-glycosylation which is known to prevent interaction of glycoproteins with calnexin. As shown in Fig. 7A, incubation of cells with TM indeed prevented the association of H2a and H2b with calnexin as seen by immunoprecipitation with anti-calnexin antibody, elution and re-immunoprecipitation with anti-h2. However, in contrast to CST, TM had a stabilizing effect on H2a as it increased three fold the stability of H2a molecules (Fig. 7B). These results indicate that dissociation of calnexin does not lead automatically to an increased degradation. The effect of TM on H2b was slightly destabilizing in contrast to that on H2a. Next we examined if the accelerated degradation in the presence of CST is due to a specific inhibition of Glc trimming or if inhibition of other processing steps as Man trimming result in a decrease in the stability of H2a as well. We incubated cells expressing H2a or H2b with dmm, an inhibitor of three ER mannosidases, ER Man 9 -mannosidase (Schweden et al., 1986), ER mannosidase I (Weng and Spiro, 1996), ER mannosidase II (Weng and Spiro, 1993) and of Golgi α- mannosidase I (Kornfeld and Kornfeld, 1985). dmm, like CST prevented the appearance of the mature form of H2b (Fig. 8, lanes 13 and 15). However, in contrast to CST, dmm increased the stability of both H2a and H2b (Fig. 8, lanes 7 and 15). Thus, inhibition of the trimming of Glc or Man residues have opposing effects on H2a degradation. DISCUSSION Fig. 7. Inhibition of N-glycosylation prevents the association of H2a to calnexin but results in an increased stability of H2a. (A) Cells expressing H2a (lanes 1,2) or H2b (lanes 3,4) were metabolically labeled for 40 minutes with [ 35 S]cysteine in the absence (lanes 1,3) or in the presence (lanes 2,4) of TM (10 µg/ml). Cells treated with TM were preincubated with the drug 2.5 hours before the labeling. The drug was also present during the starvation and labeling periods. Cell lysates were immunoprecipitated with anti-calnexin (αcx) antibody. The immunoprecipitates were then boiled in SDS and reimmunoprecipitated with anti-h2 antibody as in Fig. 5 A. The coprecipitated proteins were analyzed by SDS-PAGE and fluorography. (B) The same as in A except that cells were metabolically labeled for 20 minutes with [ 35 S]cysteine, chased in the indicated cases for 120 minutes with complete medium in the absence or in the presence of TM and cell lysates were immunoprecipitated with anti-h2 antibody and directly analyzed by SDS-PAGE followed by fluorography. Indicated on the right is the migration of a molecular mass marker in kilodaltons. The arrowhead points to Golgi-processed H2b. On the left are indicated the migrations of fully glycosylated and of nonglycosylated precursors. Between these two forms some bands representing partially glycosylated molecules can be seen. The asterisk indicates a non-specific band. On the bottom panel an average of phosphorimager quantitations of several similar experiments was plotted relative to the amounts present after pulselabeling (=100, white bars). The values were normalized to the amounts of label present after the pulse in the presence or absence of TM to compensate for the decrease of labeling in the presence of the drug. Gray bars show amounts remaining after chase in the absence and black bars in the presence of TM. Recent studies have shown that inhibition of Glc trimming causes an increased degradation of several glycoproteins, e.g. class I histocompatibility heavy chain (Moore and Spiro, 1993), the T cell receptor α chain (Kearse et al., 1994), invariant chain (Romagnoli and Germain, 1995), and influenza virus hemagglutinin (Hebert et al., 1996). Inhibition of Glc trimming by CST led to an increased degradation of ASGPR H2a under a variety of conditions (Figs 1, 5, 6). In contrast to the effect of CST, inhibition of Man trimming by dmm stabilized H2a (Fig. 8). One could speculate that as prolonged retention in the ER leads to extensive Man trimming to yield Man 6 GlcNAc 2 (Atkinson and Lee, 1984; Bischoff et al., 1986; Hebert et al., 1995) this may be recognized as a signal for degradation to occur. Inhibition of Man trimming would thus be stabilizing as was also seen for other glycoproteins (Kearse Fig. 8. Inhibition of mannose trimming results in an increased stability of H2a. Cells expressing H2a (lanes 1-8) or H2b (lanes 9-16) were metabolically labeled for 20 minutes with [ 35 S]cysteine and chased for 0 or 120 minutes with complete medium in the absence (lanes 1, 5, 9, 13) or in the presence of CST (100 µg/ml) (lanes 2, 6, 10, 14), dmm (50 µg/ml) (lanes 3, 7, 11, 15) or both of these drugs (lanes 4, 8, 12, 16). Cells treated with CST were preincubated as in Fig. 1, while dmm was present only during the starvation, labeling and chase periods. Cell lysates were immunoprecipitated with anti- H2 antibody and analyzed by SDS-PAGE followed by fluorography. The arrowhead points to Golgi-processed H2b. The asterisk indicates a non-specific band.

