BAP31 Is Involved in the Retention of Cytochrome P450 2C2 in the Endoplasmic Reticulum*

Microsomal cytochrome P450 2C2 is an integral endoplasmic reticulum (ER) membrane protein that is directly retained in the ER and excluded from transport vesicles. We have used bimolecular fluorescence complementation and co-immunoprecipitation to show that a ubiquitous ER membrane protein (BAP31) interacts with P450 2C2 in transfected COS-1 cells. A chimera containing only the N-terminal signal anchor of P450 2C1 (P450 2C1-(1–29)) also interacted with BAP31, which is consistent with interaction of the two proteins via their transmembrane domains. Down-regulation of BAP31 expression with small interfering RNA resulted in redistribution of green fluorescent protein-tagged P450 2C2 or P450 2C1-(1–29) from the ER into the nuclear membrane and compact perinuclear compartment structures as well as the cell surface in a small fraction of the cells. In Bap31-null embryonic stem cells, a significant fraction of P450 2C2 or P450 2C1-(1–29) was detected at the cell surface and nuclear envelope, but was redistributed to the ER by expression of BAP31. The expression level of P450 2C2 was significantly increased in COS-1 cells with repressed levels of BAP31. Formation of the pro-apoptotic p20 fragment of BAP31 was detected in transfected COS-1 cells expressing P450 2C2, and annexin V staining was consistent with the activation of an apoptotic pathway in these cells. Down-regulation of BAP31 with small interfering RNA partially reversed the apoptosis. These results suggest that interaction of P450 2C2 with BAP31 is important for its ER retention and expression level and that BAP31 may be involved in the regulation of apoptosis induced by the ER overload response to increased expression of P450.

Microsomal cytochrome P450 2C2 is an integral endoplasmic reticulum (ER) membrane protein that is directly retained in the ER and excluded from transport vesicles. We have used bimolecular fluorescence complementation and co-immunoprecipitation to show that a ubiquitous ER membrane protein (BAP31) interacts with P450 2C2 in transfected COS-1 cells. A chimera containing only the N-terminal signal anchor of P450 2C1 (P450 2C1-(1-29)) also interacted with BAP31, which is consistent with interaction of the two proteins via their transmembrane domains. Down-regulation of BAP31 expression with small interfering RNA resulted in redistribution of green fluorescent protein-tagged P450 2C2 or P450 2C1-(1-29) from the ER into the nuclear membrane and compact perinuclear compartment structures as well as the cell surface in a small fraction of the cells. In Bap31-null embryonic stem cells, a significant fraction of P450 2C2 or P450 2C1-(1-29) was detected at the cell surface and nuclear envelope, but was redistributed to the ER by expression of BAP31. The expression level of P450 2C2 was significantly increased in COS-1 cells with repressed levels of BAP31. Formation of the pro-apoptotic p20 fragment of BAP31 was detected in transfected COS-1 cells expressing P450 2C2, and annexin V staining was consistent with the activation of an apoptotic pathway in these cells. Down-regulation of BAP31 with small interfering RNA partially reversed the apoptosis. These results suggest that interaction of P450 2C2 with BAP31 is important for its ER retention and expression level and that BAP31 may be involved in the regulation of apoptosis induced by the ER overload response to increased expression of P450.
The mechanisms underlying direct retention of integral membrane proteins in the endoplasmic reticulum (ER) 2 are not well understood. Whether specific retention signals are involved or whether the lack of positive transport (exit) signals underlies exclusion of these proteins from further transport out of the ER remains controversial. A positive signal is required for the rapid transport from the ER of many proteins because these proteins are more concentrated in the nascent transport vesicles and the rates of transport are ϳ10-fold higher compared with proteins that do not contain known exit signals (1). The slow rate of transport of the latter proteins presumably represents bulk flow of membrane and luminal contents passively incorporated into the transport vesicle. A retention signal to prevent bulk flow must operate for proteins directly retained in the ER. The existence of an ER retention signal(s) implies that specific receptors recognizing these signals must be present in the ER compartment. For some ER membrane proteins, it has been shown that short motifs (RR at the N terminus or KKXX at the C terminus) mediate their ER retention and that this process involves receptor-mediated retrieval from a post-ER compartment (2,3). Yet ER retention of KKXX-containing proteins is often unaffected by deletion or mutation of this signal, and most of the ER-specific membrane proteins do not contain any of the known retrieval signals. The transmembrane domains (TMDs) of many proteins are usually sufficient to retain a heterologous protein in the ER (4), implying that they encode some retention signal, but a conserved motif with such a function has been not identified.
