©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
HLA-DR Chains Enter into an Aggregated Complex Containing GRP-78/BiP Prior to Their Degradation by the Pre-Golgi Degradative Pathway (*)

(Received for publication, August 16, 1994; and in revised form, November 2, 1994)

Tom Cotner (1) Donald Pious (1) (2) (3)

From the  (1)Departments of Pediatrics, (2)Immunology and (3)Genetics, University of Washington, Seattle, Washington 98195

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

HLA class II molecules are membrane proteins which are assembled in the endoplasmic reticulum shortly after synthesis of the alpha and beta and invariant chain (Ii) monomers. DRbeta chains, in the absence of DRalpha, are rapidly and completely degraded by the pre-Golgi degradative pathway. Here we have examined those factors which target DRbeta chains for degradation in a DRalpha deficient cell line, 9.22.3. The DRbeta monomers in 9.22.3 were initially incorporated into a proteinaceous complex containing BiP. With time, the DRbeta complexes were further aggregated. In wild type cells, which can assemble DRalpha-beta dimers, the secondary phase of aggregation of DRbeta was not seen. Additional evidence that aggregation of DRbeta in 9.22.3 cells was progressive was that a more mature form of DRbeta was found exclusively in the largest DRbeta complexes. Furthermore, the most highly aggregated DRbeta chains were degraded more rapidly than bulk DRbeta chains. These data suggest that DRbeta aggregates are intermediates in the pre-Golgi pathway of DRbeta degradation. They further suggest that formation of large DRbeta aggregates is a proximal event to DRbeta degradation. We conclude that DRbeta chains are targeted for degradation as a consequence of a change of state, coincident with their aggregation into slow forming, high molecular weight complexes.


INTRODUCTION

There are two well characterized pathways for the degradation of membrane proteins. The first involves the internalization of membrane proteins by the invagination of plasma membrane and the removal of a fraction of membrane proteins and their degradation in lysosomes(1, 2) . The second involves the pre-Golgi degradation of certain monomeric subunits or mutated proteins shortly after their synthesis and transfer into the endoplasmic reticulum (ER) (^1)(3, 4, 5, 6, 7, 8, 9, 10, 11, 12) . The first pathway is sensitive to lysomorphotrophic agents and inhibitors of lysosomal proteases. The second is insensitive to such agents but can be blocked by drugs or conditions which prevent the transport of the membrane proteins beyond the cis-Golgi(3, 5, 8, 9, 10, 11, 12) . The events which are responsible for targeting certain membrane or secreted proteins for pre-Golgi degradation are largely unknown. None of the components of the proteolytic machinery have been identified. There is good evidence that degradation occurs in the ER(8, 10, 13) . Studies of model mutated or truncated proteins have suggested that the pre-Golgi degradative pathway is capable of recognizing unfolded or misfolded proteins. Pre-Golgi degradation is frequently the fate of the subunits of multimeric proteins which are synthesized in unequal amounts(3, 7, 8, 12) . Degradation of model proteins in a pre-Golgi compartment is preceded by a short lag period after synthesis(3, 8, 12) . Thereafter, the half-lives of membrane proteins which are degraded by this pathway are short, usually approximately 1 h. We have studied the degradation of HLA-DRbeta monomers and have found that they are degraded by the pre-Golgi pathway(12) . Degradation of DRbeta monomers is comparable to that of several other subunits of multimeric membrane proteins, and occurs rapidly in a pre-medial Golgi compartment. The pre-Golgi pathway is insensitive to lysomorphotropic agents, but degradation can be inhibited by conditions or drugs which deplete energy stores in the ER(7) .