8 3316 M. Ayalon-Soffer, M. Shenkman and G. Z. Lederkremer et al., 1994; Liu et al., 1999; Yang et al., 1998). On the other hand prolonged ER retention may lead to persistence of the last Glc residue (by cycles of deglucosylation by glucosidase II and reglucosylation by UDP-GLC: glycoprotein glucosyltransferase (Hammond et al., 1994; Labriola et al., 1995; Parodi, 1999; Parodi et al., 1983)). The latter enzyme was shown to remain associated in certain conditions to ER retained and degraded mutant α1-antitrypsin (Choudhury et al., 1997) which might lead to its persistent reglucosylation. The continued presence of one or more Glc residues could also be a signal for degradation, explaining the destabilizing effect of Glc trimming inhibitors. The increased degradation in the presence of these inhibitors is proteasome-mediated (Fig. 2). The modulating effects of sugar trimming events on the rate of degradation occur in the lumenal side of the ER and therefore must regulate steps prior to delivery to the cytosolic proteasome. Fig. 9 illustrates a working model of how the action of the inhibitors could lead to the accumulation of stable molecules or others prone for degradation. Besides its effect on accelerating degradation, inhibition of Glc trimming also prevents the association of the glycoproteins with calnexin. Thus, it was obviously suggested that the accelerated degradation results from a lack of association to calnexin (Hebert et al., 1996; Keller et al., 1998; Liu et al., 1999). Our results presented here argue against a relation between the increased degradation of ASGPR H2a due to the effect of CST and its interaction with calnexin, for the following reasons: (a) Partial inhibition of Glc trimming by CST caused accelerated degradation of H2a, yet did not inhibit calnexin binding (Fig. 5). (b) Inhibition of N-glycosylation with tunicamycin prevented the interaction of H2a with ER "QUALITY CONTROL" SITES H2a-GlcNAc 2 Man 9 Glc 3 CST H2a-GlcNAc 2 Man 9 Glc 1 * calnexin but did not accelerate its degradation and quite the contrary resulted in an increased stability of H2a (Fig. 7), and (c) Inhibition of the dissociation of H2a from calnexin by preventing the removal of the last Glc residue also resulted in accelerated degradation of H2a (Fig. 6). Therefore we suggest that the increase in the ER degradation of H2a, caused by inhibition of Glc trimming, is independent of its binding to calnexin. This may also be true for the other glycoproteins that show the same effect (Liu et al., 1997; Romagnoli and Germain, 1995). For one such case, mutant α1-antitrypsin, there are conflicting reports on the effect of CST, which inhibited its degradation in a cell-free system (Qu et al., 1996) but accelerated the degradation in cells in vivo (Liu et al., 1997). However, in a patient with mutant α1-antitrypsin induced liver disease there was a decreased association of the protein with calnexin which correlated with a decreased rather than an increased degradation (Wu et al., 1994). In a study of the HIV/Vpu-induced degradation of CD4 in calnexindeficient cells there was no effect on the rate of degradation as compared to normal cells (Schubert et al., 1998). Two other candidates that could be linked to the effect of the inhibition of Glc trimming are the ER resident proteins calreticulin (Hebert et al., 1996; Peterson et al., 1995) and ERp57 (Oliver et al., 1997). Association to calreticulin was also shown to depend on Glc trimming. In the case of ERp57, binding to the substrate protein is not direct but through a tertiary complex with calnexin or calreticulin (Zapun et al., 1998). In the cases of tyrosinase and of T cell receptor α a very rapid degradation in certain cell types was found to correlate to a reduced association with calreticulin and not to calnexin (Bennett et al., 1998; Halaban et al., 1997). For H2a we have ER EXIT SITES H2b-GlcNAc 2 Man 9 Glc 3 CST H2b-GlcNAc 2 Man 9 Glc 1 * CST CST H2a-GlcNAc 2 Man 9,8 dmm CST * H2a-GlcNAc 2 Man 9,8 Glc 1 H2a-GlcNAc 2 Man 6 H2b-GlcNAc 2 Man 9,8 dmm H2b-GlcNAc 2 Man CST 6 H2b-GlcNAc 2 Man 9,8 Glc 1 * GOLGI PROTEASOME Fig. 9. Working model for the