Microsomal cytochromes P450 are integral ER membrane proteins inserted in the membranes via the N-terminal signal anchor sequence, which also functions as an ER retention signal (5)(6)(7). In several P450s, such as P450 2C1, 2C2, and M1, the signal anchor sequence mediates direct retention in the ER (5,6), whereas in other microsomal P450s, retention in the ER involves recycling through the intermediate compartment (6), and in some cases, P450 can be detected in the Golgi and plasma membrane (8,9). These differences may result from heterogeneity of the length and sequence of P450 signal anchors, but despite extensive studies, the specific signal encoded by these sequences or the mechanism of ER retention is not known. In addition to the signal anchor, the cytoplasmic domain of P450 2C2 is also excluded from vesicular transport when inserted into the ER membrane by a heterologous signal anchor sequence (10), suggesting that this domain contains a redundant ER retention signal. Microsomal P450s do not contain any of the known ER retention/retrieval signals, and it is not known whether their targeting and retention in the smooth ER involve a specific receptor. High concentrations of P450s in the ER attained by drug-mediated induction would require high concentrations of any specific receptor to prevent their transport out of the ER by bulk flow.
Induction of microsomal P450s also leads to proliferation of the smooth ER compartment, a process that activates both ER stress and ER overload responses in a time-dependent fashion (11)(12)(13)(14). The balance between the two signaling pathways determines whether accumulation of P450 is accommodated by expansion of the ER membrane compartment or whether the severity of the stress induces apoptosis in these cells (11). These processes must be mediated by multiple interactions of P450 with folding chaperones, quality control components, and signaling proteins of the ER. Moreover, formation of an enzymatically active P450 requires not only proper folding of P450 and its redox partners, but also homo-or hetero-oligomerization among these proteins (15), processes that may require chaperone actions. Thus, it is likely that microsomal P450s are involved in many interactions with multiple components of the ER membrane.
Recent studies indicate that a ubiquitous and evolutionary conserved protein (BAP31) is involved in the quality control and regulation of intracellular trafficking and ER retention/exit of some membrane proteins. BAP31 is a polytopic integral ER membrane protein, and it forms a hetero-oligomeric complex with a closely related protein, BAP29 (16,17). BAP31 is involved in the ER retention and/or exit of membranebound IgD (18), the cystic fibrosis transmembrane conductance regulator (19), tetraspanins (20), major histocompatibility complex class I proteins (21), and cellubrevin (22). In addition, BAP31 interacts with proteins that regulate apoptosis (procaspase-8 and Bcl-2), cytoskeleton components, and the ER chaperone calnexin, which implicates BAP31 in the regulation of multiple cellular pathways originating in the ER (16,18,20,(22)(23)(24)(25)(26). BAP31 also plays a role in apoptosis. In its full-length form, it has anti-apoptotic activity, but a caspase cleavage product of BAP31 (a transmembrane fragment called p20) promotes apoptosis (16,(27)(28)(29). It has been suggested that BAP31 has some role in the ER membrane architecture because it associates with the cytoskeletal components actin and myosin (30).
Because of its role in ER retention of several membrane proteins, it seemed possible that BAP31 might be involved in ER retention of P450. In this study, we show that BAP31 interacts with P450 2C2 in the ER of transfected cells and that reduction of BAP31 levels affects the subcellular localization and accumulation of P450 and apoptosis in response to increased P450 in the ER membrane.