Recognition of misfolded or incompletely folded proteins must occur shortly after synthesis. Studies of protein folding in the ER indicate that folding of the nascent polypeptide chain begins before translation is completed(14, 15, 16) . A class of proteins, termed chaperones, associate transiently with newly synthesized proteins and are believed to assist in the folding process. The chaperones BiP-GRP78 (hereafter referred to as BiP), calnexin, protein disulfide isomerase, and GRP94 bind a broad but select set of newly synthesized polypeptides and likely assist in their folding(16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28) . Folding is not completely efficient, however, and a fraction of proteins may be misfolded. Misfolded proteins can be detected shortly after synthesis and, in general, are retained in the ER as aggregates(15, 29, 30) . Severe misfolding of proteins can result in formation of large aggregates; precipitates may also form which can exist in the ER for several hours (15, 20, 22, 29, 30, 31) . It is not clear how these model proteins are degraded and there is no indication that such aggregated proteins are substrates of the pre-Golgi degradative pathway. In contrast, subunits of multimeric proteins which remain unassembled appear to be handled differently(29, 31) . The unassembled subunits, though retained in the ER, are usually not found in highly aggregated states and they retain the capability of assembling into multimers for long periods of time (29, 31, 32, 33) . In general, unassembled subunits appear to interact with BiP, but these interactions are less avid and more reversible than BiP interactions with highly aggregated proteins(28) .

The pre-Golgi degradative pathway is thought to degrade proteins which are misfolded, but it is unclear how unfolded proteins are recognized and once recognized, selectively sorted for disposal. It has been suggested that the ER chaperones such as BiP may play a role in the determining a protein's fate toward native conformation(16, 17, 32, 34, 35, 36) or toward misfolding and ultimately, degradation(19, 25, 31) . Although much progress has been made in understanding the functions of the ER resident chaperones, the potential role of ER resident chaperones in either identifying or targeting misfolded proteins for degradation has not been fully explored. Here we have examined the possible role of one ER stress protein, BiP, in determining the fate of DRbeta chains destined for degradation. We find that BiP initially interacts transiently with DRbeta chains shortly after their synthesis but at a time when no DRbeta degradation takes place, suggesting that binding of BiP to DRbeta chains per se does not target them for degradation. However, at later times, multiple copies of BiP bind to DRbeta monomers, resulting in the formation of tightly associated high molecular weight oligomers. The aggregation of DRbeta chains into high molecular weight BiPbulletDRbeta complexes suggests a means by which DRbeta chains are segregated within the ER and targeted for degradation.


MATERIALS AND METHODS

Antibodies

The monoclonal antibodies used were W6/32, specific for HLA class I dimers(37) ; HB10.A, specific for DRbeta monomers(38) , VI.15, specific for DR dimers(39) ; and a rat antibody specific for human BiP/GRP78(16) , which was a generous gift of Dr. David Bole, Yale University. Antibodies were centrifuged at 50,000 times g for 5 min after thawing to remove aggregated or denatured immunoglobulin that potentially would bind BiP.

Cell Lines

9.22.3 is a B cell line with a homozygous deletion of DRA but which synthesizes normal levels of DR3beta mRNA and beta chains(40, 41) . SC-JB is a DR3 homozygous cell which expresses twice the level of DR3 as the 9.22.3 progenitor 8.1.6 and was therefore used to examine the role of BiP in DR assembly(41) .

Metabolic Labeling of Cells

Pulse-chase experiments were performed (12) using [S]methionine (DuPont NEN) following a 40-min incubation in medium containing 10% dialyzed fetal calf serum and lacking methionine. Cells (10^7/ml) were labeled for 10-20 min in methionine RPMI 1640 containing 1 mCi/ml [S]methionine and then diluted to a cell concentration of 2 times 10^6/ml into RPMI 1640 and 10% fetal calf serum supplemented with 1 mM methionine. Cells were harvested at the times indicated and processed as below. Steady-state labeling conditions were carried out for 4 days in complete RPMI 1640 medium containing 0.2 mCi/ml [S]methionine using an initial concentration of 0.8 times 10^6 cells/ml.

Cell Lysates, Immunoprecipitations, and Electrophoretic Analysis

Cell lysates and immunoprecipitations were carried out as previously described(12, 42) , and the immune complexes were captured on protein A-Sepharose. Immunoprecipitates were washed in buffers without EDTA(42) , and the material was electrophoresed on 10 or 12% gels, fixed, placed in Amplify (Amersham Corp.), dried, and exposed to XAR-5 x-ray film. Computer densitometric analysis of gels was performed with a whole band analyzer (BioImage, Ann Arbor, MI). In some cases, quantitation of fixed and dried gels was performed on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) using the ImageQuant software program. The following pre-stained molecular mass markers were used: phosphorylase B (106 kDa), bovine serum albumin (80 kDa), ovalbumin (49.5 kDa), carbonic anhydrase (32.5 kDa), soybean trypsin (28.5 kDa), and lysozyme (18.5 kDa), all from Bio-Rad.

