High-level expression and purification of the human bradykinin B(2) receptor in a tetracycline-inducible stable HEK293S cell line

The B(2) bradykinin receptor belongs to the G-protein coupled receptor family. Development of new drugs for this important therapeutic target requires structural information on the receptor. The main goal of the present work was to overexpress the human B(2) receptor for future biophysical studies. Different tagged B(2) receptors were engineered and their properties were evaluated by transient expression in HEK293S cells. A B(2) receptor tagged with a hexahistidine at the N-terminus and a nonapeptide at the C-terminus was selected for high expression level and preserved ligand-binding characteristics. First, we generated a HEK293S stable cell line expressing the receptor constitutively at a level of 60pmol/mg of crude membrane protein. However, the decrease of expression level with cell passages led us to express the B(2) receptor in a HEK293S tetracycline-inducible stable cell line. Induction of expression of the B(2) receptor with tetracycline and sodium butyrate led to a level of 100pmol/mg of membrane protein, which is the highest level reported so far for this receptor. The expression level was stable with cell passages and the ligand-binding and signal transduction properties of the receptor were unaltered. The receptor was purified to near homogeneity by solubilization with n-dodecyl-beta-d-maltoside followed by a two-step purification procedure combining hydroxyapatite and immunoaffinity chromatography. Although the purified receptor is not functional, the purification of the B(2) receptor to near homogeneity from a stable cell line overexpressing this receptor pave the way for future structural studies of this receptor.     

Study of a synthetic human olfactory receptor 17-4: expression and purification from an inducible mammalian cell line

In order to begin to study the structural and functional mechanisms of olfactory receptors, methods for milligram-scale purification are required. Here we demonstrate the production and expression of a synthetically engineered human olfactory receptor hOR17-4 gene in a stable tetracycline-inducible mammalian cell line (HEK293S). The olfactory receptor gene was fabricated from scratch using PCR-based gene-assembly, which facilitated codon optimization and attachment of a 9-residue bovine rhodopsin affinity tag for detection and purification. Induction of adherent cultures with tetracycline together with sodium butyrate led to hOR17-4 expression levels of approximately 30 microg per 150 mm tissue culture plate. Fos-choline-based detergents proved highly capable of extracting the receptors, and fos-choline-14 (N-tetradecylphosphocholine) was selected for optimal solubilization and subsequent purification. Analysis by SDS-PAGE revealed both monomeric and dimeric receptor forms, as well as higher MW oligomeric species. A two-step purification method of immunoaffinity and size exclusion chromatography was optimized which enabled 0.13 milligrams of hOR17-4 monomer to be obtained at >90% purity. This high purity of hOR17-4 is not only suitable for secondary structural and functional analyses but also for subsequent crystallization trials. Thus, this system demonstrates the feasibility of purifying milligram quantities of the GPCR membrane protein hOR17-4 for fabrication of olfactory receptor-based bionic sensing device.     

The synthesis and high-level expression of a beta2-adrenergic receptor gene in a tetracycline-inducible stable mammalian cell line

High-level expression of G-protein-coupled receptors (GPCRs) in functional form is required for structure-function studies. The main goal of the present work was to improve expression levels of beta2-adrenergic receptor (beta2-AR) so that biophysical studies involving EPR, NMR, and crystallography can be pursued. Toward this objective, the total synthesis of a codon-optimized hamster beta2-AR gene suitable for high-level expression in mammalian systems has been accomplished. Transient expression of the gene in COS-1 cells resulted in 18 +/- 3 pmol beta2-AR/mg of membrane protein, as measured by saturation binding assay using the beta2-AR antagonist [3H] dihydroalprenolol. Previously, we reported the development of an HEK293S tetracycline-inducible system for high-level expression of rhodopsin. Here, we describe construction of beta2-AR stable cell lines using the HEK293S-TetR-inducible system, which, after induction, express wild-type beta2-AR at levels of 220 +/- 40 pmol/mg of membrane protein corresponding to 50 +/- 8 microg/15-cm plate. This level of expression is the highest reported so far for any wild-type GPCR, other than rhodopsin. The yield of functional receptor using the single-step affinity purification is 12 +/- 3 microg/15-cm plate. This level of expression now makes it feasible to pursue structure-function studies using EPR. Furthermore, scale-up of beta2-AR expression using suspension cultures in a bioreactor should now allow production of enough beta2-AR for the application of biophysical techniques such as NMR spectroscopy and crystallography.     

