©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Transient Aggregation of Major Histocompatibility Complex Class II Chains during Assembly in Normal Spleen Cells (*)

Michael S. Marks (1)(§), Ronald N. Germain (2), Juan S. Bonifacino (1)(¶)

From the (1) Cell Biology and Metabolism Branch, NICHD and the (2) Laboratory of Immunology, NIAID, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Many cell surface proteins exist as complexes of multiple subunits.It is well established that most such complexes are assembled within the endoplasmic reticulum (ER). However, the mechanistic details of the assembly process are largely unknown. We show here that and subunits of major histocompatibility complex class II antigens in spleen cells of normal mice pass through a transiently aggregated phase in the ER prior to assembly with the invariant chain (Ii). Aggregates form immediately after synthesis and disappear concomitantly with assembly of mature Ii complexes. In spleen cells lacking Ii, aggregates fail to be efficiently dissociated over time, implicating subunit assembly as a requirement for disaggregation. Two ER chaperones, BiP and calnexin, bind to newly synthesized class II MHC chains but do not contribute appreciably to the large size of the aggregates. Our observations suggest that some subunits of multisubunit complexes pass through a transient, dynamic high molecular weight aggregate phase during the physiological process of assembly. The results further suggest a novel role for Ii in promoting stable dissociation of preformed aggregates containing and subunits rather than in preventing their formation.


INTRODUCTION

Transmembrane proteins destined for distal compartments of the secretory pathway or the cell surface must be properly folded in order to be competent for exit from the endoplasmic reticulum (ER)()(1) . For most multisubunit protein complexes, the process of achieving transport competence requires the assembly of the various constituents by a mechanism that probably involves ER-resident chaperones (1, 2) . Unassembled subunits or incomplete complexes are generally retained within the ER and, in some cases, degraded. The quality control mechanisms that ensure that unassembled subunits are recognized as such and retained within the ER prior to assembly are still poorly defined.

We have been studying the retention of unassembled subunits in the ER using major histocompatibility complex (MHC) class II molecules as a model system. MHC class II molecules consist of a noncovalent complex of two transmembrane glycoproteins, and , that function in presenting to CD4 T cells antigenic peptides derived from proteins encountered largely within the endocytic pathway (3) . In the ER, newly synthesized and chains associate with preformed homotrimers of a third transmembrane glycoprotein called the invariant chain (Ii) (4) to form a nine-subunit (Ii) complex (5) . Only the complete nonameric complex is fully transport competent; complexes that are incomplete are retained within the ER (6) . Recent studies have demonstrated that an ER chaperone, calnexin (p88; IP90), remains bound to incomplete Ii complexes (7, 8) ; based on other systems (9, 10) , calnexin is likely to play a role in retention and/or further assembly of such complexes. In addition to partial Ii complexes, and chains that are expressed in the absence of Ii, either by virtue of Ii-targeted gene disruption in transgenic mice or by transfection of and chain cDNAs without Ii into fibroblasts, exit the ER inefficiently at best (11, 12, 13, 14, 15, 16, 17) .

We have previously shown that, like many misfolded or aberrantly processed polypeptides, and chains expressed in the absence of Ii are incorporated into large, heterogeneous aggregates that are associated with the hsp70-like chaperone, BiP (18) . Both the aggregation and association with BiP, a resident ER protein, may have contributed to retention of the incompletely assembled and chains in the ER. These studies thus demonstrated how, in the absence of one subunit, the remaining subunits of a complex could accumulate in a transport-incompetent form. Several questions, however, remained unanswered. First, it was unclear whether aggregation would only occur under the unusual circumstances in which and subunits were inappropriately expressed in the absence of Ii or whether it could also occur to some extent when Ii was present in normal class II-expressing cells. Second, it was not established whether aggregates represented irreversible, aberrant products of alternative folding side reactions or whether aggregated subunits remained competent for assembly into normal complexes. Finally, it was unclear whether aggregation was related in any way to the association with the ER chaperone calnexin.