Sucrose Gradient Centrifugation

Detergent-soluble extracts of both unlabeled or S-labeled proteins were analyzed on 5-30% sucrose gradients. Samples containing 100 µl of extract (20-40 times 10^6 unlabeled cells or 4 times 10^6 labeled cells) were overlaid on a 4.6-ml 5-30% sucrose gradient containing 0.2% Nonidet P-40, 10 mM Tris-HCl, pH 7.5, and centrifuged at 47,000 rpm in a SW 50.1 rotor for 16 h at 4 °C. Nineteen fractions (0.25 ml) were collected from the bottom, and labeled extracts were analyzed by immunoprecipitation. The following marker proteins (20-100 µg) were centrifuged in parallel, and the resulting fractions were analyzed by Coomassie staining of SDS gels: cytochrome c (1.83 S), bovine serum albumin (4.5 S), rabbit muscle aldolase (7.35 S), catalase (11.3 S), and alpha(2)-macroglobulin (20 S), all from Sigma.


RESULTS

BiP Associates with DRbeta Chains in a DRalpha-deficient Cell Line

To investigate whether the ER resident protein BiP plays a role in determining the fate of DRbeta chains, the DRalpha-deficient 9.22.3 cell line was metabolically labeled for 3 h with [S]methionine and immunoprecipitated with antibodies to either DRbeta or to BiP. The immunoprecipitates were analyzed on SDS-polyacrylamide gel electrophoresis. The anti-DRbeta monoclonal antibody, in addition to precipitating the DRbeta monomer, also coprecipitated the Ii chain (M(r) 31,000) and a strong band of M(r) 78,000 with a mobility identical to that of BiP (Fig. 1). With long labeling periods, DRbeta migrates as two bands (29,000 and 27,500, hereafter referred to as p29 and p27), corresponding in size to the two forms of DRbeta found in 9.22.3 cells(12) . We have previously demonstrated, using pulse-chase analysis, that a precursor-product relationship existed between the 29,000 and 27,000 forms of DRbeta(11) . The 27,000 form appeared to be generated from the 29,000 form by the action of one or more endogenous glycosidases, since endoglycosidase H treatment completely converted both forms to a single, 26,000-27,000 band (see Fig. 1A, Fig. 2, and Fig. 5of (11) ). In short labeling periods, only p29 is evident, whereas p27 is quite prominent with the long labeling period employed in Fig. 1. The p27 form of DRbeta probably reflects the age of the molecule and may not be a necessary intermediate of pre-Golgi degradation. Importantly, analysis of anti-BiP immunoprecipitates revealed that the p27 form of DRbeta was more prominently coprecipitated by anti-BiP antibody than p29. This suggests BiP either preferentially binds to or is more stably associated with the smaller, more mature form of DRbeta.


Figure 1: BiP associates strongly with DRbeta monomers in a DRalpha-deficient cell line. 9.22.3 cells (20 times 10^6) (lanes 1-7) or 8.1.6 cells (4 times 10^6) (lane 8) were labeled for 3 h with [S]methionine, and extracts were reacted with anti-BiP (lanes 2 and 4), anti-DRbeta (lanes 1 and 3), anti-class I (lanes 5 and 6), normal mouse IgG (lane 7), or VI.15 (anti-DR dimer) (lane 8). Immunoprecipitates were washed six times, including twice with buffers including either 0.65 M NaCl (lanes 1, 2, 5, 7, and 8) or 0.5 M LiCl (lanes 3, 4, and 6). Note that anti-BiP preferentially coprecipitated the p27 form of DRbeta.




Figure 2: BiP binding to DRbeta is biphasic. 9.22.3 cells were pulsed with [S]methionine for short periods (A) or longer periods (B) and chased for the times indicated. Cells were pulsed for 8 or 10 min in (A) or for 20 min in (B). Samples were immunoprecipitated with HB10.A and the BiP and DRbeta regions of the gel were quantitated by PhosphorImager-based analysis. The BiP/DRbeta ratios, uncorrected for methionine content, were normalized to the BiP/DRbeta ratios found at 0 chase time. The initial decline in BiP/DRbeta ratio was seen in five of five experiments and the extent of decline appeared to relate to the length of the pulse. It is likely that with the longer pulse time shown in B, the secondary increase of BiP binding tends to mask the initial decline in BiP/DRbeta ratio.




