©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Association of the Invariant Chain with Major Histocompatibility Complex Class I Molecules Directs Trafficking to Endocytic Compartments (*)

(Received for publication, August 26, 1994; and in revised form, October 19, 1994)

Masahiko Sugita (§) Michael B. Brenner (¶)

From the Lymphocyte Biology Section, Department of Rheumatology and Immunology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Major histocompatibility complex (MHC) class I and class II molecules have been shown to present peptides of different origin to alphabeta T cells. Most peptides presented by class I molecules are derived from endogenously synthesized proteins, whereas most peptides presented by class II molecules are from exogenous sources. This functional dichotomy can largely be achieved by the preferential intracellular association of the invariant chain (Ii) with MHC class II molecules, which may inhibit binding of endogenous peptides to class II molecules and direct them to endocytic compartments where extracellularly derived peptides can be sampled. Here, we show that Ii also can associate with a subset of MHC class I molecules and direct them to endocytic compartments. Ii was coprecipitated with class I molecules after lysis of human lymphocytes in mild detergent such as 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid or digitonin, and the association was more clearly visualized by the use of dithiobis[succinimidylpropionate], a homobifunctional chemical cross-linker. The class IbulletIi complex was reconstituted in Ii negative cells by transfection of corresponding cDNA clones and was found to be transported through the Golgi to acidic endocytic compartments. These observations may explain how some exogenous antigens can be presented by MHC class I molecules and how MHC class II molecules can bind self peptides derived from MHC class I molecules in endocytic compartments.


INTRODUCTION

Major histocompatibility complex (MHC) (^1)class I and class II molecules are integral membrane glycoproteins whose primary function is to present bound antigenic peptides to T cells expressing alphabeta T cell receptor (1) . MHC class I molecules bind peptides of intracellular origin, such as those derived from endogenously synthesized viral proteins, and present them to CD8 T cells, whereas MHC class II molecules bind exogenous antigens and present them to CD4 T cells(2, 3) . This functional difference is believed to be explained by the capability of class II molecules to bind intracellularly the invariant chain (Ii), which directs them to endocytic vesicles where they encounter exogenously derived peptides(4, 5, 6) .

Ii is a nonpolymorphic type II transmembrane glycoprotein that exists in humans in four forms, designated according to their molecular sizes as p31, p33, p41, and p43 (note that p31 and p33 are referred to as p33 and p35, respectively, by some authors)(7, 8) . These different forms of Ii are generated by the use of alternative translation initiation codons and alternative mRNA splicing(9) . Amino acid residues 12-15 (measured from the Ii p31 N-terminal cytoplasmic tail), which are shared among all the four forms of Ii, have been identified as critical residues for endosomal targeting(10) .

Assembly of MHC class II alpha and beta chains with Ii occurs in the endoplasmic reticulum (ER). Newly synthesized Ii associates as a trimer with calnexin(11) , an ER resident molecular chaperone(12, 13, 14) . Newly synthesized class II alpha and beta chains also bind to calnexin and dissociate when a complete nonamer complex, containing three alphabeta dimers and an Ii trimer, is formed(11) . Subsequently, the class IIbulletIi complex exits the ER and is transported through the Golgi to a specialized endocytic compartment, distinct from early/late endosomes or dense lysosomes, where Ii is proteolytically cleaved and class II molecules acquire antigenic peptides of exogenous origin(15, 16, 17, 18, 19) . Following dissociation of Ii and binding of peptides, class II molecules are transported to the cell surface for recognition by CD4 T cells(20) .

Newly synthesized class I heavy chains also bind to calnexin during their biosynthesis and assembly(21, 22) . We recently demonstrated in human cells that, following association with beta(2)m, class I heavy chains dissociate from calnexin and subsequently bind peptides of endogenous origin(22) . These peptides are transported from the cytoplasm by TAP molecules, and their association with nascent class I chains in the ER might be facilitated by physical association of the class I heavy chainbulletbeta(2)m heterodimer with TAP molecules (23) . Fully assembled class I complexes leave the ER and are transported through the Golgi to the cell surface without intersecting the endocytic route(24) . Segregation of class I and class II takes place in the trans-Golgi reticulum, where class I molecules traffic to the plasma membrane while class II molecules are sorted to endocytic compartments(15) . Thus, the different origin of peptides presented by class I and class II molecules may reflect different intracellular pathways through which these molecules traffic in the cell, which apparently result from the ability of Ii to direct class II molecules away from the default secretory pathway by virtue of endosomal targeting/retention signals(25, 26, 27) .