In this study, we show that aggregates of newly synthesized class II and chains exist transiently in normal class II-expressing spleen cells and are therefore not limited to cells in which Ii is absent or and chains are overexpressed. Aggregates formed early after synthesis and disappeared over time without concomitant degradation of chains. This finding implicates aggregation as a dynamic intermediate phase in the assembly of mature Ii complexes. In addition, Ii is shown to be required for rescue of and chains from aggregates, indicating that Ii does not necessarily prevent aggregation but rather promotes the stable dissociation of previously formed aggregates.


EXPERIMENTAL PROCEDURES

Mice and Antibodies

Normal C57BL/6 mice (H-2; purchased from Charles River Laboratories, Raleigh, NC) and Ii-deficient mice bred to the C57BL/6 background (maintained at BioQual, Rockville, MD) were maintained as described previously (11) . Cell suspensions of freshly isolated spleens from 2-6-month-old mice were prepared by disruption in DMEM containing 10% fetal bovine serum and used immediately for metabolic labeling experiments. Rabbit antisera specific for the cytoplasmic tails of A and A chains (19) and for murine calnexin (8) , and monoclonal antibodies specific for the Ii cytoplasmic tail (In-1 (20) ) and lumenal domain (P4H5 (21) ), for H-2K (Y3 (22) and AF6-88.5.3 (23) ), for CD45 (M1/89.18.7.HK (24) ), and for BiP (25) have been described. The calnexin-specific antiserum and the anti-BiP monoclonal antibody were generous gifts of Drs. D. McKean (Mayo Clinic) and D. Bole (University of Michigan), respectively.

Metabolic Labeling

Spleen cell suspensions were washed once with DMEM, 10% fetal bovine serum and then incubated in 60-mm dishes for 60-90 min at 37 °C in 10 ml/4 spleen equivalents of leucine-free DMEM containing 5% dialyzed fetal bovine serum and antibiotics. Cells were harvested and then incubated with periodic agitation at 37 °C for various times in the same medium containing 2-3 mCi/ml [3,4,5-H]leucine (DuPont NEN). For pulse-chase experiments, a 10-fold (v/v) excess of ice-cold DMEM containing 10% fetal bovine serum and a 15-fold (w/v) excess of unlabeled leucine was added to samples, cells were harvested and divided into aliquots, and chase was continued by the addition of warm DMEM, 10% fetal bovine serum with excess leucine. In some experiments, methionine/cysteine-free DMEM, TranS-label (ICN Radiochemicals, Irvine, CA), and excess methionine and cysteine were substituted for the respective leucine and leucine-free reagents. For 24-h labeling, spleen cells were cultured in methionine-free DMEM supplemented with 10% fetal bovine serum, antibiotics, and 50 µg/ml lipopolysaccharide containing 0.6 mg/liter unlabeled methionine and 62 µCi/ml [S]methionine for 24 h. At the end of the labeling/chase periods, cells were harvested and resuspended in phosphate-buffered saline containing 20 mM N-ethylmaleimide and a mixture of protease inhibitors (see below) for 15-20 min on ice (18) . Cells were harvested and either extracted immediately or frozen on dry ice.

Cell Lysis, Preclearing, and Preprecipitation with Anti-calnexin Antibody

Detergent lysates were prepared as described previously (18) using lysis buffer (50 mM Tris, 300 mM NaCl, 1% (w/v) Triton X-100) containing 20 mM iodoacetamide and a mixture of protease inhibitors (0.25 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 0.1 mM N- p-tosyl-L-lysine chloromethyl ketone, 0.1 mM N-tosyl-L-phenylalanine chloromethyl ketone, 5 µg/ml pepstatin A, 5 µg/ml E-64, 10 µg/ml leupeptin, 33 µg/ml aprotinin). In some experiments, 5 mM ATP and 5 mM MgCl were included in the lysis buffer (26) , as indicated. Lysates were clarified by high speed centrifugation and precleared twice with rabbit anti-mouse immunoglobulin coupled to protein A-Sepharose, once with normal rabbit serum coupled to Pansorbin, and once with protein A-Sepharose alone prior to either specific immunoprecipitation or sedimentation on sucrose gradients.