Figure 5: Size distribution of DRbeta complexes in wild type cells. 8.1.6 cells (8 times 10^6) were pulsed for 15 min (Panel A) or pulsed and chased for 2 h (Panel B) and analyzed in sucrose gradients by immunoprecipitation with HB10.A as in Fig. 3. In A, note that not much assembly of DR dimers has occurred during the pulse. In A, Ii is the most prominent band, followed by DRbeta, with small amounts of DRalpha seen only in fractions 8-11. Quantitation of DRbeta is shown in C. Note that the average size DRbeta complex in wild type cells becomes smaller with time.




Figure 3: Aggregation of DRbeta chains is progressive in DRalpha deficient cells. Sucrose gradient and immunoprecipitation analysis of 9.22.3 cells which were pulsed with [S]methionine for 20 min and then chased for 0 (A), 1 h (B), or 2 h (C) or pulsed and cross-linked with 500 µg/ml dithiobis(succinimidyl propionate) for 30 min at 25 °C (D). Panels A-D were immunoprecipitated with HB10.A, whereas Panel E was immunoprecipitated with a mixture of W6/32 (anti-class I) and HB10.A (E). For Panels A-D, fractions 2, 12, 14, 16, and 17 were analyzed on a separate gel and the PhosphorImaging quantitation of DRbeta is shown in Fig. 5. Because of the brief exposure time (4 h) in Panel E, only MHC class I is visible. The above experiment was performed three times with essentially identical results.



The coprecipitation of BiP by anti-DRbeta antibody does not appear to be simply the gratuitous interaction with an abundant ER protein. Evidence that the interaction was not gratuitous was that binding exhibited both a high degree of specificity and was strong. No BiP was immunoprecipitated with normal mouse IgG (lane 7), indicating that coprecipitation of BiP with anti-DR antibody was not due to the well known ability of BiP to bind to free or partially denatured immunoglobulin heavy chains(16) . Moreover, BiP was not detected in immunoprecipitates using antibodies to fully assembled DR dimers from wild type cells (lane 8). Trace amounts of BiP, constituting some 4% of that seen in anti-DRbeta immunoprecipitates, was coprecipitated from 9.22.3 cells with antibodies to MHC class I molecules (lanes 5 and 6). The coprecipitation of BiP by anti-DRbeta antibodies was quite strong; it did not require the use of chemical cross-linkers, and BiPbulletDRbeta complexes were resistant to stringent wash conditions of either 0.5 M LiCl or 0.65 M NaCl (Fig. 1, compare lanes 1 and 2 with lanes 3 and 4).

DRbeta was a prominent band in the anti-BiP immunoprecipitate, comprising 6% of the total non-BiP material. This value seems unexpectedly high, given that DRbeta chains make up only 0.1% of the total mRNA and protein in wild type B cell lines(41) . Thus DRbeta chains, when they exist as monomers, are more likely to bind to or interact more stably with BiP than the average membrane protein. The corollary of this is that some membrane proteins would be expected either not to interact with BiP or to interact only very transiently. In support of this, neither proteins the size of Ii or MHC class I were evident in anti-BiP immunoprecipitates (Fig. 1, lanes 2 and 4). An estimate by another method, quantitative Western blotting, of the BiP found in anti-DRbeta immunoprecipitates compared to the BiP found in the starting cellular extract indicated that, under steady state conditions, 3% of the total BiP was complexed with DRbeta monomers in DRalpha-deficient cells (data not shown).

The Stoichiometry of BiPbulletDRbeta Complexes in 9.22.3 Cells

Preliminary experiments indicated that BiP association with DRbeta monomers was biphasic. BiP binding to DRbeta appeared to peak during the pulse, followed by a decline at early chase times (Fig. 2A). This was followed by a secondary 2.3-4 fold increase in the BiP/DRbeta ratio over the next 45-120 min. The initial decline in BiP associated with DRbeta was most pronounced during short pulses (Fig. 2A). With longer pulse times, the initial decline in BiP associated with DRbeta was not as evident, probably because the longer pulse time encompassed the time the secondary wave of BiP binding occurred (Fig. 2B). The secondary increase in BiP binding to DRbeta was only noted in DRalpha deficient 9.22.3 cells; it was not seen in wild type cells, suggesting that only those DRbeta monomers destined for degradation become increasingly complexed with BiP.