Tetraspanins CD9 and CD81 modulate HIV-1-induced membrane fusion

Protein organization on the membrane of target cells may modulate HIV-1 transmission. Since the tetraspanin CD81 is associated to CD4, the receptor of HIV-1 envelope protein (Env; gp120/gp41), we have explored the possibility that this molecule may modulate the initial steps of HIV-1 infection. On the other hand, CD81 belongs to the tetraspanin family, which has been described as organizers of protein microdomains on the plasma membrane. Therefore, the role of CD81 and other related tetraspanin, CD9, on the cell-to-cell fusion process mediated by HIV-1 was studied. We found that anti-tetraspanin Abs enhanced the syncytia formation induced by HIV-1 envelope proteins and viral entry in human T lymphoblasts. In addition, anti-CD81 Abs triggered its clustering in patches, where CD4 and CXCR4 were included. Moreover, the knocking down of CD81 and CD9 expression resulted in an increase in syncytia formation and viral entry. Accordingly, overexpression of CD81 and CD9 rendered cells less susceptible to Env-mediated syncytia formation. These data indicate that CD9 and CD81 have an important role in membrane fusion induced by HIV-1 envelope.     

Functional expression of milligram quantities of the synthetic human serotonin transporter gene in a tetracycline-inducible HEK293 cell line

The serotonin transporter (SERT), a member of the solute carrier 6 family, is responsible for reuptake of the monoamine neurotransmitter serotonin (5-hydroxytryptamine) from the synaptic cleft on the neural cells, and a vital target for several antidepressants. To investigate biophysical studies of this pharmacologically relevant transporter, we developed a mammalian expression system with tetracycline-inducible HEK293 cells using synthetic human SERT genes produced by PCR-based self-assembly method. Codon-optimization of this de novo constructed genes and construction of stable cell lines improved expression 3.5-fold and single-step immunoaffinity purification with FLAG-epitope tag yielded around one milligram functional SERT per liter culture medium assessed by [(3)H] imipramine ligand binding. Some characterizations including electrospray ionization MS/MS analysis, subcellular localization and cellular-uptake assay demonstrated that expressed human SERT was properly expressed, folded and fully functional. The long cytosolic N-terminal of SERT was predicted as containing 'intrinsically disordered region (IDR)' (～85 residues) by DISOPRED2 program. We engineered this salient region by step-wise truncation and ligand binding assay determined that dissociation constant for a series of de novo designed truncation constructs was close to the one for full-length wild type SERT. Our expression platform using synthetic codon-optimized gene and mammalian stable cell lines is feasible to produce milligram-scale functional membrane transporter for further biophysical and biochemical studies.     

Profiling of the tetraspanin web of human colon cancer cells

Tetraspanins are integral membrane proteins involved in a variety of physiological and pathological processes. In cancer, clinical and experimental studies have reported a link between tetraspanin expression levels and metastasis. Tetraspanins play a role as organizers of multimolecular complexes in the plasma membrane. Indeed each tetraspanin associates specifically with one or a few other membrane proteins forming primary complexes. Thus, tetraspanin-tetraspanin associations lead to a molecular network of interactions, the "tetraspanin web." We performed a proteomic characterization of the tetraspanin web using a model of human colon cancer consisting of three cell lines derived from the primary tumor and two metastases (hepatic and peritoneal) from the same patient. The tetraspanin complexes were isolated after immunoaffinity purification using monoclonal antibodies directed against the tetraspanin CD9, and the associated proteins were separated by SDS-PAGE and identified by mass spectrometry using LC-MS/MS. This allowed the identification of 32 proteins including adhesion molecules (integrins, proteins with Ig domains, CD44, and epithelial cell adhesion molecule) (EpCAM), membrane proteases (ADAM10, TADG-15, and CD26/dipeptidyl peptidase IV), and signaling proteins (heterotrimeric G proteins). Importantly some components were differentially detected in the tetraspanin web of the three cell lines: the laminin receptor Lutheran/B-cell adhesion molecule (Lu/B-CAM) was expressed only on the primary tumor cells, whereas CD26/dipeptidyl peptidase IV and tetraspanin Co-029 were observed only on metastatic cells. Concerning Co-029, immunohistofluorescence showed a high expression of Co-029 on epithelial cells in normal colon and a lower expression in tumors, whereas heterogeneity in terms of expression level was observed on metastasis. Finally we demonstrated that epithelial cell adhesion molecule and CD9 form a new primary complex in the tetraspanin web.     