For analysis of calnexin-bound material, cells were lysed in digitonin lysis buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 1% (w/v) digitonin, 0.02% NaN, 20 mM iodoacetamide with protease inhibitors). After preclearing, lysates were subjected to two rounds of immunoprecipitation with antiserum to calnexin bound to protein A-Sepharose. Immunoprecipitates were washed 3 times with digitonin wash buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 0.1% (w/v) digitonin) and then resuspended in lysis buffer containing 1% (w/v) Triton X-100 and incubated at 25 °C for 30 min, as described previously (7) . Unbound lysate fractions were similarly incubated with 1% Triton X-100. Following a final round of pre-clearing with protein A-Sepharose, eluate and unbound material were fractionated and analyzed by sedimentation. Only the results of fractionation of the bound and eluted material is shown; results with the unbound fraction were not significantly different from those obtained using lysates that had not been incubated with the antiserum to calnexin, perhaps due to inefficient immunodepletion.

Sedimentation Velocity Analysis

Precleared lysates (or calnexin-eluted material) (0.5 ml) were applied to the top of 12-ml linear 5-20% (w/v) sucrose gradients, subjected to centrifugation for 16 h at 4 °C in a SW41 rotor at 39,000 rpm (188,000 g), and fractionated as described previously (18) . Each of the 15 fractions collected from each gradient was sequentially immunoprecipitated with antisera or monoclonal antibodies prebound to protein A-Sepharose or Gammabind G Sepharose (Pharmacia Biotech Inc.). Generally, anti-A and anti-A immunoprecipitations were performed first, followed by immunoprecipitations with anti-Ii, where appropriate. Peak fractions for the migration of H-2K (60 kDa with -microglobulin) and CD45 (220 kDa) molecules were identified by a final round of immunoprecipitation to provide internal size standards for integral membrane proteins. Estimates of molecular mass were calculated as described previously (27) .

Immunoprecipitation and SDS-PAGE

Sequential immunoprecipitations were performed for 2 h at 4 °C with protein A-Sepharose or Gammabind G Sepharose that had been prebound to antisera or monoclonal antibody supernatants and washed 2 times with wash buffer (50 mM Tris, 300 mM NaCl, 0.1% (w/v) Triton X-100). Immunoprecipitates were washed 4 with wash buffer and once with phosphate-buffered saline prior to elution with SDS sample buffer at 95 °C for 10 min. SDS-PAGE was as described previously (28) using 13% acrylamide gels cross-linked to GelBond with AcrylAide, and fluorography was with 1 M sodium salicylate. Quantitation of bands was done by scanning laser densitometry of autoradiographs or by PhosphorImager analysis on a Molecular Dynamics system. Calculations of percent A and A chains present in aggregate fractions was based on densitometry from three independent experiments.


RESULTS

Prior analyses of spleen cells from mice deficient in Ii expression (Ii-/- mice) showed that a large fraction of newly synthesized MHC class II A and A chains remained unassociated (11) and were incorporated into high molecular weight aggregates (HMWA) as determined by sedimentation on sucrose density gradients (see Ref. 18 and below). In contrast, most A and A chains synthesized in the presence of Ii in spleen cells from wild-type C57BL/6 (WT) mice were assembled into discrete complexes that migrated as a 200-300 kDa species (18) , as expected for a nonameric Ii complex of 30-35-kDa subunits (5) . Interestingly, prolonged exposure of autoradiograms of size-fractionated class II chains from metabolically labeled WT spleen cells revealed a small amount of unassembled A (Fig. 1 a, bracket) or A (Fig. 1 b, bracket) chain in fractions that corresponded to M of 300,000-1,000,000. These high molecular weight forms of A and A chains, although present in much smaller amounts, were reminiscent of the HMWA observed in spleen cells that do not express Ii.