The detection of a secondary increase in DRbeta-BiP complexes after 10-20 min of DRbeta synthesis suggested that the ratio of BiP to DRbeta might be increasing during this time. Therefore we measured the ratio of DRbeta to BiP as a function of time following DRbeta synthesis in DRalpha-deficient 9.22.3 cells. The BiP to DRbeta ratio was measured at three different times: during a 10-min pulse, at 3 h when DRbeta but not BiP is labeled to equilibrium, and at 48 h, a time when the labeling of BiP approached equilibrium. (^2)The amounts of DRbeta and BiP coprecipitated by anti-BiP and anti-DRbeta, respectively, were quantitated by densitometry and corrected for the relative methionine content of the two proteins as previously described(15) . The BiP to DRbeta ratio went from 0.2 after a 10-min pulse, to 0.9 after 3 h and increased to an average (in three experiments) of 2.2 at 48 h. These data indicate that, at equilibrium in the absence of DRalpha chains, the average DRbeta molecule is in a complex with about 2 BiP molecules. The intensity of the BiP band coprecipitated by anti-DRbeta antibody probably significantly underestimates the stoichiometry of the DRbetabulletBiP complex for short labeling periods because it does not account for the large amount of unlabeled BiP present in the anti-DRbeta immunoprecipitate. The kinetics of BiP binding to DRbeta in DRalpha deficient cells and a BiP/qDRbeta ratio of about 2 at steady state suggested that, in the absence of DRalpha chains, DRbeta monomers increasingly enter into a complex with BiP.

DRbeta Chains Are Found in Large Aggregates at the Time of Maximal DRbeta Degradation

The fact that the initial association/dissociation of BiP with DRbeta chains appears equivalent in DRalpha deficient and wild type cells at very early times suggests that some other signal must be employed to target misfolded or incompletely folded DRbeta chains for degradation. One possibility is that DRbeta chains undergo a change of state. An abrupt change of state would result if DRbeta chain became aggregated. To examine this possibility, we compared the aggregation state of DRbeta chains from 9.22.3 cells which were either pulsed briefly or pulsed and chased for either 1 or 2 h. Extracts were prepared and centrifuged on 5-30% linear sucrose gradients and then analyzed by immunoprecipitation with antibodies to DRbeta chains or, as an internal control, antibodies to MHC class I. During the pulse, DRbeta chains from 9.22.3 sedimented heterogeneously with an average complex of 5-6 S, with only 25% larger than 7S (Fig. 3; see Table 2). Very little DRbeta sedimented in monomeric form (<4 S) in any experiments and only 13% of DRbeta sedimented at 10 S or greater (Fig. 3A; see Table 2). By 1 h, the percentage of DRbeta complexes sedimenting faster than 10 S had increased from 13 to 28% and by 2 h over 40% of DRbeta sedimented faster than 10 S ( Fig. 3and Fig. 4; Table 1). One possibility to explain the kinetics of formation of 10 S DRbeta complexes is that DRbeta chains enter into the 10 S complex soon after synthesis but that the initial complex is labile and that partial dissociation of the complexes occurs during the time of the sucrose gradient, causing it to sediment as a 5-6 S complex. To determine if DRbeta entered into a 10 S complex shortly after synthesis, pulse-labeled extracts from 9.22.3 cells were chemically cross-linked with dithiobis(succinyl propionate) prior to sucrose gradient analysis. Cross-linking resulted in a shift of the 5-6 S region to 6.5-7 S. However, cross-linking did not change the distribution of DRbeta in fractions 7 S or greater (Fig. 4, fractions 1-9). The fact that no increase in large (i.e. 10S) DRbeta complexes was detected after extensive cross-linking argues against the notion that DRbeta enters into a large, supramolecular complex shortly after synthesis.