Cd81 Interacts with the T Cell Receptor to Suppress Signaling

CD81 (TAPA-1) is a ubiquitously expressed tetraspanin protein identified as a component of the B lymphocyte receptor (BCR) and as a receptor for the Hepatitis C Virus. In an effort to identify trans-membrane proteins that interact with the T-cell antigen receptor (TCR), we performed a membrane yeast two hybrid screen and identified CD81 as an interactor of the CD3delta subunit of the TCR. We found that in the absence of CD81, in thymocytes from knockout mice, TCR engagement resulted in stronger signals. These results were recapitulated in T cell lines that express low levels of CD81 through shRNA mediated silencing. Increased signaling did not result from alterations in the levels of TCR on the surface of T lymphocytes. Although CD81 is not essential for normal T lymphocyte development, it plays an important role in regulating TCR and possibly pre-TCR signal transduction by controlling the strength of signaling. CD81 dependent alterations in thymocyte signaling are evident in increased CD5 expression on CD81 deficient double positive (DP) thymocytes. We conclude that CD81 interacts with the T cell receptor to suppress signaling.     

In situ chemical cross-linking on living cells reveals CD9P-1 cis-oligomer at cell surface

Tetraspanins are integral membrane proteins involved in a variety of physiological and pathological processes. They associate with each other in multimolecular complexes containing numerous membrane proteins. As a first step towards the study of the supramolecular organization of tetraspanin complexes, we have implemented a proteomic approach based on in situ protein cross-linking on living cells followed by affinity purification of tetraspanin complexes. This allowed observing the presence of high molecular weight protein complexes that were characterized as containing CD9P-1/CD315 using LC-MS/MS. Western blot analyses and the use of different tags demonstrated the presence of CD9P-1 oligomer in cis-association at cell surface. A significant amount of CD9P-1 oligomer was observed on various cell types. We have shown that CD9P-1 self-associates independently from its association with tetraspanins. However, the expression level of CD9 or CD81 that associate directly and specifically with CD9P-1, positively modulates the cross-linking efficiency of CD9P-1. Thus, tetraspanins can play a role on CD9P-1 oligomerization status.     

Scavenger receptor BI boosts hepatocyte permissiveness to Plasmodium infection

Infection of hepatocytes by Plasmodium falciparum sporozoites requires the host tetraspanin CD81. CD81 is also predicted to be a coreceptor, along with scavenger receptor BI (SR-BI), for hepatitis C virus. Using SR-BI-knockout, SR-BI-hypomorphic and SR-BI-transgenic primary hepatocytes, as well as specific SR-BI-blocking antibodies, we demonstrate that SR-BI significantly boosts hepatocyte permissiveness to P. falciparum, P. yoelii, and P. berghei entry and promotes parasite development. We show that SR-BI, but not the low-density lipoprotein receptor, acts as a major cholesterol provider that enhances Plasmodium infection. SR-BI regulates the organization of CD81 at the plasma membrane, mediating an arrangement that is highly permissive to penetration by sporozoites. Concomitantly, SR-BI upregulates the expression of the liver fatty-acid carrier L-FABP, a protein implicated in Plasmodium liver-stage maturation. These findings establish the mechanistic basis of the CD81-dependent Plasmodium sporozoite invasion pathway.     

In situ chemical cross-linking on living cells reveals CD9P-1 cis-oligomer at cell surface

Tetraspanins are integral membrane proteins involved in a variety of physiological and pathological processes. They associate with each other in multimolecular complexes containing numerous membrane proteins. As a first step towards the study of the supramolecular organization of tetraspanin complexes, we have implemented a proteomic approach based on in situ protein cross-linking on living cells followed by affinity purification of tetraspanin complexes. This allowed observing the presence of high molecular weight protein complexes that were characterized as containing CD9P-1/CD315 using LC-MS/MS. Western blot analyses and the use of different tags demonstrated the presence of CD9P-1 oligomer in cis-association at cell surface. A significant amount of CD9P-1 oligomer was observed on various cell types. We have shown that CD9P-1 self-associates independently from its association with tetraspanins. However, the expression level of CD9 or CD81 that associate directly and specifically with CD9P-1, positively modulates the cross-linking efficiency of CD9P-1. Thus, tetraspanins can play a role on CD9P-1 oligomerization status.     