Figure 1: A portion of newly synthesized A and A chains are found in high molecular weight aggregates in normal spleen cells. Spleen cells from WT C57BL/6 mice were metabolically labeled for 30 min with [H]leucine, and detergent lysates were fractionated by sedimentation in sucrose density gradients. Individual gradient fractions were immunoprecipitated with antisera to the cytoplasmic tails of A ( a) or A ( b) and analyzed by SDS-PAGE and fluorography. As internal size standards for integral membrane proteins, the MHC class I antigen H-2K-microglobulin complex (60 kDa) and the CD45 antigen (220 kDa) were also immunoprecipitated from gradient fractions. Gradient fraction numbers, from top to bottom, are indicated at the bottom. The peak fractions for the H-2K (fractions 2 and 3) and CD45 (fraction 5) markers are indicated at the top. The bracket indicates fractions defined as HMWA. The migration of protein molecular weight standards (10 M) by SDS-PAGE is indicated on the left, and the positions of , , and Ii chains and a degradation product of Ii ( Ii`) are indicated on the right. The Ii complex migrates as a single peak in fractions 4- 6 in this gradient. Bands present in fraction 1 (and most likely some in fractions 2 and 3) represent material that did not enter or partially entered the gradient. Fig. 1 a is an overexposure of an autoradiogram similar to one shown in Fig. 8 of Ref. 18.



The HMWA observed in WT cells could have represented either the product of a distinct dead-end pathway for a minor amount of unassembled and/or misfolded A or A chains, or alternatively, an intermediate in the normal assembly process for MHC class II molecules. To distinguish between these possibilities, WT spleen cells were pulse-labeled with [H]leucine for only 5 min and chased for various times prior to detergent lysis and analysis by sedimentation and immunoprecipitation. As shown in Fig. 2, a large fraction (42.1 ± 0.7%) of A chains ( left, bracket) and a smaller but significant fraction (10.6 ± 3.3%) of A chains ( right, bracket) were present in HMWA after the 5-min pulse. In contrast, less than 1% of Ii directly immunoprecipitated from the same gradient was observed in fractions corresponding to HMWA (Fig. 3); rather, Ii was found predominantly in fractions 4 and 5, corresponding in size to Ii trimers and partial Ii complexes. Similarly, mature H-2 K and CD45, used as molecular size markers in our experiments, were never observed in HMWA (data not shown). These observations suggested that aggregation was an intrinsic characteristic of unassembled class II chains.


Figure 2: Pulse-chase analysis of aggregation and disaggregation. Spleen cells from WT mice were pulse-labeled for 5 min with [H]leucine and then chased with excess unlabeled leucine for 10, 30, or 180 min at 37 °C. Lysates were fractionated by sucrose density gradient centrifugation, individual fractions were immunoprecipitated sequentially with antibodies to A ( left) and A ( right), and immunoprecipitates were separated by SDS-PAGE. Only the portion of the gels corresponding to the relevant chains are shown; no specific bands were visible elsewhere in the gels. The bracket indicates fractions defined as HMWA. The positions of the , , and Ii chains and of a degradation product of Ii ( Ii`) are indicated. The band above chain in fractions containing Ii corresponds to Iip41, the product of an alternatively spliced form of Ii (51). The Ii observed in fraction 6 at the 180-min chase most likely represents excess Ii that was synthesized in the pulse and associated with A and A chains synthesized during the chase.




Figure 3: Pulse-chase analysis of the aggregation state of Ii. Ii was isolated by immunoprecipitation from the same gradient fractions described in Fig. 2 after sequential immunoprecipitation of A and A. The positions of the Ii chain, a degradation product of Ii ( Ii`) and Iip41 are indicated. Notice the absence of aggregated species of Ii in fractions that contain aggregated A and A chains ( bracket).