EXPERIMENTAL PROCEDURES
Materials-Tissue culture materials were purchased from Invitrogen; the pEGFP-N1 vector was from Clontech; and Tran 35 S-label from MP Biomedicals. The yellow fluorescent protein (YFP) fragments encoding plasmids pBiFCYN and pBiFCYC were kindly provided by Dr. T. Kerpolla (University of Michigan, Ann Arbor, MI). Antibodies against green fluorescent protein (GFP) and GM130 were from Santa Cruz Biotechnology, Inc. and BD Biosciences, respectively, and rhodamine-conjugated anti-mouse antibody was obtained from Jackson ImmunoResearch Laboratories, Inc. For BAP31 detection, either anti-BAP31 antibody (kindly supplied by Dr. Gordon Shore, McGill University, Montreal, Canada) or a commercial antibody against BAP31 (Affinity BioReagents) was used.
Cell Culture and Transfection-COS-1 cell culture, transfection with Lipofectamine 2000 reagent, biosynthetic labeling of the transfected cells, immunoprecipitation, and Western blotting were done as described (6,11). Anti-GFP antiserum was used to detect chimeric proteins containing YN and YC. Bap31-null mouse differentiated embryonic stem (ES) cells were obtained from Dr. Gordon Shore and were maintained as described (20,21,26).
Apoptosis Assay-Apoptosis in transfected cells was assayed 48 h after transfection using the fluorescein isothiocyanate (FITC)-conjugated annexin V apoptosis detection kit (BioVision Inc.) following the manufacturer's protocol.
BiFC Analysis by Flow Cytometry and Fluorescence Microscopy-For BiFC analysis by fluorescence microscopy, cells were grown on coverslips in 6-well plates; and 18 -20 h after transfection, the cells were incubated for 5 h at 30°C and imaged with a Zeiss LSM510 confocal microscope using YFP excitation at 514 nm and detection at 535-545 nm as described (32,33). For flow cytometry, 18 -20 h after transfection, cells were incubated for 5 h at 30°C, gently trypsinized, washed with Dulbecco's modified Eagle's medium, and resuspended in 1 ml of Dulbecco's modified Eagle's medium. The cells were analyzed with a Coulter Epics XL-MCL flow cytometer using an excitation wavelength of 488 nm and Summit Version 3.1 software (Cytomation, Inc.) as described (32). Only cells exhibiting forward and side scatter typical of live cells were included in the analysis. A threshold fluorescence intensity was selected such that Ͻ1% of the control cells (mock-transfected or expressing a chimera with YN or YC only) were above the threshold. The percentage of cells above the threshold and the average intensity of these cells were determined for the experimental samples.

Analysis of BAP31 Interaction with Cytochrome P450 2C2 by BiFC-
The BiFC assay is based on recombination of the non-fluorescent N-terminal (YN) and C-terminal (YC) fragments of YFP to produce a fluorescent protein (34). The interaction between two proteins can be analyzed by coexpressing in cells chimeras of one protein fused to YN and the other protein to YC. If the two proteins interact with each other, YC and YN are brought together and irreversibly recombine to form fluorescent YFP, whereas if the two proteins do not interact, fluorescence is not observed. The interaction between YN and YC does not contribute to the potential interaction between the chimeric proteins (34). The intensity of the fluorescence resulting from BiFC is dependent on both the affinity of the interaction of the two proteins and the relative positions of the two YFP fragments in the protein complex. The intensity of fluorescence therefore does not directly correlate with the strength of the protein interaction, particularly if the interaction of two proteins of differing sizes, such as P450 2C2 and its N-terminal fragment P450 2C1-(1-29), with a third protein is studied.
We have recently used BiFC to detect oligomerization of P450 2C2 and its interaction with P450 reductase in live cells (32). A fluorescent signal was generated in the ER of cells transfected with chimeras of P450 2C2 or its 29-amino acid N-terminal signal anchor sequence attached to YN and YC, consistent with oligomerization of P450 2C2 mediated by its N-terminal signal anchor sequence. Here, we used these P450 2C2 chimeras to detect potential interactions of P450 2C2 with BAP31 tagged at its C terminus with the YFP fragments. We have shown pre- FEBRUARY 17, 2006 • VOLUME 281 • NUMBER 7 viously that the ER membrane localization and topology of either fulllength P450 2C2 or its N-terminal signal anchor only are not affected by attaching GFP or YFP to the C terminus (31). However, BAP31 contains a KKXX ER retention signal at its C terminus (16,17). Although mutation of the KKXX sequence does not alter the ER distribution of BAP31 (18), it was still important to establish that attachment of YN or YC to the C terminus does not affect its ER retention. Immunofluorescence of COS-1 cells transfected with untagged BAP31 (BAP31/cytomegalovirus) and detected with anti-BAP31 antibody showed the expected ER localization (Fig. 1B). A similar pattern of fluorescence was observed in cells transfected with BAP31/YN and detected with an antibody against GFP that recognizes YN (Fig. 1A), indicating that attachment of the YFP fragment does not affect BAP31 ER retention. Cotransfection of untagged BAP31 (Fig. 1B) and P450 2C2/GFP (Fig. 1C) showed the expected co-localization of these proteins in the ER compartment (Fig. 1D).