The major CD9 and CD81 molecular partner. Identification and characterization of the complexes

By associating with specific partner molecules and with each other, the tetraspanins are thought to assemble multimolecular complexes that may be especially relevant with respect to metastasis. We have previously identified a 135-kDa molecule (CD9P-1) as a major molecular partner of CD9 in cancer cell lines. This molecule was identified, after immunoaffinity purification and mass spectrometry analysis, as the protein encoded by the KIAA1436 gene and the human ortholog of a rat protein known as FPRP. Cross-linking experiments detected a complex of the size of CD9 plus CD9P-1, showing that these glycoproteins directly associate with each other, probably in the absence of any other molecule. The use of chimeric CD9/CD82 molecules revealed the role of the second half of CD9, comprising the large extracellular loop and the fourth transmembrane domain. CD9P-1 was also shown to form separate complexes with CD81 and with an unidentified 175-kDa molecule. It also associated with other tetraspanins under conditions maintaining tetraspanin/tetraspanin interactions. The identification of a protein strongly linked to the tetraspanin web and the production of a specific monoclonal antibody will help to further characterize the role of this "web" under physiological and pathological conditions.     

Hepatitis C virus entry and the tetraspanin CD81

CD81, a member of the tetraspanin integral membrane protein family, has been identified as an essential receptor for HCV (hepatitis C virus). The present review highlights recent published data on the role that CD81 plays in HCV entry, including the importance of actin-dependent lateral diffusion of CD81 within the cell membrane, CD81 endocytosis and the CD81-Claudin-1 receptor complex in HCV internalization. Additional functions for CD81 in the viral life cycle and the role of HCV-CD81 interactions in HCV-induced B-cell and CNS (central nervous system) abnormalities are discussed.     

EWI‐2wint promotes CD81 clustering that abrogates Hepatitis C Virus entry

CD81 is a major receptor for Hepatitis C Virus (HCV). It belongs to the tetraspanin family whose members form dynamic clusters with numerous partner proteins and with one another, forming tetraspanin‐enriched areas in the plasma membrane. In our study, we combined single‐molecule microscopy and biochemistry experiments to investigate the clustering and membrane behaviour of CD81 in the context of cells expressing EWI‐2wint, a natural inhibitor of HCV entry. Interestingly, we found that EWI‐2wint reduces the global diffusion of CD81 molecules due to a decrease of the diffusion rate of mobile CD81molecules and an increase in the proportion of confined molecules. Indeed, we demonstrated that EWI‐2wint promotes CD81 clustering and confinement in CD81‐enriched areas. In addition, we showed that EWI‐2wint influences the colocalization of CD81 with Claudin‐1 – a co‐receptor required for HCV entry. Together, our results indicate that a change in membrane partitioning of CD81 occurs in the presence of EWI‐2wint. This study gives new insights on the mechanism by which HCV enters into its target cells, namely by exploiting the dynamic properties of CD81.     

The transferrin receptor and the tetraspanin web molecules CD9, CD81, and CD9P-1 are differentially sorted into exosomes after TPA treatment of K562 cells

Here we show that treatment of K562 cells with the phorbol ester TPA induces the down-modulation of various surface antigens. Among them, the transferrin receptor (TfR), the tetraspanin CD81, and a CD81-associated protein, CD9P-1, were unique in that their expression levels were lower after 24 h incubation than after 3 h. We demonstrated that like the TfR, CD81 was internalized at early times, and was less synthesized at latter times. Despite the association of a fraction of the TfR with CD81, these two molecules were subjected to different fates. TPA increased targeting of CD81 and CD9P-1 into exosomes but strongly reduced the localization of the TfR in these vesicles. Using this model we have shown that a fraction of CD81 and CD9P-1 in exosomes comes from a surface pool and that these molecules remain associated in exosomes. However, CD9P-1 could be targeted to exosomes in the absence of CD81 and of another tetraspanin, CD9. The targeting of CD9 into exosomes did not require palmitoylation of the protein. J. Cell. Biochem. 102: 650-664, 2007. (c) 2007 Wiley-Liss, Inc.     