With increasing chase times, the fraction of A and A chains in HMWA decreased, such that by 30 min no detectable A chains and a small fraction (5%) of A chains remained aggregated (Fig. 2). Concomitantly, there was an increase in the level of chains detected in fractions corresponding to the mature Ii complex (fractions 5-7). Quantitation of direct immunoprecipitates from lysates in multiple experiments indicated no decrease in the amount of labeled or chains from the pulse to the 30-min chase period (data not shown), suggesting that the chains were not significantly degraded over the course of the experiment. These data were consistent with a large fraction of the A chains, and perhaps A chains, in the Ii complexes originating from precursor HMWAs. Similar results were obtained upon pulse-chase and sedimentation analysis of HLA-DR and chains from a HLA-DR5-expressing human B-lymphoblastoid cell line (data not shown), suggesting that transient aggregation is a general phenomenon during assembly of class II chains. By 180 min of chase, no A or A-containing aggregates were observed, and Ii had dissociated from most of the Ii complexes, leaving predominantly dimers that migrate as a discrete 60 kDa species (Fig. 2). This is consistent with proteolysis and removal of Ii upon transport into a late endosomal compartment, releasing Ii-free peptide-bound dimers for transport to the cell surface (29, 30, 31, 32) .

A similar pulse/chase analysis of A and A chains from Ii-/- spleen cells (Fig. 4) showed that while aggregates also formed immediately, disaggregation was much less efficient than in WT cells. Even by 180 min of chase, the vast majority of A and A chains remained in HMWA, rather than in fractions expected for mature AA dimers (fraction 3, co-migrating with H-2K). These data suggest that in the absence of Ii, newly synthesized and chains are incorporated into HMWA as they do in the presence of Ii, but become arrested at the aggregate phase and fail to be efficiently disaggregated.


Figure 4: Aggregates containing A and A fail to be dissociated in the absence of Ii. Spleen cells from Ii-/- mice were pulse-labeled for 5 min with TranS-label and then chased with excess unlabeled methionine and cysteine for 30 or 180 min at 37 °C. Lysates were fractionated by sucrose density gradient centrifugation; individual fractions were immunoprecipitated sequentially with antibodies to A, A, H-2K and CD45; and immunoprecipitates were separated by SDS-PAGE. The positions of and chains and of a degradation product of (`) chain are indicated. Identification of ` as a proteolytic fragment of A, possibly generated subsequent to solubilization, was by reprecipitation with anti-A antibodies following elution with SDS and boiling (18).



The higher amount of chain relative to chain present in aggregates may be a consequence of a larger pool of free chains than that of free chains. This difference in pool sizes could be due to the fact that spleen cells synthesize A chains in excess of A chains (data not shown; the same phenomenon was noted for HLA-DR5 and chains in a human B lymphoblastoid cell line). In pulse-chase experiments, this uneven rate of synthesis results in the assembly of labeled chains with chains synthesized prior to the pulse and of labeled chains with chains synthesized during the chase (results not shown). It should also be noted that co-precipitation of aggregated chains with chain was observed in some experiments ( e.g. see Fig. 4) but not others ( e.g. see Fig. 2 ). Co-aggregation of with was reminiscent of results in transfected fibroblasts in which the chains were overexpressed (18) and may reflect heterogeneity of aggregate content (see ``Discussion'').