relation between sugar trimming events on H2a/H2b and their degradation. H2a and H2b receive the N-linked oligosaccharide GlcNAc 2Man 9Glc 3 cotranslationally. Sorting would then occur to ER quality control sites (for H2a which contains a retention signal, black circle) or to ER exit sites (for H2b). Man and Glc trimming reactions then take place followed by reglucosylation by UDP-Glc: glycoprotein glucosyltransferase. These reactions create monoglucosylated substrates that bind to calnexin (marked with a blue asterisk). Inhibition of Glc trimming by CST would lead to the accumulation of H2a species particularly prone for degradation (in red). Inhibition of Man trimming by dmm would lead to the accumulation of relatively stable molecules containing GlcNAc 2Man 9,8 (in green). Otherwise Man trimming leads to molecules containing GlcNAc 2Man 6 targeted to degradation. The scheme takes into consideration the reported ideal substrates for UDP-Glc: glycoprotein glucosyltransferase (GlcNAc 2Man 9,8; Sousa et al., 1992), dmm insensitive ER mannosidase (GlcNAc 2Man 9; Bischoff et al., 1986), dmm sensitive ER mannosidases I and II (GlcNAc 2Man 9; Weng and Spiro, 1993; Weng and Spiro, 1996) and dmm sensitive Man 9-mannosidase (GlcNAc 2Man 9-7; Schweden et al., 1986).

9 Man and Glc trimming in ER degradation 3317 found insignificant binding to calreticulin (data not shown), which argues against a role of this chaperone in the effect of Glc trimming inhibition. Another possibility is that the continued presence of the Glc residues leads to a structural instability of H2a in spite of the fact that its general folding is not affected (Fig. 3). However, we have pulse-labeled cells expressing H2a in the presence or absence of CST, lysed them in non-denaturing conditions and incubated the cell lysates at 37 C for several time periods and no difference was observed in the rates of in vitro degradation of the glucosylated versus the non-glucosylated species (data not shown). Inhibition of Glc trimming had a differential effect on the stability of H2a and H2b. Whereas it accelerated the degradation of H2a, it did not affect the stability of H2b (Figs 1, 4, 6). The only difference between H2a and H2b is an extra charged pentapeptide (EGHRG) present in the ectodomain of H2a next to the transmembrane domain. These five amino acids were shown to act as an ER retention signal (Shenkman et al., 1997). Here we demonstrate an additional effect of these five amino acids in determining the increased instability of H2a in the absence of Glc trimming. Singly expressed H2b can exit the ER with about 30% efficiency as opposed to the complete retention of H2a (Lederkremer and Lodish, 1991). However, the differential degradation of H2a and H2b in the presence of CST cannot be explained by a mechanism of escape from the ER of H2b. After treatment with CST both H2a and H2b were found to be in the ER (Fig. 4) although only H2a showed an accelerated degradation. One possible explanation for this could be that upon inhibition of Glc trimming both are in a pre-golgi compartment but not in the exact same location. As illustrated in the model of Fig. 9 the ER retention of H2a could conceivably involve its targeting to specific locations, ER quality control sites for degradation. As for H2b it would be transported to ER exit sites where it would fold and become competent for exit to the Golgi. H2b molecules that persistently fail to mature would undergo Man trimming and become rerouted to degradation. In view of current models for participation of the ubiquitin/proteasome machinery in ER degradation (Bonifacino and Weissman, 1998; Sommer and Wolf, 1997) and of our own evidence of involvement of this machinery in the degradation of H2a, the ER quality control sites might be the ER translocon itself through which the protein could be reverse translocated and targeted to degradation. We are grateful to Ari Helenius for antibodies, Hidde Ploegh for ZL 3VS, Rachel Ehrlich for helpful discussions and Nathan Sharon, Shoshana Bar Nun and Drorit Neumann for critically reading the manuscript. 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