From the
Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel,
¶ Department of Biological Sciences, Stanford University, Stanford, California 94305
Received for publication, August 26, 2002
, and in revised form, February 25, 2003.
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ABSTRACT |
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INTRODUCTION |
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Quality control refers to the process by which misfolded proteins or unassembled subunits of oligomeric complexes are recognized and prevented from deployment to distal compartments of the secretory pathway (6). These aberrant proteins are dislocated across the ER membrane to be ultimately degraded by the cytoplasmic proteasome via a ubiquitin-dependent process known as the ER-associated degradation (ERAD) (7, 8, 9). Although ERAD is widely viewed as a system for eliminating aberrant proteins, this mechanism is also responsible for regulated degradation of correctly folded ER proteins in response to metabolic cues (10, 11, 12). In the current study we investigate the possibility that selective ERAD is also crucial for regulating the IgM expression during B cell differentiation.
The degradation of most mammalian ERAD substrates is not affected by brefeldin A (BFA) (13, 14). Conversely, as indicated above, the degradation of sIgM depends on vesicular transport, as demonstrated by the marked stabilization of sIgM in BFA-treated (2, 4) or permeabilized (3) 38C B cells. It could have indicated that µ heavy chains are not recognized as an ERAD substrate in B cells. This possibility is ruled out by the vesicular transport-independent degradation of both µs and µm, provided that these heavy chains are either unassembled because of light chain deficiency (2) or disassembled from light chains upon thiol treatment (3). Clearly, the degradation of these free µ heavy chains is sensitive neither to BFA nor to cell permeabilization. Nonetheless, upon association with light chain, the µs is diverted to a distinct degradation process, which depends on vesicular transport.
Because expression of conventional light chains is the hallmark of differentiation of pre-B to B cells, the striking effect of the light chain on the intracellular fate of its partner, the µ heavy chain, prompted us to investigate the nature of this developmental change in the mode of µ degradation. Such a developmental switch may be one of the many events that comprise differentiation. Alternatively, it may reflect an exclusive impact of the light chain in the course of pre-B to B cell differentiation. To distinguish between these possibilities, we made use of the 70Z/3 pre-B cell line. When stimulated with lipopolysaccharides (LPS), theses cells can differentiate in culture from a light chain-negative pre-B cell into a
-expressing B cell (15). To bring about a light chain expression without causing differentiation, we expressed
light chain ectopically. We show that both conventional light chains assemble with µ heavy chains and divert these assembled µ from a transport-independent degradation process to a vesicular transport-dependent one. Nonetheless, our data reveal that both routes converge at the ubiquitin-proteasome degradation pathway.
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EXPERIMENTAL PROCEDURES |
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Ectopic Expression in 70Z/3 Cells of Light Chain Using a Vaccinia Virus/T7 RNA Polymerase Hybrid SystemThe vaccinia virus/T7 RNA polymerase overexpression hybrid system (16) was used.
I cDNA was generated by reverse transcribing RNA from COS-7 cells transiently transfected with pJDE
I genomic
I vector (kindly provided by Y. Argon), followed by PCR using PWO DNA polymerase (Roche Applied Science) and these forward (5'-CCATGCCATGGCCTGGATTTCACTTATAC-3') and reverse (5'-TCTCCCCCGGGCTAGGAACAGTCAGCACGG-3') primers. The
I cDNA was cloned between the NcoI and SmaI sites of pTM1 (17), and its authenticity was verified by DNA sequencing.
Preparation of I-encoding vaccinia virus was according to Current Protocols in Molecular Biology (18). Confluent HuTK-143B cells, maintained in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum, 50 units/ml penicillin, and 50 µg/ml streptomycin, were infected with WR wild-type vaccinia virus and 4 h later were transfected with the pTM1-
I. Thirty minutes later, the cells were covered with fresh medium that was also supplemented with 5 µg/ml bromodeoxyuridine and 100 µg/ml Neutral Red (Sigma) and in which low melting Agarose (Amresco) was dissolved to 1%. Recombinant bromodeoxyuridine-resistant plaques were obtained and tested by PCR for the presence of
I cDNA with the primers described above. Positive clones were enriched and purified by three additional cycles of infection, plaque selection, and PCR testing. Large scale preparation of
I-encoding vaccinia virus were made in HeLa cells 3 days post-infection, and viral titers were estimated by serial dilution and infection of CV-1 cells, followed by crystal violet staining. Viral stocks were stored at -80 °C.