Accumulating evidence has shown that the dichotomy in presentation of antigen from endogenous and exogenous origin is not absolute. Some endogenous antigens can be presented by class II molecules(28, 29, 30) , whereas exogenous antigens are in some cases presented by class I molecules(31, 32, 33, 34) . The exact mechanisms accounting for these observations are not well understood. In this paper, we show that Ii can associate with a subset of class I molecules and direct them into acidic endosomal compartments. This observation suggests a mechanism that could explain how exogenous antigens can be presented by class I molecules and how self class I-derived peptides gain access to endosomal compartments for binding to class II molecules.


EXPERIMENTAL PROCEDURES

Cells

The human T times B hybrid T1 and T2 cell lines (35) were cultured in RPMI 1640 (Life Technologies, Inc.) supplemented with 10% bovine calf serum (Hyclone, Logan, UT). The human epithelial cell line, HeLa(5) , was grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% bovine calf serum.

Antibodies

Monoclonal antibodies (mAbs) W6/32 (specific for beta(2)m-associated HLA class I heavy chain)(36) , ME.1 (specific for HLA-B27)(37) , BBM.1 (specific for human beta(2)m)(38) , L243 (specific for HLA-DR)(39) , and P3 (negative control) (40) were obtained from the American Type Culture Collection. mAb HC10, which recognizes beta(2)m-unassociated HLA class I heavy chain(41) , was provided by Dr. Hidde Ploegh (Massachusetts Institute of Technology, Cambridge, MA). Anti-human Ii mAb PIN.1 (42) was a generous gift from Dr. Peter Cresswell (Yale University, New Haven, CT). Anti-human calnexin antibody, AF8, was generated in our laboratory(12) . mAb UPC10, a negative control antibody, was purchased from Sigma.

Plasmids and Transfection

Human Ii p31 cDNA in expression vector pCMU4 (5) was a kind gift from Dr. Per Peterson (The Scripps Research Institute, La Jolla, CA). HLA-B2705 cDNA as a 1.1-kilobase SalI-HindIII insert in phagemid pT7T3-18H was provided by Dr. William Biddison (National Institutes of Health, Bethesda, MD). A SalI-HindIII fragment of HLA-B27 cDNA was first inserted into pBluescript SK to generate a SalI-XbaI fragment, which was then subcloned into pSRalpha-neo(43) . Following transfection of HeLa cells with HLA-B27 cDNA in pSRalpha-neo either with pCMU4 vector or with Ii p31 cDNA in pCMU4 by the calcium phosphate precipitation method(44) , HLA-B27-positive cells with or without Ii p31 expression were cloned from the G418 (0.5 mg/ml, Sigma)-resistant cell population.

Metabolic Labeling, Immunoprecipitation, and Electrophoresis

Metabolic labeling of cells with [S]methionine was performed as described(22) . Radiolabeled cells were lysed in 0.3% CHAPS in phosphate-buffered saline, pH 8, either in the presence or absence of 0.1 mM dithiobis[succinimidylpropionate] (DSP) (Pierce) and incubated on ice for 30 min. Chemical cross-linking was quenched by the addition of 10 mM glycine. The nuclei were removed by centrifugation, and the lysates were precleared overnight at 4 °C with Staphylococcusaureus Cowan strain I (Pansorbin, Calbiochem, La Jolla, CA). Immunoprecipitation was performed with indicated mAbs, followed by incubation with protein A-Sepharose CL-4B (Pharmacia Biotech, Inc.). Pellets were washed 5 times with 0.3% CHAPS in Tris-buffered saline, resuspended in 1 times sample buffer containing 1.5% SDS and 5% 2-mercaptoethanol, and boiled for 5 min to cleave DSP. The samples were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) under reducing conditions. The gels were fluorographed using Me(2)SO/2,5-diphenyloxazole, dried, and exposed to Kodak XAR film. For peptide mapping, the gel slices containing Ii p31 after separation on an SDS-PAGE gel were overlaid with 0.5 µg of V8 protease (Promega, Madison, WI) and were electrophoresed on a 15% SDS-PAGE gel. Endoglycosidase H (endo H) digestion was performed as described(45) .