BAP31 and P450 ER Retention
As a positive control for the BiFC assay, we cotransfected cells with chimeras P450 2C1-(1-29)/YN and P450 2C1-(1-29)/YC. In these cells, strong fluorescence was detected in a pattern consistent with ER membrane localization of the P450 chimeras that homo-oligomerize (Fig.  1E). Cotransfection of COS-1 cells with BAP31/YN and P450 2C2/YC also generated yellow fluorescence in live cells in a typical ER pattern (Fig. 1G), indicating that these proteins interacted in this compartment. To test whether the interaction of P450 2C2 with BAP31 is mediated by the TMD of P450 in the N-terminal signal anchor sequence, as has been shown for other proteins (17,18), we used the chimera P450 2C1-(1-29)/YC. In cells transfected with BAP31/YN and P450 2C1-(1-29)/YC, a fluorescence intensity and pattern of distribution similar to those for BAP31/YN and full-length P450 2C2/YC were seen (Fig. 1F), consistent with the interaction of P450 with BAP31 being mediated by the 29-amino acid N-terminal signal anchor sequence. For both chimeras, a pattern of reconstituted YFP fluorescence in an ER-like distribution was observed in most cells, and a distribution of fluorescence characteristic of Golgi or plasma membrane localization was not detected in any cells. These results indicate that the P450 2C2/YC and BAP31/YN interaction occurs within the ER network.
The interaction of some proteins with BAP31 requires a heterodimer of BAP31 with its partner BAP29 (18,27). To determine whether increased expression of BAP29 would enhance the BAP31-P450 2C2 interaction, we also tested the effect of coexpressing BAP29/YN in transfected cells. No enhancement of fluorescence intensity was observed in cells when BAP29/YN was cotransfected with P450 2C2/YC and BAP31/YN (Fig. 1H), which suggests that BAP29 is not required for complex formation between BAP31 and P450 2C2. However, it remains possible that BAP29 is part of the BAP31 complex with P450 2C2, but that saturating amounts of endogenous BAP29 mask any effects of exogenously expressed BAP29.
The interaction of P450 2C2 and BAP31 was further studied by detection of BiFC with flow cytometry, which analyzes a large population of transfected cells rather than individual cells as is done by confocal microscopy. As a negative control, cells transfected with only one YFP fragment chimera (P450 2C1-(1-29)/YC, P450 2C1-(1-29)/YN, or BAP31/YN) were analyzed, and a threshold of fluorescence was chosen such that 0.1% of the control cells were above the threshold ( Fig. 2A). As a positive control, we again measured the fluorescence of live cells expressing the YC and YN chimeras of P450 2C1-(1-29), which are known to homo-oligomerize. As expected, in these cells, BiFC was detected in 22% of the cells above the threshold fluorescence (Fig. 2B). For cells cotransfected with BAP31/YN and either full-length P450 2C2/YC or P450 2C1-(1-29)/YC, BiFC was detected in 19 and 21%, respectively, of the cells above the threshold fluorescence (Fig. 2, C and  To test whether P450 2C2 association with BAP31 is related to its direct ER retention, we also analyzed BAP31 binding to the chimera P450 2C1-(1-21)/YC, which contains only the TMD of the signal anchor sequence without the juxtamembrane linker residues 22-29. P450 2C1-(1-21)/GFP was shown in our previous study (35) to be transported from the ER and retained by the retrieval pathway rather than directly retained in the ER. Although BiFC was detected in cells transfected with BAP31/YN and P450 2C1-(1-21)/YC (Fig. 2E), only 7.3% of the cells were above the threshold compared with 21% for P450 2C1-(1-29)/YC, suggesting a weaker interaction of P450 2C1-(1-21)/YC with BAP31/YN. Similar results were obtained when BiFC was detected by confocal microscopy (data not shown). Optimal binding of P450 2C2 to BAP31 thus requires both the TMD (residues 1-20) and linker region (residues 21-29) of the signal anchor sequence.