In vivo gene silencing of CD81 by lentiviral expression of small interference RNAs suppresses cocaine-induced behaviour

The tetraspanin CD81 is induced in the mesolimbic dopaminergic pathway after cocaine administration. To further investigate its role, a regulatable lentivirus (Lenti-CD81) bearing the CD81 gene under the control of a tetracycline-inducible promoter and lentiviruses expressing short hairpin RNA (shRNA) targeted against CD81 (Lenti-CD81-shRNAs) have been prepared. Infection of HEK293T cells in vitro with Lenti-CD81-shRNAs resulted in 96.5% gene silencing (from quantitative real-time PCR(qRT-PCR) and immunocytochemistry). In vivo delivery of Lenti-CD81-shRNA into the nucleus accumbens or ventral tegmental area resulted in 91.3 and 94% silencing of endogenous CD81, respectively. Stereotaxic injection of Lenti-CD81 into these regions, resulting in CD81 overexpression, induced a four- to fivefold increase in locomotor activity after chronic cocaine administration, which returned to basal levels when Lenti-CD81-shRNA had been coinjected or when CD81 expression was blocked by doxycycline. Furthermore, silencing endogenous CD81 in vivo resulted in a significant decrease in locomotor activity over controls, again suppressing cocaine-induced behaviour.     

Single-step immunoaffinity purification and characterization of dodecylmaltoside-solubilized human neutrophil flavocytochrome b

Flavocytochrome b (Cyt b) is a heterodimeric, integral membrane protein that serves as the central component of an electron transferase system employed by phagocytes for elimination of bacterial and fungal pathogens. This report describes a rapid and efficient single-step purification of Cyt b from human neutrophil plasma membranes by solubilization in the nonionic detergent dodecylmaltoside (DDM) and immunoaffinity chromatography. A similar procedure for isolation of Cyt b directly from intact neutrophils by a combination of heparin and immunoaffinity chromatography is also presented. The stability of Cyt b was enhanced in DDM relative to previously employed solubilizing agents as determined by both monitoring the heme spectrum in crude membrane extracts and assaying resistance to proteolytic degradation following purification. Gel filtration chromatography and dynamic light scattering indicated that DDM maintains a predominantly monodisperse population of Cyt b following immunoaffinity purification. The high degree of purity obtained with this isolation procedure allowed for direct determination of a 2:1 heme to protein stoichiometry, confirming previous structural models. Analysis of the isolated heterodimer by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry allowed for accurate mass determination of p22(phox) as indicated by the gene sequence. Affinity-purified Cyt b was functionally reconstituted into artificial bilayers and demonstrated that catalytic activity of the protein was efficiently retained throughout the purification procedure.     

Transfected HEK293 Cells Expressing Functional Recombinant Intercellular Adhesion Molecule 1 (ICAM-1) – A Receptor Associated with Severe Plasmodium falciparum Malaria

Intercellular adhesion molecule 1 (ICAM-1) is a membrane-bound glycoprotein expressed on endothelial cells and cells of the immune system. Human ICAM-1 mediates adhesion and migration of leucocytes, and is implicated in inflammatory pathologies, autoimmune diseases and in many cancer processes. Additionally, ICAM-1 acts as receptor for pathogens like human rhinovirus and Plasmodium falciparum malaria parasites. A group of related P. falciparum erythrocyte membrane protein 1 (PfEMP1) domains, the DBLβ, mediates ICAM-1 binding of P. falciparum-infected erythrocytes. This ICAM‑1-binding phenotype has been suggested to be involved in the development of cerebral malaria. However, more studies identifying cross-reactive antibody and ICAM-1-binding epitopes and the establishment of a clinical link between DBLβ expression and e.g. cerebral malaria are needed before the DBLβ domains can be put forward as vaccine candidates and go into clinical trials. Such studies require availability of functional recombinant ICAM-1 in large quantities. In this study, we compared recombinant ICAM-1 expressed in HEK293 and COS-7 cells with mouse myeloma NS0 ICAM-1 purchased from a commercial vendor in terms of protein purity, yield, fold, ability to bind DBLβ, and relative cost. We present a HEK293 cell-based, high-yield expression and purification scheme for producing inexpensive, functional ICAM‑1. ICAM-1 expressed in HEK293 is applicable to malaria research and can also be useful in other research fields.     