Immunofluorescence Labeling

HeLa transfectant cells grown on glass coverslips were fixed in 3.7% paraformaldehyde in phosphate-buffered saline for 10 min, permeabilized in 0.1% digitonin (Aldrich), and incubated with mAb ME.1 followed by rhodamine B-conjugated goat anti-mouse antibodies (Tago, Burlingame, CA). After blocking free antigen binding sites of the goat antibodies with isotype-matched mouse control antibodies, cells were incubated with FITC-conjugated PIN.1 antibody. In some experiments, transfectant cells, incubated for 2 h with 1 mg/ml of Texas red-conjugated ovalbumin (Molecular Probes, Inc., Eugene, OR), were labeled with mAb ME.1 followed by detection with FITC-conjugated goat anti-mouse antibodies (Tago). The cells were viewed and photographed using a Nikon Optiphot-2 fluorescence microscope (Melville, NY) with FITC and rhodamine filter sets.


RESULTS

Association of Ii p31 with MHC Class I Molecules Demonstrated by Chemical Cross-linking

T2 is a mutant cell line with a homozygous deletion in the MHC on chromosome 6, including all functional class II genes and the TAP-1 and TAP-2 peptide transporter genes. Thus, T2 cells completely lack the expression of MHC class II molecules and have impaired assembly of class I molecules due to restricted availability of peptides in the ER resulting in reduced class I expression on the cell surface. Class I heavy chains that are assembled with beta(2)m were detected either by immunoprecipitation with conformation-dependent antibody, W6/32, or by coimmunoprecipitation with beta(2)m-specific BBM.1 antibody (Fig. 1, lanes6 and 4, respectively). We observed that mAb BBM.1 immunoprecipitated 12-kDa beta(2)m and 43-kDa class I heavy chain and coprecipitated a scant amount of 33-kDa protein barely detected on this gel exposure (see arrow, Fig. 1, lane4). This association was more clearly visualized by the use of the thiol-cleavable, bifunctional chemical cross-linker, DSP (Fig. 1, lane10). Radiolabeled protein that comigrated exactly with the 33-kDa protein was also detected by coimmunoprecipitation with the anti-calnexin AF8 antibody and with mAb HC10, which recognizes beta(2)m-unassociated class I heavy chains (see arrow, Fig. 1, lanes8 and 11, respectively).


Figure 1: Association of a 33-kDa protein with MHC class I molecules. T2 cells radiolabeled for 4 h with [S]methionine were lysed in 0.3% CHAPS in the absence (lanes1-6) or presence (lanes7-12) of 0.1 mM DSP. Immunoprecipitation was performed with mAbs P3 (control), AF8 (anti-calnexin), L243 (anti-HLA-DR), BBM.1 (anti-beta(2)m), HC10 (anti-class I heavy chain unassociated with beta(2)m) and W6/32 (anti-class I heavy chain associated with beta(2)m) followed by resolution on a 12% SDS-PAGE gel under reducing conditions. The positions of class I heavy chain (HC), beta(2)m, and 33-kDa protein (arrow) are indicated to the right. Molecular markers are shown to the left.



mAb PIN.1 is an anti-Ii antibody that was generated by immunization with a synthetic peptide corresponding to a sequence located in the cytoplasmic domain of all forms of Ii (residues 12-28, measured from the Ii p31 N terminus)(42) . Ii p31 is the most abundant form, and it was visualized prominently by immunoprecipitation with mAb PIN.1 (Fig. 2A, lane4). Importantly, the 33-kDa protein associated with class I (Fig. 2A, lanes2 and 3) or calnexin (lane1) comigrated with Ii p31. The identity of this 33-kDa protein with Ii p31 was confirmed by peptide mapping with V8 protease. The peptide map of either class I-associated or calnexin-associated 33-kDa protein was exactly the same as that of Ii p31 (Fig. 2B). Thus, we concluded that Ii p31 was associated with class I molecules in T2 cells.