Figure 4: Quantitation of the DRbeta complex size in sucrose gradients after chemical cross-linking or as a function of time. The amount of DRbeta in each sucrose gradient fraction of pulse-chased DRbeta immunoprecipitates of 9.22.3 in Fig. 3, A-D, was analyzed by PhosphorImaging. The DRbeta in each fraction is plotted and was calculated as the percent of the total DRbeta in the entire gradient.





The observation that DRbeta chains are found in increasingly aggregated states in DRalpha deficient cells contrasted sharply with the kinetics of DRbeta complex formation in wild type cells. Pulse-chase and sucrose gradient analysis of DRbeta complex formation in wild type 8.1.6 cells revealed that DRbeta was initially found as a 6 S complex (Fig. 5, fractions 9-10). After 2 h of chase, the peak size of DRbeta complexes decreased (Fig. 5, B and C, fractions 12-13). Thus in both wild type and DRalpha-deficient cells newly synthesized DRbeta chains are incorporated into a 5-6 S complex, probably composed of one or more members of the ER stress response family. Thereafter the fate of DRbeta chains in mutant and wild type cells diverges.

The large size (7-12 S) of late forming DRbeta complexes in DRalpha-deficient cells cannot be explained by the contribution of bound detergent because other membrane proteins of comparable size sediment much more slowly in sucrose gradients. Mature MHC class I molecules (M(r) 57,000) for example, sedimented at 5.3 S, just ahead of the albumin marker (Fig. 3E). Moreover, 2 h after synthesis the size of mature DR heterodimers from wild type cells sedimented at 4.5-5 S (Fig. 5B). These data indicate that the size of DRbeta complexes in sucrose gradients must be due to the binding of other proteins to DRbeta chains.

BiP appears to be a major component of the large, late forming DRbeta complexes found in DRalpha-deficient cells. A 78-kDa band which co-migrated with BiP was found in fractions 1-9 (6.5-20 S) after 1 h of chase (Fig. 2B). In the fastest sedimenting fractions, the intensity of the BiP band approached that of DRbeta, in spite of the fact that only a fraction (<10%) of the cellular BiP was labeled during the short pulse (Table 2). Conversely, little or no BiP was coprecipitated in fractions which sedimented at 5-6 S. The lightest fractions (14, 15, 16, 17) had little or no BiP and very little Ii chain (Table 2). The Ii chain was found in both intermediate and large DRbeta complexes, but interestingly, appeared to be relatively absent from the largest DRbeta complexes (Table 2, fractions 1-4).

The Mature Form of DRbeta, p27, Sediments as a 15 S Complex

Sucrose gradient analysis of DRbeta complexes from 9.22.3 indicated that the size of DRbeta-BiP aggregates increased with time. Therefore it was of interest to examine the behavior of p27, the more mature form of DRbeta chains. p27 is often poorly represented in pulse-chase experiments because of its rapid turnover (see below). 9.22.3 cells were therefore labeled for longer periods, a procedure which has consistently labeled p27. Extracts were analyzed by sucrose gradient fractionation and immunoprecipitation. p27 was found exclusively in the fastest sedimenting fractions (Fig. 6, A and B, Table 1). All p27 sedimented at 7.5-18 S, with the peak occurring at 15-16 S (Fig. 6, fraction 3). One striking observation was that the largest p27 fractions appeared to be devoid of Ii. The relative absence of Ii chain from p27 complexes is in agreement with the decreased Ii presence in the larger, late forming p29 complexes (Table 2). Together these observations suggest that Ii may be actively displaced by BiP or other members of the ER stress response family during the late stages of the aggregation process.


Figure 6: The size distribution of p27 in DRalpha- deficient cells. 9.22.3 cells (4 times 10^6 cells) were pulsed for 4 h and analyzed by sucrose gradient fractionation and immunoprecipitation with HB10.A (A). Note that p27 is confined exclusively to the densest fractions. Quantitation of p27 and total DRbeta (p29 + p27) is plotted in (B). p27 was confined exclusively to the densest functions in three independent experiments.