The interaction between BAP31 and P450 2C2 was also analyzed by co-immunoprecipitation. COS-1 cells were transfected with P450 2C1-(1-29)/YN and BAP31 and labeled 24 h later with Tran 35 S-label for 2 h. Equal amounts of cell lysates were then immunoprecipitated with an antibody against either BAP31 or GFP (which recognizes YN in P450 2C1-(1-29)/YN). BAP31 was co-immunoprecipitated with anti-GFP antibody; and conversely, anti-BAP31 antibody coprecipitated P450 2C1-(1-29)/YN (Fig. 3). In both cases, only a fraction of the proteins coprecipitated compared with the amount of protein precipitated with the homologous antisera. Similar results were obtained with full-length P450 2C2 (data not shown). This suggests either that the interaction is not completely stable under the conditions of the immunoprecipitation or that the interaction between BAP31 and P450 is only transient, and only a fraction of the proteins remains in a complex at any time point.
These observations further support the conclusion from microscopic and flow cytometry studies that P450 2C2 binds to BAP31 in transfected COS-1 cells and that the interaction is mediated by the 29-amino acid signal anchor sequence, which is responsible for direct ER retention. Decreased binding by the P450 2C1-(1-21) chimera, which is not directly retained in the ER but undergoes recycling through the retrieval pathway, suggests that BAP31 binding may have a role in the direct retention of P450 2C2 in the ER.
Effect of Suppression of BAP31 Expression on P450 2C2 Localization-To obtain a better understanding of the function and significance of the interaction of P450 2C2 with BAP31, we constructed a BAP31 siRNA expression vector (pSUPER/T1). Western blot analysis showed that, in COS-1 cells transfected with this plasmid, the level of BAP31 protein was significantly decreased by ϳ40% (Fig. 4A). The transfection efficiency in these experiments is ϳ50%, so a better estimate of the inhibition of BAP31 expression in the transfected cells is ϳ80%.
To test whether BAP31 has a role in P450 2C2 ER retention, we cotransfected COS-1 cells with the BAP31 siRNA vector and the P450 2C2/GFP expression plasmid. Similar to native P450 2C2, the chimera P450 2C2/GFP localized to the ER membrane (Fig. 4B, panel a), where it is directly retained and does not undergo retrieval from the intermediate compartment (6). Remarkably, expression of BAP31 siRNA resulted in a dramatic change in the pattern of fluorescence for both the fulllength P450 2C2 (Fig. 4B, panels b and d) and P450 2C1-(1-29) (panel c) chimeras from a reticular pattern (typical of ER distribution) to accumulation of fluorescence in the nuclear envelope and in a perinuclear region. The perinuclear pattern was similar to that observed for Golgi localization (36), so the transfected cells were also immunostained with an antibody against the Golgi protein GM130 (Fig. 4B, panel e). A significant co-localization of P450 2C2/GFP fluorescence with that of the Golgi marker was observed in the presence of BAP31 siRNA (Fig. 4B,  panel f). The size of the Golgi-like region was, however, larger than the Golgi regions in the untransfected cells, so it was not clear whether the perinuclear fluorescence was present in the Golgi or in another compartment that overlaps with the Golgi. P450 2C2 was detected on the cell surface in a minor fraction of cells with down-regulated BAP31 (data not shown). Thus, in the absence of sufficiently high concentrations of BAP31 protein, P450 2C2 escapes ER retention and is redistributed, possibly to Golgi-like organelles, the nuclear envelope, and the cell surface.  These data were further confirmed using Bap31-null ES cells (20,21). In these cells, P450 2C1-(1-29)/GFP showed a prominent staining of the nuclear membrane; and in many cells, the protein was detected in the plasma membrane (Fig. 5A). However, exogenous expression of BAP31 in these cells resulted in redistribution of fluorescence to an ER-like distribution (Fig. 5B). The full-length chimera P450 2C2/GFP, which was poorly expressed in Bap31-null cells, was also detected in the nuclear and plasma membranes of Bap31-null cells; however, a significant fraction of this protein was still located in the ER (Fig. 5C). Cotransfection with BAP31 further increased the reticular ER localization of full-length P450 2C2 (Fig. 5D). The difference between P450 2C1-(1-29) and full-length P450 2C2 in the ES cells may be related to the direct ER retention property of the cytoplasmic domain of P450, independent of the N-terminal signal anchor (10). The differences in localization in the BAP31-deficient COS-1 cells compared with the Bap31-null differentiated ES cells could reflect cell-specific differences in localization or the complete absence of BAP31 in the ES cells compared with a partial decrease in COS-1 cells. Nevertheless, these results with the Bap31-null ES cells confirmed COS-1 cell studies with BAP31 siRNA and suggest that the interaction of BAP31 with P450 2C2 is involved in its retention in the ER membrane.