A 20 multiplicity of infection of I or WR ("mock") viruses along with a 10 multiplicity of infection of T7 RNA polymerase viruses were mixed with an equal volume of trypsin (0.25 mg/ml; Worthington). The mixtures were incubated for 30 min at 37 °C, sonicated in a bath sonicator on ice (three bursts of 10 s each), and then immediately added to 70Z/3 cells (107 cells in 0.5 ml of RPMI supplemented with 2.5% fetal calf serum). After3hof continuous agitation at 37 °C, the cells were diluted 20-fold to 106 cells/ml with the same medium, and the infected cells were assayed 1013 h later.
Biosynthetic Labeling, Cell Permeabilization, and in Vitro IncubationThe cells were pulse-labeled with [35S]methionine and either chased in vivo (1) or permeabilized with 35 µg/ml digitonin (Calbiochem), resuspended in growth medium, and chased in vitro (3). Where indicated, BFA, N-acetyl-leucyl-leucyl-norlecinal (ALLN), carboxybenzyl-leucyl-leucyl-leucinal (MG-132), carboxybenzyl-leucyl-leucyl-leucine vinyl sulfone (Z-L3 VS), or dithiothreitol were added. The ALLN (100 µM; Calbiochem), MG-132 (5 µM; Calbiochem), BFA (5 µg/ml; Epicentre), or dithiothreitol (5 mM) was added to the chase medium, whereas Z-L3 VS (50 µM) was present during the starvation, pulse, and chase.
Immunoprecipitation, Immunoblotting, Lectin Blotting, and Treatment with Endoglycosidase HIgM was immunoprecipitated from lysates obtained from an identical number of intact or permeabilized cells, normalized for equal cell protein and equal 35S incorporated into total cell protein. To ensure immunoprecipitation of the entire IgM, an excess of goat anti-mouse IgM antibodies was used, followed by protein A-Sepharose (Repligen). Immunoprecipitated µ or total cell proteins were resolved by reducing or nonreducing SDS-PAGE and electroblotted onto nitrocellulose, and the blots were probed with the indicated antibodies. When radiolabeled proteins were resolved, the gels were either directly autoradiographed or first electroblotted, and then the blots were autoradiographed and subsequently probed. The latter protocol, which is routinely used and is represented here in Fig. 5B, confirms that at each time point throughout the chase, identical steady-state amounts of µ protein are immunoprecipitated, which include the radiolabeled µ. This indicates that the decrease with time in radiolabeled µ reflects a net loss of intracellular µ. Incubation of immunoprecipitated IgM with endoglycosidase H (New England Biolabs) was as previously described (1). Assembly of µ with either or
was verified by the positive staining of anti-IgM immunoprecipitates resolved by nonreducing SDS-PAGE with both anti-µ and either anti-
or anti-
antibodies. Assembly efficiency of µ with
,
, or both (mixed population) was based on densitometry of the various assembly species detected by anti-µ, anti-
, or anti-
antibodies, respectively. Each assembly species was calculated as a percentage of the sum of all of the assembly species within the same lane detected by a certain antibody. Ubiquitination of µ was verified by probing anti-IgM immunoprecipitates with an anti-ubiquitin antibody. The antibodies used were: horseradish peroxidase (HRP)-conjugated anti-mouse µ (Sigma), rabbit anti-µs (19), mouse anti-BiP (StressGen), and mouse anti-ubiquitin (clone P4D1, BabCO). The appropriate HRP-conjugated secondary antibodies were from Sigma. Galactosylation of immunoprecipitated IgM was detected by lectin blotting with biotin-conjugated Ricinus communis agglutinin, as previously described (5). HRP was visualized by ECL reaction.
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RESULTS |
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The expression of and its assembly with µ altered the intracellular fate of the latter, resulting in considerable maturation and cell surface expression of the membrane µm. This was indicated by the retarded mobility (Fig. 1B, lane 2), the increase from 18 to 42% of µ that acquired resistance to endoglycosidase H (Fig. 1C, compare lanes 1 and 3), the galactosylation of µ in the trans-Golgi detected by lectin blotting only in LPS-stimulated 70Z/3 cells (Fig. 1D, compare lanes 1 and 2), and the increase in surface membrane form of IgM from 14% in unstimulated 70Z/3 cells to 76% in LPS-stimulated 70Z/3 cells, as detected by cytofluorometric analysis (Fig. 1E). This indicated that only in the presence of
, did a substantial fraction of µm traverse the entire secretory pathway to be displayed at the cell surface, where it served as a B cell receptor (20, 21, 22, 23).