Aggregation of DRbeta Is a Proximal Event to DRbeta Degradation

The data presented above are consistent with the initial incorporation of DRbeta chains into a proteinaceous complex which progressively enters into a more aggregated state. The observation that p27 was found exclusively in the largest DRbeta aggregates prompted us to investigate whether these large DRbeta aggregates were more susceptible to degradation. The turnover of p27 was examined by pulsing 9.22.3 cells with [S]cysteine for 90 min and chasing for various times. The DRbeta was immunoprecipitated, and the p27 and p29 forms of DRbeta were resolved on 13.5% acrylamide gels. p27 was found to be degraded with a half-time of 17.5 min (three determinations), some three times faster than bulk DRbeta chains (Fig. 7, A and B). The p27 turnover is likely to be faster than 17 min because we have not corrected for any conversion of p29 to p27 which occurred during the chase. These results indicate that the largest DRbeta aggregates are more likely to be degraded than smaller aggregates and that the largest DRbeta aggregates may be immediate precursors to DRbeta degradation.


Figure 7: The degradation of p27 is rapid. 9 times 10^6 922.3 cells were labeled for 90 min with 1.5 mCi of [S]cysteine in RPMI medium lacking cysteine. Cells were chased by the addition of RPMI with 1 mML-cysteine, and 1.5 times 10^6 cells were removed at 0, 10, 20, 30, 45, and 60 min of chase. Material immunoprecipitated with HB10.A was electrophoresed on 13.5% acrylamide gels, the gels were dried, and radioactivity was captured by PhosphorImaging (A). The p29 and p27 forms were resolved by the use of the software quantitation program, ImageQuant. Note the relative absence of Ii from cysteine labeled cells. B, linear regression of p27 turnover. The p27 half-life in this experiment was 21.0 min and the half-life of total DRbeta (p27 + P29) was 54.0 min; the correlation coefficients were 0.91 and 0.98, respectively. The p27 half-life was 17.5 ± 3.0 min in three experiments. Note that the longer labeling period necessary to label p27 results in the immediate onset of DRbeta degradation without the 15-20-min lag normally seen. Consequently the half-life of DRbeta in this experiment is shorter (54 min), by some 16-18 min than previously reported(12) .




DISCUSSION

In this report we have demonstrated that DRbeta chains destined for degradation by the pre-Golgi degradative pathway enter into an aggregated complex containing multiple copies of BiP. Our data demonstrate that aggregation of unassembled DRbeta chains occurs prior to their degradation and raises the possibility that complex formation with BiP and aggregation may be the common fate of other membrane-anchored substrates of the pre-Golgi degradative pathway(25) . Aggregation appears to be a proximal event in the degradation of DRbeta chains. This conclusion stems from two observations that correlate the highly aggregated DRbeta state with faster DRbeta turnover. The first observation is that p27, which we have previously shown to be a more mature form of DRbeta, is found exclusively in a highly aggregated state. Furthermore, p27 was found to be degraded about three times faster than the p29 form of DRbeta chains. Taken together these observations suggest that aggregation is progressive but that large DRbeta aggregates are continually being removed by degradation.

It is not known whether formation of large aggregates is a universal step for all substrates of the pre-Golgi degradation pathway. Studies of one soluble, non-membrane anchored substrate, a variant of alpha-antitrypsin, indicated that although formation of small complexes occurred, the complexes did not get progressively larger with time (10) . The equilibrium size of aggregates is likely to reflect the complex dynamics of their formation and degradation. This is likely to vary depending on abundance of the protein, its half-life and such factors as whether it is soluble or membrane-bound. Studies comparing membrane proteins with their anchorless variants have demonstrated differences in the kinetics of both folding, and when observed, aggregate formation(43) .

The formation of DRbeta aggregates reported here is similar in nature to the slow forming aggregates previously described for a mutated herpes gB glycoprotein (25) and the alpha subunit of the acetylcholine receptor (31) . In each case, aggregate formation was relatively slow and BiP was a prominent component of the complex. Like DRbeta, both the unassembled alpha subunit of the acetylcholine receptor and the mutant gB protein are aggregated in the ER and are ultimately degraded. However, the half-lives of the these two proteins were apparently several hours, leaving some doubt as to whether they are degraded by the same pathway as unassembled DRbeta chains.