Effect of siRNA Inhibition of BAP31 Expression on the Expression Level of P450 2C2-The level of fluorescent P450 2C2/GFP appeared consistently higher in cells expressing BAP31 siRNA (Fig. 4B), suggesting that down-regulation of BAP31 might lead to increased accumulation of P450. To better quantitate the increased fluorescence, transfected COS-1 cells were analyzed by flow cytometry. In cells expressing BAP31 siRNA compared with cells transfected with P450 2C2/GFP and the pSUPER empty vector, the percentage of cells above the threshold was increased by almost 2-fold, and the median fluorescence intensity of the cells above the threshold was 2-fold higher (Fig. 6), demonstrating that P450 2C2 expression was increased by at least 2-fold. The increased level of P450 2C2 could result from increased biosynthesis or decreased degradation of P450 2C2; but because expression of P450 2C2 is driven by a strong cytomegalovirus promoter, it is most likely that degradation of P450 2C2 is affected by decreased levels of BAP31.
Effect of siRNA Inhibition of BAP31 Expression on Apoptosis Induced by P450 Expression-Expression of P450 2C2 in COS-1 cells induces both ER stress and ER overload responses, eventually resulting in the activation of apoptotic signals (11). Because a role for BAP31 in the regulation of apoptosis is well established, we considered the possibility that it might be also involved in apoptosis associated with P450 accumulation. Activation of caspase-8 leads to BAP31 cleavage and formation of the p20 membrane protein, which activates pro-apoptotic signals (16,(27)(28)(29). Western analysis of lysates of COS-1 cells expressing either P450 2C2/YN or P450 2C1-(1-29)/YN (or untagged P450 2C2) showed that anti-BAP antibody precipitated a protein of the size expected for p20 (Fig. 7A). Thus, it is possible that ER stress induced by increased P450 expression results in BAP31 processing, which would contribute to the activation of the pro-apoptotic pathway.
We then compared the apoptotic response in cells expressing P450 2C2 in the presence and absence of BAP31 siRNA. Apoptosis was assayed using FITC-conjugated annexin V staining, which detects apoptotic changes in cell membranes (37). Flow cytometry analysis of cells transfected with P450 2C2 showed an increase in the population of cells with higher fluorescence intensity, consistent with increased binding of FITC-conjugated annexin V and increased apoptosis (Fig. 7B). However, coexpression of BAP31 siRNA partially reversed the increased fluorescence, with a smaller fraction of cells having greater fluorescence compared with mock-transfected cells (Fig. 7B). These results are consistent with the hypothesis that BAP31 contributes to the activation of apoptosis in COS-1 cells that is induced by increased expression of P450 2C2.