The large quantities of BiP that co-precipitated with µ (Fig. 1B, lane 3) suggested that, also in 70Z/3 pre-B cells, the intracellular retention of unassembled µ was mediated to a large extent by the interaction of µ with BiP. Indeed, upon expression and its assembly with µ, the levels of BiP that co-precipitated with µ markedly decreased (Fig. 1B, lane 4), even though the cellular levels of BiP were hardly affected (Fig. 1B, lanes 5 and 6). Inasmuch as
expression was also correlated with the intracellular transport of µm to the cell surface, our results were consistent with the notion that upon differentiation, the expressed
assembled with µ and displaced BiP.
Expression, the Hallmark of Pre-B to B Cell Differentiation, Diverts µ from a Vesicular Transport-independent to a Vesicular Transport-dependent DegradationBecause differentiation of 70Z/3 pre-B cells to
-expressing B cells was manifested by modified assembly patterns, maturation, and surface expression of µm and displacement of BiP (Fig. 1), we anticipated that this developmental passage would be accompanied by a conversion in the degradation mode of the µ heavy chains. When µ stability was followed in pulse-chase experiments, t
of
55 min was measured in unstimulated 70Z/3 cells (Fig. 2, lanes 13 and open circles), reflecting rapid degradation of both µs and µm. The addition of BFA during chase hardly affected µ degradation (t
of
50 min; Fig. 2, lanes 46 and open squares). In the LPS-stimulated cells, µ chains, which were predominantly assembled with
(Fig. 1A), still disappeared as rapidly, with a measured t
of
55 min (Fig. 2, lanes 1012 and closed circles). Because assembled µm is a stable molecule (1), this half-life probably reflected degradation of assembled µs as well as of µs and µm that remained unassembled. That unassembled µs and µm represented a minor proportion of µ was indicated by the nearly arrested degradation of µ in the presence of BFA in these LPS-stimulated cells. This extension of t
to >9 h (Fig. 2, lanes 1315 and closed squares) reflected that the majority of µs was indeed assembled with
and was therefore diverted to the vesicular transport-dependent degradation.
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The change in the mode of µ degradation that accompanied pre-B to B cell differentiation was corroborated by experiments in permeabilized cells in which vesicular transport was severely hampered (3). When pulse-labeled 70Z/3 cells were permeabilized with digitonin and incubated in vitro, the stability of µ heavy chains correlated with the stage of differentiation and the expression of light chain. In unstimulated
-negative cells, µ chains were rapidly degraded during the in vitro incubations (t
= 54 min; Fig. 2, lanes 79 and open triangles), with kinetics very similar to those measured in intact cells. Remarkably, permeabilization of LPS-stimulated
-expressing cells resulted in stabilization of µ during the in vitro incubation, with t
significantly extended to 3.7 h (Fig. 2, lanes 1618 and closed triangles). Taken together, these results demonstrate that upon differentiation into
-expressing B cells,
-assembled µ heavy chains are diverted from a BFA-insensitive transport-independent degradation to a BFA-sensitive vesicular transport-dependent process.
Ectopically Expressed Light Chain Assembles with µ Heavy Chains and Diverts Them to a Vesicular Transport-dependent DegradationThe striking effect of the pre-B to B differentiation on the intracellular fate of µ heavy chains (Fig. 2) was not necessarily the consequence of
expression and its evident assembly with µ but could be simply the result of the differentiation stage, regardless of light chain expression. To test whether the mere contribution of the pre-B to B cell differentiation to the intracellular fate of µ was to provide the light chain, the latter was ectopically expressed in pre-B cells by infection with a light chain-encoding vaccinia virus. It allowed us to achieve light chain expression while circumventing differentiation into B cells. To verify that indeed ectopic light chain expression does not trigger differentiation into
-expressing B cells, we chose to express
rather than
. Moreover, it allowed us to generalize our observations vis à vis the effect of conventional Ig light chains on the intracellular fate of their partner µ heavy chains. Pulse-chase experiments following mock infection revealed a prolonged t
of µ chains from
1 h (Fig. 2) to 2.5 h (Fig. 3A, lanes 14 and open circles), with BFA exerting only a slight effect (t
= 3.5 h; Fig. 3A, lanes 58 and open squares). In 70Z/3 cells infected with
-encoding virus, µ chains disappeared with a somewhat extended t
of 3.7 h (Fig. 3A, lanes 912 and closed circles). However, when BFA was added to these
-expressing 70Z/3 cells, the µ chains were markedly stabilized with half-life extended to >9.5 h (Fig. 3A, lanes 1316 and closed squares).