Efficient expression screening of human membrane proteins in transiently transfected Human Embryonic Kidney 293S cells

It is often an immense challenge to overexpress human membrane proteins at levels sufficient for structural studies. The use of Human Embryonic Kidney 293 (HEK 293) cells to express full-length human membrane proteins is becoming increasingly common, since these cells provide a near-native protein folding and lipid environment. Nevertheless, the labor intensiveness and low yields of HEK 293 cells and other mammalian cell expression systems necessitate the screening for suitable expression as early as possible. Here we present our methodology used to generate constructs of human membrane proteins and to rapidly assess their suitability for overexpression using transiently transfected, glycosylation-deficient GnT I-HEK 293 cells (HEK 293S). Constructs, in the presence or absence of a C-terminal enhanced green fluorescence protein (EGFP) molecule, are made in a modular manner, allowing for the rapid generation of several combinations of fusion tags and gene paralogues/orthologues. Solubilization of HEK 293S cells, using a range of detergents, followed by Western blotting is performed to assess relative expression levels and to detect possible degradation products. Fluorescence-detection size exclusion chromatography (FSEC) is employed to assess expression levels and overall homogeneity of the membrane proteins, to rank different constructs for further downstream expression trials. Constructs identified as having high expression are instantly suitable for further downstream large scale transient expression trials and stable cell line generation. The method described is accessible to all laboratory scales and can be completed in approximately 3 weeks.     

Membrane-Associated RING-CH proteins associate with Bap31 and target CD81 and CD44 to lysosomes

Membrane-associated RING-CH (MARCH) proteins represent a family of transmembrane ubiquitin ligases modulating intracellular trafficking and turnover of transmembrane protein targets. While homologous proteins encoded by gamma-2 herpesviruses and leporipoxviruses have been studied extensively, limited information is available regarding the physiological targets of cellular MARCH proteins. To identify host cell proteins targeted by the human MARCH-VIII ubiquitin ligase we used stable isotope labeling of amino-acids in cell culture (SILAC) to monitor MARCH-dependent changes in the membrane proteomes of human fibroblasts. Unexpectedly, we observed that MARCH-VIII reduced the surface expression of Bap31, a chaperone that predominantly resides in the endoplasmic reticulum (ER). We demonstrate that Bap31 associates with the transmembrane domains of several MARCH proteins and controls intracellular transport of MARCH proteins. In addition, we observed that MARCH-VIII reduced the surface expression of the hyaluronic acid-receptor CD44 and both MARCH-VIII and MARCH-IV sequestered the tetraspanin CD81 in endo-lysosomal vesicles. Moreover, gene knockdown of MARCH-IV increased surface levels of endogenous CD81 suggesting a constitutive involvement of this family of ubiquitin ligases in the turnover of tetraspanins. Our data thus suggest a role of MARCH-VIII and MARCH-IV in the regulated turnover of CD81 and CD44, two ubiquitously expressed, multifunctional proteins.     

The tetraspanin CD81 regulates the expression of CD19 during B cell development in a postendoplasmic reticulum compartment

CD81 is a widely expressed tetraspanin that associates in B cells with CD19 in the CD19-CD21-CD81 signaling complex. CD81 is necessary for normal CD19 expression; cd81(-/-) B cells express lower levels of CD19, especially cd81(-/-) small pre-BII cells, which are almost devoid of surface CD19. The dependence of CD19 expression on CD81 is specific to this particular tetraspanin since cd9(-/-) B cells express normal levels of CD19. Furthermore, expression of human CD81 in mouse cd81(-/-) B cells restored surface CD19 to normal levels. Quantitative analysis of CD19 mRNA demonstrated normal levels, even in cd81(-/-) pre-BII cells. Analysis of CD19 at the protein level identified two CD19 glycoforms in both wild-type and cd81(-/-) B cells. The higher M(r) glycoform is significantly reduced in cd81(-/-) B cells and is endoglycosidase H (endo-H) resistant. In contrast, the low M(r) glycoform is comparably expressed in cd81(-/-) and in wild-type B cells and is endo-H sensitive. Because endo-H sensitivity is tightly correlated with endoplasmic reticulum localization, we suggest that the dependency of CD19 expression on CD81 occurs in a postendoplasmic reticulum compartment where CD81 is necessary for normal trafficking or for surface membrane stability of CD19.