Because of the rapid turnover of DRbeta chains in DRalpha-deficient cells (T of 70 min) the high molecular weight BiPbulletDRbeta complexes do not accumulate and can only be observed during a relatively narrow kinetic window. However, several properties of the late forming BiPbulletDRbeta complexes allow their detection and the independent examination of their role as intermediates in the DRbeta degradative pathway. The large BiPbulletDRbeta complexes observed at 30-120 min after DRbeta synthesis are seen only in the DRalpha-deficient cells. Second, the stoichiometry of BiPbulletDRbeta in the late forming complexes is high (approximately 2). Finally, p27 is found exclusively in large aggregates (7-16 S) and is degraded much more rapidly than the p29 form of DRbeta. These findings suggest that the late forming complexes represent DRbeta chains destined for degradation.

The kinetics of BiP association with DRbeta provide insights into the way DRbeta monomers may be targeted for degradation. BiP interacts with a given DRbeta monomer not once, but in multiple cycles. Shortly after DRbeta synthesis, BiP interacts transiently with DRbeta monomers and then dissociates (Fig. 2A). At later times, the ratio of the BiP to DRbeta increases so that at steady state it is approximately 2. This suggests that the dissociation rate of BiP from DRbeta has decreased, probably because DRbeta chains, in the absence of DRalpha chains, cannot achieve a stable, native conformation. As a consequence, they increasingly enter into a misfolded state, probably exposing hydrophobic surfaces with which BiP is known to tightly interact(35, 36) . BiP can exist as a homodimer(44) , but it is not known whether BiP is functionally divalent and capable by itself of cross-linking misfolded DRbeta chains. Our data suggest that the binding of multiple BiP molecules results in the incorporation of DRbeta into a supramolecular complex. Thus the signal which targets DRbeta monomers for degradation may be a physical change of state, the cross-linking of the monomer or their aggregation into large, oligomeric complexes with BiP. BiP is the only protein (other than Ii) which we have identified in DRbeta complexes, but other, unidentified proteins of M(r) of 90,000-94,000 and 68,000-72,000 are also present in various amounts in the high molecular weight complexes. The identity of these other proteins is currently under study.

The selective aggregation of misfolded proteins may constitute a means by which misfolded proteins can be concentrated and selectively sorted from other properly folded secreted and membrane proteins. Selective aggregation of misfolded proteins might be a necessary step in segregating proteins destined for degradation and transporting them to either a specialized subcompartment of the ER or possibly for vesicular transport to another pre-Golgi location. It is interesting to note that in pancreatic exocrine cells and other regulated secretory systems, sorting of regulated secreted proteins into dense granules is thought to involve their selective aggregation(45, 46) . Moreover, the delay in degradation which is characteristic of the pre-Golgi degradative system is a distinctive feature of many aggregation or nucleation-dependent biological systems(47) . Thus aggregation is a general biological phenomenon which may play a necessary role in both the recognition and differential sorting of misfolded proteins within the endoplasmic reticulum.

It is surprising that the endoplasmic reticulum should be both a site of protein folding/subunit assembly and of protein degradation. The potential for disruption of subunit assembly by proteolytic fragments would seem to be great if such fragments were allowed to exist in a freely soluble state at the site of assembly. Therefore, the cell must take measures to ensure that either proteolysis takes place at a spatially segregated site or that proteolytic fragments do not become soluble. The ER resident chaperones such as BiP are a class of proteins which have been demonstrated to interact with a range of newly synthesized protein subunits and therefore are the likeliest candidates to function in recognizing misfolded proteins. BiP and other chaperones bind to a newly synthesized protein and prevent it from aggregating until it achieves a folded state(33, 34, 35, 36) . However, the failure of a protein to fold correctly results in its stable association with BiP, and ultimately, its incorporation into large aggregates(19, 25) . Thus BiP and other chaperones are well suited to act at the decision point in a protein's fate, either by temporarily preventing the misfolding of newly synthesized proteins or by facilitating the aggregation of misfolded proteins.


FOOTNOTES

*
The research was supported in part by Royalty Research Grant (to T. C.) and by National Institutes of Health Grant AI16689 (to D. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The abbreviations used are: ER, endoplasmic reticulum; MHC, major histocompatibility complex.

(^2)
T. Cotner, unpublished results.


ACKNOWLEDGEMENTS

We thank Benjamin Arp for helpful discussions and Dan Hill for preparation of the manuscript.


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