DISCUSSION
BAP31 is a ubiquitous ER membrane protein that has been implicated in the regulation of export of membrane proteins from the ER (18 -22).  The data presented in this study show that BAP31 forms a complex with cytochrome P450 2C2 or with its 29-amino acid signal anchor sequence and that this interaction affects both the localization and expression level of P450 2C2. The results are consistent with the hypothesis that BAP31 has a role in mediating the direct retention of P450 2C2 in the ER. The major support for this hypothesis comes from studies with COS-1 cells in which BAP31 expression was reduced by siRNA expression and in Bap31-null ES cells. In COS-1 cells with reduced expression of BAP31, there was a redistribution of P450 2C2 from a normal reticular ER-like pattern to prominent fluorescence in the nuclear membrane and compact perinuclear structures. In some of these cells, fluorescence was observed in the plasma membrane. The perinuclear fluorescence partially overlapped with structures that were immunostained with antibodies to the Golgi marker protein GM130 but that were different in size from the Golgi structures in untransfected cells and may not be normal Golgi complexes. It is also possible that this is a pre-Golgi quality control compartment, which has been detected in cells accumulating substrates for the proteasomal degradation pathway (38). If BAP31 assists in P450 folding, in its absence, misfolded P450 could be transported to this quality control compartment. In Bap31null cells, P450 2C1-  was observed mostly in the nuclear and plasma membranes, whereas a smaller fraction of full-length P450 2C2 escaped ER retention. Exogenous expression of BAP31 restored an ER fluorescent pattern. The altered localization of P450 2C2 or P450 2C1-  in both Bap31-null ES cells and COS-1 cells expressing BAP31 siRNA strongly supports a role for BAP31 in P450 ER retention. The restoration of ER localization by exogenous expression of BAP31 in the Bap31-null ES cells demonstrates that the absence of BAP31 is responsible for the abnormal distribution of P450 2C2 in these cells.
The interaction of BAP31 with the signal anchor sequence is decreased if the linker sequence (residues [22][23][24][25][26][27][28][29] is deleted, which also results in loss of direct ER retention, although the truncated signal anchor still mediates ER localization via the retrieval pathway (35). This would be consistent with a principal role of BAP31 in the direct ER retention of P450 2C2, but reduction of BAP31 also appears to prevent efficient retrieval because the P450 2C1-(1-29) chimera should be competent for retrieval, as is the P450 2C1-(1-21) chimera. Thus, with reduced concentrations of BAP31, P450 2C2 escapes ER retention and probably reaches an early Golgi/intermediate compartment.
BAP31 binds to the P450 2C2 N-terminal signal anchor sequence, which is known to mediate direct ER retention and contains the only TMD (residues 3-20) in P450 2C2. Consistent with this observation, interactions of BAP31 with other ER-retained proteins have also been shown to involve TMDs of both BAP31 and the interacting partner (17). On the other hand, because binding of P450 2C2 to BAP31 is decreased in the absence of the short linker sequence (residues [22][23][24][25][26][27][28][29], residues flanking the hydrophobic TMD might also contribute to the interaction with BAP31. The cytoplasmic domain of BAP31, which has been shown by glutathione S-transferase pull-down experiments to interact with the CD11b/CD18 integrin (24), could potentially interact with the linker sequence of P450 2C2. Mutagenesis studies suggested that polar amino acids of the interacting TMDs plays an important role in the binding of some proteins to BAP31 (17). Interestingly, our previous study (35) indicated that polar amino acids of the P450 2C1 TMD are important for direct ER retention as well, which provides a correlation between the structural requirement for direct ER retention and the interaction of P450 2C2 with BAP31.
The N-terminal signal anchor sequence of P450 2C2 also mediates the homo-oligomerization of P450 2C2 (33). This raises the question of whether the interaction of BAP31 with the signal anchor sequence competes for the homo-oligomerization or whether BAP31 can interact with the homo-oligomers of the signal anchor. Because only a fraction of P450 2C2 appears to be bound to BAP31, it is further possible that homo-oligomers and BAP31-P450 2C2 complexes coexist. The present data cannot distinguish among these possibilities.
BAP31 forms a large heteromeric complex containing the closely related protein BAP29 and binds to membrane-bound IgD in the ER of live cells (18). In contrast, BAP31 (and not BAP29) co-immunoprecipitates with cellubrevin (22), which suggests that BAP31 may also be present in complexes that do not contain BAP29. Coexpression of BAP29/YN with BAP31/YN and P450 2C2/YC did not enhance BiFC over that which was observed with BAP31/YN alone. Furthermore, BAP31 was the major protein that was co-immunoprecipitated with antisera to P450 2C2/YN. These results suggest that BAP29 is not involved in the interaction of BAP31 with P450 2C2.