Figure 2: Association of Ii p31 with MHC class I molecules in T2 cells. A, T2 cells radiolabeled for 4 h with [S]methionine were lysed in 0.3% CHAPS with 0.1 mM DSP. Immunoprecipitation was carried out with mAbs AF8 (anti-calnexin), BBM.1 (anti-beta2 m), HC10 (anti-class I heavy chain unassociated with beta2 m), and PIN.1 (anti-Ii), followed by resolution on a 12% SDS-PAGE gel under reducing conditions. The position of Ii p31 is indicated with an arrow. B, 33 kDa bands were cut out from each lane in A and subjected to digestion with V8 protease followed by resolution on a 15% SDS-PAGE gel.



To rule out the possibility that this novel association might be achieved only in class II negative, mutant T2 cells with impaired class I assembly, we examined wild type T1 cells. Unlike T2 cells, T1 cells have normal class I assembly, which was manifested by the appearance of abundant assembled class I heavy chains detected with mAb W6/32 (Fig. 3, lane4) and fewer beta(2)m-unassociated heavy chains detected with mAb HC10 (lane3). Importantly, mAb W6/32 coimmunoprecipitated a protein comigrating with Ii p31 (Fig. 3, lane4, shown with an arrow). The identity of this class I-associated protein to Ii p31 was confirmed by resolution on nonequilibrium pH gradient electrophoresis/SDS-PAGE two-dimensional gels (not shown). This association was also observed in a human B cell line, JY (data not shown). Thus, we concluded that the association of Ii p31 with class I molecules was not attributable to the abnormal class I assembly in T2 cells but was a more general phenomenon that also occurred in cells with normal class I assembly and class II expression.


Figure 3: Association of Ii p31 with MHC class I molecules in T1 cells. T1 cells radiolabeled for 4 h with [S]methionine were lysed in 0.3% CHAPS with 0.1 mM DSP. Immunoprecipitation was performed with mAbs UPC10 (control), BBM.1 (anti-beta2 m), HC10 (anti-class I heavy chain unassociated with beta2 m), W6/32 (anti-class I heavy chain associated with beta2 m), and PIN.1 (anti-Ii), followed by resolution on a 12% SDS-PAGE gel under reducing conditions. The position of Ii p31 is indicated with an arrow.



Reconstitution of Class IbulletIi p31 Association by Transfection of Ii p31 into Ii-negative Cells

In order to confirm the association of Ii p31 with class I molecules and to determine the intracellular transport and distribution of the complex, we reconstituted the association in HeLa cells, an Ii-negative cervical epithelial carcinoma cell line. HeLa cells were transfected with an HLA-B27 cDNA with or without an Ii p31 cDNA. After selection with G418, stable transfectant clones expressing either HLA-B27 alone (HeLaB27 mock) or both HLA-B27 and Ii p31 (HeLaB27Iip31) were obtained. No PIN.1-reactive material was immunoprecipitated from radiolabeled HeLaB27 mock cells (Fig. 4, lane5), and class I-associated 33-kDa protein was not detected in these cells (lanes2-4). In contrast, transfected Ii p31 was abundantly expressed in HeLaB27Iip31 cells (Fig. 4, lane10, shown at an arrow), and the association of Ii p31 with class I molecules could be demonstrated in these cells (lanes7-9, see arrow).


Figure 4: Reconstitution of class IbulletIi p31 complex in HeLa cells. Stable transfectant clones of HeLa expressing either HLA-B27 alone (HeLaB27 mock) or both HLA-B27 and Ii p31 (HeLaB27Iip31) were radiolabeled for 20 min with [S]methionine and lysed in 0.3% CHAPS with 0.1 mM DSP. Immunoprecipitation was carried out with mAbs L243 (anti-HLA-DR), BBM.1 (anti-beta2 m), HC10 (anti-class I heavy chain unassociated with beta2 m), W6/32 (anti-class I heavy chain associated with beta2 m), and PIN.1 (anti-Ii), followed by resolution on a 12% SDS-PAGE gel under reducing conditions. The positions of class I heavy chain (HC), beta(2)m, and Ii p31 (arrow) are indicated to the right.