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This BFA-dependent stabilization of µ correlated with its assembly with (Fig. 3B). In mock infected 70Z/3 cells, expression of the
light chain was not detected (lane 9), and the majority of µ heavy chains were found either free, assembled into µ2 homodimers, or assembled into
5 SLC-containing hemimers (lane 3; see also Fig. 1A, lane 3). When cells were infected with the
-encoding virus, assembly into predominant µ2
2 monomers was detected, because these assembly species stained positive with both anti-µ (lane 4) and anti-
(lane 10) antibodies. Importantly,
ectopic expression did not trigger differentiation into
-expressing cells, based on the negative reactivity with an anti-
antibody (lane 16). The assembly of µ into µ2
2 monomers was significant in
-infected cells (
50% of all µ-containing species within lane 4). On the other hand, when calculated relative to all the
-containing species within the same lane (lane 10), the assembly into µ2
2 reached only 15%. This was not due to inefficient assembly into µ2
2, because
was in excess (lane 10) and also the assembly of µ into µ2
2 monomers in LPS-treated cells did not exceed 50% when calculated relatively to all µ-containing species within the same lane (lane 5). Note that the apparent intense band of µ2
2 (lane 5) reflected the
2-fold increase in µ expression upon LPS stimulation. Interestingly, when 70Z/3 cells were first stimulated by LPS and then infected with
-encoding virus, we could detect inherent differences between the capacity of pre-B and B cells to promote assembly of µ with
into µ2
2 monomers (compare lanes 10 and 12). This
2-fold improvement took place despite the vast amount of
(lane 18) and the apparent preference of µ to assemble with
in B cells. This preference was manifested by the poor assembly of µ and
when
was introduced into the 38C B cells, which constitutively express abundant
and µ (lane 8). Nonetheless, whenever
or
conventional light chains are expressed, a stabilization of µ by BFA is observed (Figs. 2 and 3). We necessarily interpret these findings as the competence of conventional light chains to assemble with the µ heavy chain and divert it from a BFA-insensitive, vesicular transport-independent degradation to a BFA-sensitive, vesicular transport-dependent degradation.
Both Modes of µ Degradation Are ProteasomalThe first indication that the loss of intracellular 35S-labeled µ was the consequence of proteasomal degradation was the prevention of this loss by ALLN, a relatively nonspecific proteasome inhibitor. This applied to the vesicular transport-independent degradation of unassembled (24) or disassembled (3) µ heavy chains, as well as to the vesicular transport-dependent degradation of µs in -expressing 38C B cells (4). To elucidate the role of the proteasome in these two degradative processes, we compared the effect of the proteasome inhibitors ALLN, MG-132 (25), and Z-L3 VS. The latter compound is a more specific irreversible inhibitor that was shown to bind covalently to a critical active site threonine in the proteasomal
subunits (26). The data in Fig. 4 revealed that the transport-independent degradation of unassembled µ in unstimulated 70Z/3 pre-B cells was more (>7-fold) sensitive to ALLN than to Z-L3 VS; the t
of µ was extended from
40 min (Fig. 4A, open triangles) to >20 h (Fig. 4A, closed diamonds) in the presence of ALLN but only to
160 min (Fig. 4A, closed triangles) in the presence of Z-L3 VS. The reverse phenomenon was observed in the
-expressing LPS-stimulated 70Z/3 cells, where the vesicular transport-dependent degradation of µs was >4-fold more sensitive to Z-L3 VS than to ALLN; the t
of µs increased from
40 min (Fig. 4B, open circles)to >10 h in the presence of Z-L3 VS (Fig. 4B, closed circles) but only to
140 min with ALLN (Fig. 4B, closed diamonds). For comparison, we investigated the differential inhibitory effects exerted by pretreatment with Z-L3 VS on the
-expressing 38C cells (Fig. 4C), where the assembly state of µ could be altered by treating cells with thiols (3). Clearly, upon pretreatment with Z-L3 VS, the vesicular transport-independent degradation of disassembled µ was 2.4-fold slower, extending t
from 35 min (Fig. 4C, open triangles) to 85 min (Fig. 4C, closed triangles). Yet Z-L3 VS pretreatment completely blocked the vesicular transport-dependent degradation of
-assembled µs in the untreated 38C cells (t
extended from 80 min (Fig. 4C, open circles) to >10 h (Fig. 4C, closed circles)). These differential effects of proteasome inhibitors further illustrate the differences between the two modes of µ degradation. Nevertheless, these results strongly suggest that both modes of µ degradation are proteasomal.