The large size of the HMWA observed transiently in WT cells and more stably in Ii-/- cells could be due either to an agglomeration of multiple newly synthesized chains or to an association of a few chains with major ER chaperones that were not efficiently labeled during the short metabolic pulses used above. In order to determine whether other major proteins were associated with A or A chains under the conditions of our experiments, spleen cells from WT mice were metabolically labeled for 24 h prior to immunoprecipitation. Only a 78-kDa protein was specifically co-precipitated in significant amounts with A chain (Fig. 5, lane4). A similar band was observed in anti-A chain immunoprecipitates and in anti-A chain immunoprecipitates from Ii-/- spleen cell lysates (data not shown). This band corresponded to BiP/GRP78 since it co-migrated with a protein precipitated by an anti-BiP antibody ( lane3) and was absent in anti-A immunoprecipitates from lysates treated with 5 mM ATP ( lane6), consistent with the well characterized ATP-dependent dissociation of BiP from substrate proteins (2, 33) . Since no other bands were observed, it is unlikely that the large size of the HMWA is due to a contribution from some other major bound protein.


Figure 5: Immunoprecipitation of class II chains from WT spleen cells labeled for 24 h. Spleen cells from WT mice were labeled for 24 h with TranS-label. Detergent lysates were prepared with or without 5 mM ATP ( ± ATP), and immunoprecipitated sequentially with nonspecific rabbit serum ( NS) and then either a monoclonal antibody to BiP, an antiserum to calnexin ( Cnx), or an antiserum to A, as indicated. Because of the limited availability of anti-BiP and anti-calnexin antibodies, immunoprecipitation of BiP and calnexin was not quantitative. The migration of protein molecular weight standards (10 M) by SDS-PAGE is indicated at the left, and the positions of , , Ii, BiP and calnexin are indicated at the right. Similar results were obtained when cells were labeled for 16 h with [H]leucine (not shown). The lower band co-precipitated with A is most likely the Ii` degradation product of Ii.



In order to assess a possible contribution of bound BiP to the size of the HMWA, we analyzed the sedimentation characteristics of newly synthesized A and A chains before and after treatment of lysates with 5 mM ATP. As shown in Fig. 6, the sedimentation of A and A chains from pulse-labeled WT spleen cells was virtually unchanged by ATP treatment. Identical results were obtained with A and A chains from Ii-/- spleen cells (data not shown). These data suggested that BiP did not substantially contribute to the size of the - and -containing HMWA and was therefore most likely present in substoichiometric amounts.


Figure 6: Dissociation of BiP does not affect migration of HMWA. Spleen cells from WT mice were metabolically labeled with TranS-label for 5 min and extracted in lysis buffer that did or did not contain 5 mM ATP. Lysates were fractionated on sucrose density gradients, and fractions were sequentially immunoprecipitated with antibodies to A ( left), A ( right), H-2K, and CD45; separated by SDS-PAGE; and analyzed by fluorography. Only the portion of the gels containing relevant bands are shown. The peak fractions of migration for CD45 and H-2K are indicated at the top; fraction numbers are indicated at the bottom; the positions of the , , and Ii chains and a proteolytic product of Ii ( Ii`) are indicated to the left and right.



Two recent publications have described the association of another ER-resident chaperone, calnexin (Ip90; p88), with assembling MHC class II chains (7, 8) . We did not detect a band co-migrating with calnexin in immunoprecipitates from long term labeled cells (Fig. 5), suggesting that calnexin was not responsible for the large size of the HMWA. The inability to detect bound calnexin in our system was not surprising, since the detergent and salt conditions used during cell solubilization in our experiments would be expected to have largely dissociated calnexin from class II subunits (7) .

Even though calnexin was not a component of the aggregates observed under our solubilization conditions, we were interested in knowing the characteristics of A and A chains that bound to calnexin under more favorable conditions. To this end, we took advantage of the reported stability of the calnexin/class II interaction in digitonin at 4 °C and its instability in Triton X-100 at room temperature (7) . Pulse-labeled proteins from WT spleen cells were immunoprecipitated from digitonin-solubilized cells with an anti-calnexin antiserum. Calnexin-associated proteins were then eluted with Triton X-100 at room temperature, fractionated on sucrose gradients, and reimmunoprecipitated with antibodies to A or A. As shown in Fig. 7, the A and A chains that had been bound to and eluted from calnexin were enriched in species that sedimented even more slowly than the mature Ii complex, as would be expected for or chain monomers or small oligomers. These species may include partially assembled Ii complexes as previously observed (7) , particularly those containing radiolabeled A chain associated with unlabeled pre-existing A and Ii chains, both of which are synthesized in excess in normal spleen cells. These data suggest that calnexin interacts primarily with a pool of monomeric class II chains or incomplete complexes. Due to the low sensitivity of the assay, however, we cannot exclude that calnexin binds to the aggregates to some extent.