The transport of P450 2C2 out of the ER in cells with reduced expression of BAP31 coincided with increased accumulation of P450 2C2 in the cells. Similarly, it has been shown that antisense inhibition of BAP31 expression increases expression of the cystic fibrosis transmembrane conductance regulator (19). Degradation of many P450s, like the misfolded cystic fibrosis transmembrane conductance regulator (39), involves the proteasomal pathway (40 -42), which would require retrotranslocation of P450 2C2 from the ER membrane into the cytosol. One explanation for the increased levels of the cystic fibrosis transmembrane conductance regulator and P450 2C2 in cells deficient in BAP31 could therefore be that, after transport from the ER, the proteins are no longer accessible to endoplasmic reticulum-associated degradation. Alternatively, BAP31 may act as a quality control chaperone that directs excess and misfolded proteins to endoplasmic reticulum-associated degrada- tion; and if the BAP31 concentration is decreased, excess levels of P450 2C2 accumulate, which can escape the ER. This second possibility seems less likely because, first, BAP31 appears to act as a quality control protein that delays exit from the ER until maturation of the proteins rather than targeting proteins to the degradation pathway. In fact, BAP31 is required for maintaining detectable levels of PTPLB (43), suggesting that it acts primarily as a chaperone protein. Second, although P450 2C2 concentrations increased by only 2-fold, nearly all of the P450 2C2 in cells deficient in BAP31 was present in the nuclear membrane and juxtanuclear compartment (Golgi-like structures). Even if excess protein escapes the ER, there should still be strong fluorescence in a reticular pattern as well as the Golgi-like pattern. It seems therefore likely that the increased P450 2C2 in cells deficient in BAP31 results from transport of the protein from the ER away from endoplasmic reticulum-associated degradation.
BAP31 functions in apoptosis as a substrate of caspase-8, which cleaves the cytoplasmic tail of BAP31 to form p20, which activates proapoptotic signals (16,(27)(28)(29). We observed the formation of pro-apoptotic p20 fragments of BAP31 in cells expressing P450 2C2, and our previous study (11) showed that ER stress is activated in COS-1 cells in response to P450 accumulation in the ER, which ultimately leads to apoptosis. This raises the possibility that the interaction of P450 2C2 with BAP31 may be involved in the apoptotic response of the cells to increased expression of P450 2C2 in the ER. One possibility is that, in cells deficient in BAP31, P450 2C2 exits the ER rather than accumulating, which reduces the stimulus for the ER overload response and the initiation of the apoptotic pathway. Alternatively, the interaction of excess ER protein, like P450 2C2, with BAP31 could act as a stimulus for initiation of the apoptotic process. It is also possible that the interaction of P450 2C2 with BAP31 does not directly affect BAP31, but instead BAP31 is part of the apoptotic signaling pathway in response to ER stress. In this case, the apoptotic role of BAP31 would be independent of its interaction with P450 2C2. BAP31 could act as an ER retention "receptor" for P450 2C2 by forming a stable complex with P450 2C2 and retaining it in the ER. However, a transient interaction would be consistent with the quality control function of BAP31 for the other ER membrane proteins with which it interacts and which it prevents from entering the transport vesicles until maturation or oligomerization. The coprecipitation of only a fraction of the total P450 2C2/YN by anti-BAP31 antisera in cells overexpressing BAP31 would be consistent with a transient interaction. It appears therefore more likely that BAP31 interacts with P450 2C2 only transiently during its synthesis, folding, oligomerization, or interaction with other proteins to prevent it from exiting the ER. This process could either result in the interaction of P450 2C2 with another ER retention receptor or target the P450 to a subdomain of the ER away from the protein exit sites. If ER retention of P450 results from its association with a specific retention receptor, enzymatic partners, and/or some other integral membrane proteins, BAP31 may mediate this interaction, for example, by assisting in complex formation between the TMDs of the interacting proteins.