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Ubiquitination of µ Heavy Chains Requires Vesicular Transport in -expressing B Cells but Not in Pre-B CellsEvidently, the two degradation routes of µ heavy chains, which at their early stages were segregated into vesicular transport-dependent and -independent pathways, merged at the ultimate proteasomal degradation. To determine where along this proteasomal pathway these two routes converged, we chose to monitor the ubiquitination of µ with respect to events that required vesicular transport. Clearly, µ underwent ubiquitination along both degradation routes, as in 38C B cells (Fig. 5A), as well as in untreated or LPS-stimulated 70Z/3 cells (Fig. 5B), we detected µ heavy chains that were immunoprecipitated with anti-IgM antibody and stained positive when probed with an anti-ubiquitin antibody. As expected, ubiquitinated µ was detected in both cell types only when accumulated because of inhibition of proteasomal degradation with ALLN (Fig. 5, A, lane 2, and B, lanes 3 and 7). Conversely, when BFA was added to these proteasome-inhibited cells, this vesicular transport blocker exerted very different effects on the ubiquitination of µ in the different cell types. In the
-expressing 38C B cells, BFA strongly attenuated the ubiquitination of µ (Fig. 5A, lanes 2 and 4), as it did to the degradation of µs (2, 3, 4), without affecting ubiquitin conjugation to cellular proteins in general (Fig. 5A, lanes 58). In contrast, BFA hardly affected the ubiquitination of unassembled µ in the
-negative 70Z/3 pre-B cells (Fig. 5B, top panel, lanes 3 and 4), but again, BFA attenuated the ubiquitination of µ upon LPS-stimulated
-expression in these cells (Fig. 5B, top panel, lanes 7 and 8). As shown in Fig. 5B (compare lanes 3 and 7 with lanes 4 and 8), the attenuating effect of BFA on µ ubiquitination (top panel) did not result from counteracting the proteasomal inhibition by ALLN, as indicated by 35S-labeled µ (middle panel), or steady-state levels of µ (bottom panel), or an effect of BFA on ubiquitination of cellular proteins in general (lanes 916). Similar to the situation in the 38C cells, also in the 70Z/3 cells, the effect of BFA on the ubiquitination of µ paralleled the effect of this transport blocker on the degradation of µ (Fig. 2). To conclude, only in light chain-expressing cells, where µ heavy chains were diverted to the BFA-sensitive degradation route, was the vesicular transports a prerequisite also for the ubiquitination of these heavy chains. This indicates that the vesicular transport-dependent degradation route of light chain-assembled µ converges with the vesicular transport-independent degradation route of unassembled µ subsequent to the vesicular transport but prior to the ubiquitination of µ, eventually funneling both routes into the ubiquitin-proteasome pathway.