Figure 7: Calnexin binds primarily to low molecular weight class II species. Spleen cells from WT mice were metabolically labeled for 20 min with [H]leucine and then lysed in buffer containing 1% (w/v) digitonin as described previously (7). Following pre-clearing, lysates were immunoprecipitated with an antiserum to calnexin, and calnexin-bound polypeptides were eluted with 1% Triton X-100 at 25 °C (7). Eluted material was fractionated on sucrose density gradients. Fractions were sequentially immunoprecipitated with antibodies to A, A, H-2K, and CD45, and immunoprecipitates were analyzed by SDS-PAGE and fluorography. Relevant bands and peak fractions are indicated.




DISCUSSION

Our observations suggest that MHC class II (and perhaps ) chains pass through a transient physiological intermediate phase, prior to assembly, in which they are present in high molecular weight aggregates. These transient aggregates are observed in normal spleen cells expressing typically low levels of the class II polypeptides and thus are not a result of overexpression or a property of transformed cells. The fact that no degradation of chains is observed over the time course in which aggregates are dissembled favors the argument that aggregates are a preassembly intermediate rather than a predegradation intermediate. The eventual release of class II chains over time demonstrates that aggregates can be dynamic complexes, the assembly and disassembly of which must be regulated by resident ER proteins.

The HMWA that we observed are most likely composed predominantly of unassembled or partially folded class II or chains, bound to substoichiometric quantities of BiP and perhaps other ER chaperones (15, 17) . Aggregation is likely to occur in vivo, based on our earlier observations with transfected COS cells. In the COS cell system, A and A chains were found to aggregate together, but only when co-expressed in the same cells; no association of aggregated A and A chains was observed when cells that expressed each chain individually were mixed prior to cell lysis. In either case, the transient aggregation observed in our experiments minimally represents a transient tendency to aggregate, most likely due to an immature folding state. It is not clear whether the aggregates are products of homotypic associations between like chains or represent an agglomeration of multiple newly synthesized polypeptides, as would be consistent with the previously observed co-aggregation of two misfolded proteins in vivo(34) or of unassembled subunits in vitro(35) . Indeed, the aggregates may contain, besides A and A chains, complex mixtures of additional polypeptides such as other folding and/or assembly intermediates; the heterogeneity of these polypeptides might render them undetectable by SDS-PAGE. This hypothesis is consistent with the ability to co-precipitate aggregated and chains in some experiments, particularly those in which their level of expression may have been elevated ( e.g. co-precipitation of excess aggregated DR chain with DR chain was observed consistently in an Epstein-Barr virus-transformed human B cell line in which class II chains are relatively highly expressed).

Various chaperones have been shown to bind to folding and assembly intermediates in the ER. Our data suggest that two of these chaperones, BiP and calnexin, may have distinct and perhaps sequential functions, as has been suggested by recent data analyzing the folding and assembly of the vesicular stomatitis virus G protein (36) . BiP was previously shown to bind to aggregated class II species in the ER of transfected COS cells (18) . We were unable to detect BiP in our gradients of pulse-labeled spleen cells, but this was likely due to slower synthetic and turnover rates for BiP in spleen cells relative to those in COS cells in which overexpression of ER-retained class II chains may have induced higher BiP expression. BiP was observed to co-immunoprecipitate with class II chains in spleen cells after 24 h of metabolic labeling and likely binds to transient aggregates. Supporting this, BiP was co-precipitated in higher amounts relative to the A chain in spleen cells from Ii-/- mice than from normal mice (data not shown), in which aggregates would be expected to constitute a higher proportion of the steady state level of class II chains. Based on these observations and our previous observations in COS cells (18) , BiP may bind to aggregated species and function either in blocking exposed patches on the surface of the aggregates or in promoting their dissociation (2, 37) .