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DISCUSSION |
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The onset of expression is only one of multiple changes that 70Z/3 cells undergo upon LPS stimulation, a complex process that culminates in pre-B to B cell differentiation (20, 21, 22, 23, 28). However, the indistinguishable intracellular fate of µ heavy chains in the thiol-treated 38C B cells (3), the
-deficient 38C-derived EH cells (2), or the NS0 cells transfected with µ (24) strongly suggests that
light chain plays a key role in this process. This notion is confirmed and extended to conventional light chains in general by our observations in 70Z/3 cells, where the
light chain is ectopically expressed. Thus, in this context, the differentiation into B cells primarily contributes conventional light chains, which in turn affect the intracellular fate of their partner µ heavy chains. Our results clearly show that assembly of µ with either LPS-induced
or ectopically expressed
is tightly correlated with µ stabilization under conditions that block vesicular transport. Yet we cannot exclude B cell-specific components, other than conventional light chains, that might contribute to intracellular transport, retention, or assembly of µ heavy chains. Actually, it has been shown that transfection of
in pre-B cells failed to fully restore the transport of the µm pre-B cell receptor complex to the cell surface (28). Likewise, we show here that the inherent capacity to promote assembly improves upon differentiation from pre-B to B cells, as judged by the increased assembly of µ with the ectopically expressed
upon LPS stimulation. This improvement occurs despite the onset expression of the endogenous
light chain, which is shown here to preferentially assemble with µ. Most importantly, ectopic light chain expression does not trigger endogenous light chain expression. It argues that conventional light chains, rather than other B cell-specific components, play a key role in diverting the route of µ heavy chains in the course of pre-B to B cell differentiation.
The indistinguishable intracellular fate of µ that is assembled with either or
stands in contrast with the effects exerted by the SLC. Both SLC and
interact with µ through its Cµ1 domain, which, because of its stable interaction with BiP (29, 30, 31, 32, 33), probably directs free µ or µ2 homodimers to the vesicular transport-independent ERAD. Therefore, it is anticipated that either SLC or
would equally counteract Cµ1-mediated retention and displace BiP. Indeed, µs assembled with SLC is secreted from 70Z/3 cells, provided that its additional retention signal in the µs tail piece (µtpCys) is neutralized (34). Moreover, µm associated with SLC, Ig
, and Ig
forms the pre-B cell receptor visualized on the surface of several pre-B cell lines (28, 35, 36, 37), where it plays a critical role in development of B-lymphocyte lineage (22, 23, 34). Yet we could detect only slight maturation of µm in the unstimulated 70Z/3 pre-B cells expressing either endogenous SLC or
ectopically. Similarly, only after enrichment could Brouns et al. (28) detect a minute fraction of lectin-bound transported µm in NALM-6 pre-B cells. Thus, the assembly of µ with either SLC or
may reflect a quantitative difference between the relative amounts of these chains. Indeed, very low levels of µ-SLC hemimers are detected in naive 70Z/3 cells, and µ remains bound to BiP mostly as free µ or µ2 homodimers. Hence, if these minute amounts of µ-SLC complexes could exit the ER, they would hardly affect the mode of µs degradation or the extent of BiP association, and mature µm would be just at the lowest level of detection. Thus, one facet of the pre-B to B cell differentiation may be simply to provide excess
chains. This would improve assembly into µ
hemimers and µ2
2 monomers, depleting the pools of free µ and µ2 homodimers and decreasing the levels of µ-associated BiP. On the other hand, the assembly of µ with either SLC or
may also reflect a qualitative differences between these chains with respect to affinity, bond stability, BiP displacement, and Cµ1 masking. This notion is corroborated by the distinct antigen specificity of the same µ heavy chain when paired with either SLC or conventional light chains (34). Thus, the
light chain could replace SLC because of either its larger quantities or its higher affinity to µ. Indeed, µ
5 hemimers are still detected in stimulated 70Z/3 cells that express limited amounts of
, whereas none are observed in 38C cells that express
in abundance. Finally, the assembly of µ with either SLC or
may also reflect an inherent difference between pre-B and B cells in the capacity to promote assembly, as indicated here by the improved assembly of µ with the ectopically expressed
upon LPS-stimulated differentiation.
The involvement of the proteasome in the degradation of Ig chains has been reported only for unassembled chains such as nonsecreted light chain (38), mutant light chains (39), membrane
2b heavy chain (40), and µ heavy chain (24). In this study we show that degradation of unassembled µ in light chain-negative cells, and also of assembled µ in cells expressing conventional light chains, involves the proteasome as the proteolytic system and ubiquitin as the tagging mechanism. The unassembled or disassembled µ heavy chains resemble other mammalian ERAD substrates, because they are targeted to degradation independently of vesicular transport. On the other hand, upon assembly with either
or
, µ is diverted to a BFA-sensitive, vesicular transport-dependent degradation process. Nonetheless, these two distinct routes to degradation converge at the ubiquitin-proteasome pathway. This is demonstrated by the marked stabilization and accumulation as polyubiquitinated species of both unassembled and assembled µ in light chain-negative and
-expressing cells, respectively, provided the proteasome is blocked.
Our data clearly show that vesicular transport is an obligatory step that is a prerequisite for ubiquitination of µ only in -expressing cells. Although the function of this vesicular transport remains to be elucidated, recent studies in yeast demonstrate that vesicular transport-dependent degradation is neither restricted to µ heavy chain nor to B-lymphocytes. Genetic data show that degradation of luminal ERAD substrates such as CPY* and PrA* requires vesicular trafficking between the ER and Golgi (41, 42, 43). The ubiquitination and elimination of µs by the cytosol-oriented components of the ubiquitin-proteasome pathway indicates that µs must dislocate from the ER lumen back to the cytosol. In
-expressing cells, µs should be transported from the ER by vesicles prior to its dislocation, ubiquitination, and degradation. Hence, µs may dislocate to the cytosol from a distal compartment. If µs is handled like other ERAD substrates and its dislocation is mediated by the Sec61p translocon (44, 45, 46, 47), it may dislocate through the Sec61p located in ER-Golgi intermediate compartment (48). Involvement of compartments distal to the ER in quality control is suggested by immunolocalization to the Golgi apparatus and pre-Golgi intermediates of deglucosylating endomannosidase, UDP-glucose:glycoprotein glucosyltransferase, glucosidase II, and calreticulin (49, 50), key components of the protein quality control machinery.
Alternatively, dislocation to the cytosol may take place from the ER, provided that the degradation substrate is retrograde-transported to this compartment. Again, this mechanism is supported by genetic data from yeast that demonstrate anterograde and retrograde vesicular trafficking in the ER-Golgi system as a prerequisite for the degradation of CPY* and PrA*, two well established luminal ERAD substrates (41, 42). Nevertheless, even if the retrograde-transported substrate is dislocated from the ER, its elimination is not necessarily executed through ERAD. A quality control mechanism that is independent of HRD/DER has been reported in yeast (51). Recently, this HRD/DER-independent proteasomal degradation, designated HIP, was shown to depend on vesicular transport and to involve Rsp5p as E3 and possibly Ubc4p and Ubc5p as E2 (43). It has been suggested that saturation of the vesicular transport-independent ERAD machinery is responsible for diverting overexpressed quality control substrates (e.g. CPY*) to this vesicular transport-dependent HIP pathway (43). This situation, however, does not apply to the µs heavy chain in B cells, where it is the assembly with conventional light chains that diverts µs to the transport-dependent pathway. Interestingly, a very recent report from yeast suggests that the Sar1p/COPII machinery is required for sequestration of the membrane ERAD substrate CFTR into ER subdomains before its proteasomal degradation, but this function is independent of the role of Sar1p/COPII machinery in ER-Golgi vesicular transport (52).
The developmental transition, from the vesicular transport-independent route to ERAD of unassembled µ heavy chains in pre-B cells to the vesicular transport-dependent route to degradation by the proteasome-ubiquitin pathway of µs in -expressing B cells, adds new perspective to the post-translational regulation of the immune response and the differentiation of lymphocytes of the B lineage. Moreover, our study extends the degradation along the secretory pathway beyond the quality control elimination of aberrant proteins. In response to developmental cues, two routes lead proteins selected by their assembly state to the ubiquitin-proteasome pathway.
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FOOTNOTES |
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Both authors contributed equally to this work.
|| To whom correspondence should be addressed. Tel.: 972-3-6408984; Fax: 972-3-6406834; E-mail: sbnun{at}post.tau.ac.il.
1 The abbreviations used are: sIgM, secretory form of IgM; ALLN, N-acetyl-leucyl-leucyl-norlecinal; BFA, brefeldin A; ER, endoplasmic reticulum; ERAD, endoplasmic reticulum-associated degradation; HRP, horseradish peroxidase; LPS, lipopolysaccharides; MG-132, carboxybenzyl-leucyl-leucyl-leucinal; SLC, surrogate light chain; Z-L3 VS, carboxybenzyl-leucyl-leucyl-leucine vinyl sulfone; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase.
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ACKNOWLEDGMENTS |
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REFERENCES |
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