Calnexin, another ER chaperone, has been previously shown to bind to class II assembly intermediates (7) . Our data confirm and extend these results, suggesting that calnexin binds primarily to more mature intermediates, such as monomers of and chains, in addition to small oligomers that may include partially assembled Ii complexes as described previously (7) . This finding is consistent with observations for other calnexin-bound proteins (38, 39, 40) . We speculate that calnexin binds to free class II chains that have dissociated from aggregates or assists in dissociation from aggregates, playing a role in folding or stabilizing the chains in a conformation that is permissive for assembly with cognate chains (41) .

Invariant chain has been suggested to play several roles in the function of MHC class II molecules, including enhancing assembly and ER egress (11, 12, 13, 14, 15, 16, 17, 42) , blocking peptide binding (43, 44) , and transporting dimers to a late endosomal compartment (45, 46, 47) . The fact that aggregation of and chains is transient in WT cells and more stable in Ii-/- cells suggests that Ii enhances assembly by facilitating the stable release of and chains from aggregates rather than by preventing aggregation from occurring. This function might involve promoting proper folding (15) or sequestering properly folded monomers of and subunits. In either case, Ii would relieve aggregation that results from an incompletely or incorrectly folded structure of newly synthesized chains. Alternatively, it is possible that aggregation of and chains occurs by interaction of partial peptide-binding domains of individual subunits (48) with exposed peptide-like regions of other proteins within the ER; the ability of Ii to inhibit peptide binding by class II molecules could thus sequester dissociated chains away from the aggregates.

In conclusion, our observations demonstrate that aggregation of newly synthesized subunits is a dynamic early event in the physiological assembly process for a heteromeric protein complex, and not merely a dead-end pathway for stably unassembled subunits. This phenomenon may be related to that reported for the folding of several highly expressed proteins, such as thyroglobulin (49) and vesicular stomatitis virus G protein (50) , which also proceeds via aggregated, BiP-associated intermediates. Two unique aspects of the system examined in this study are that 1) the class II chains are expressed at much lower levels than thyroglobulin or viral proteins, showing that aggregation is not simply a result of high expression, and 2) dissociation of the aggregated chains cannot be completed unless all of the constituent subunits are available in the ER. The different characteristics of proteins shown to undergo transient aggregation in the ER suggests that this process might represent a common mechanistic step in the folding and assembly of many newly synthesized proteins.


FOOTNOTES

*
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.

§
Supported in part by a National Research Service Award.

To whom correspondence should be addressed: Cell Biology and Metabolism Branch, NICHD, Bldg. 18T, Rm. 101, NIH, Bethesda, MD 20892. Tel.: 301-496-6368; Fax: 301-402-0078.

The abbreviations used are: ER, endoplasmic reticulum; MHC, major histocompatibility complex; Ii, invariant chain; DMEM, Dulbecco's modified Eagle's medium; PAGE, polyacrylamide gel electrophoresis; HMWA, high molecular weight aggregates; WT, wild-type C57BL/6 mice.


ACKNOWLEDGEMENTS

We thank Drs. E. Bikoff and E. Robertson and C. Huang for generating and generously providing Ii-/- mice; Drs. D. Bole, M. Brenner, D. McKean, K. Schreiber, P. Romagnoli, E. Long, and P. Roche for generous gifts of antibodies; Dr. P. Cresswell for communication of results prior to publication; and Drs. E. Long, P. Roche, P. Cresswell, E. Bikoff, R. Klausner, and members of the Bonifacino lab for critical review of the manuscript.


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