Class IbulletIi p31 Complexes Exited the ER and Were Transported to Acidic Subcellular Compartments

The intracellular transport of the class IbulletIi p31 complex was analyzed by pulse-chase experiments in which the susceptibility of the Ii to endo H digestion was examined. These experiments were performed either in the presence or absence of concanamycin B, a vacuolar proton ATPase inhibitor(46) . Following a 10-min [S]methionine pulse labeling of HeLaB27Iip31 cells, endo H-sensitive (S) radiolabeled Ii p31 decreased during the chase period, and endo H-resistant (R) Ii p31 appeared after a chase of 60 min (Fig. 5A, upperpanel). This endo H-resistant form of Ii p31 disappeared after a chase of 240 min in the absence of concanamycin B, while in its presence, persistence of the endo H resistant species was observed (Fig. 5A, lowerpanel). This could be explained by concanamycin B-mediated, impaired acidification of vacuolar organelles such as endosomes(47) , where Ii is proteolytically degraded by proteases that require an acidic environment for their activity(48, 49, 50) . Thus, we reasoned that if the class IbulletIi p31 complex exits the ER and is transported to acidic compartments, class I molecules might remain associated with Ii p31 that acquires resistance to endo H, and the association should be prolonged in the presence of concanamycin B. To examine this possibility, immunoprecipitation with mAb W6/32, specific for assembled class I molecules, was performed at each chase time (Fig. 5B). This experiment revealed that class I molecules associated with an endo H-sensitive form of Ii p31 (approximately 33 kDa) during earlier chase periods, and this became an endo H-resistant form of Ii p31 (35 kDa) after a 1-h chase (Fig. 5B, lanes1-4). Moreover, prolonged association of class I molecules with the endo H-resistant species occurred in the presence of concanamycin B (Fig. 5B, lanes5-8). Thus, we concluded that, following assembly of class I molecules with Ii in the ER, the complex left the ER and was transported through the Golgi to acidic compartments in which acidification depended on the function of vacuolar proton ATPases.


Figure 5: Prolonged association of class I molecules with Ii p31 in the presence of concanamycin B. HeLaB27Iip31 cells were pulse labeled for 10 min with [S]methionine and chased in cold media either in the presence or absence of 10 nM concanamycin B (ConB). A, at each chase time, radiolabeled cells were lysed in 0.5% Triton X-100 in Tris-buffered saline without DSP. Ii p31 was immunoprecipitated with mAb PIN.1, and was subsequently subjected to digestion with endo H (+) or mock digested(-). The samples were resolved on a 12% SDS-PAGE gel under reducing conditions. Endo H resistant (R) and sensitive (S) species are indicated to the right. B, at each chase time, radiolabeled cells were lysed in 0.3% CHAPS with 0.1 mM DSP. Immunoprecipitation was performed with mAb W6/32, specific for class I heavy chain associated with beta(2)m, followed by resolution on a 12% SDS-PAGE gel under reducing conditions. The positions of class I heavy chain (HC), beta(2)m, and Ii p31 are shown to the right.



Colocalization of Class I with Ii p31 in an Endocytic Compartment

Immunofluorescence microscopy was carried out to determine the subcellular localization of class IbulletIi p31 complex. Staining of permeabilized HeLa transfectant cells expressing both HLA-B27 and Ii p31 (HeLaB27Iip31) with an HLA-B27-specific antibody revealed the presence of HLA-B27 in large vesicles as well as on the cell surface (Fig. 6A, left). In double-label experiments, the Ii-specific PIN.1 antibody also stained these vesicles (Fig. 6A, right), which indicated that HLA-B27 and Ii were colocalized in these intracellular compartments. These vesicles observed when Ii p31 is expressed in Ii-negative cells have been shown to correspond to endosomal compartments(5, 51) . We examined if these vesicular structures belonged to the endocytic system by incubating HeLaB27Iip31 cells in media containing Texas red-conjugated ovalbumin. Exogenously added ovalbumin is endocytosed into the cell and transported intracellularly via endocytic pathways so that endocytic compartments are labeled with Texas red(52) . With this method, the large vesicles were clearly visualized (Fig. 6B, right) and, importantly, were costained with the HLA-B27-specific antibody (Fig. 6B, left). Such a striking colocalization of HLA-B27 with endocytosed ovalbumin was not observed in Ii-negative HeLa cells (data not shown). Thus, we concluded that class IbulletIi p31 complexes were transported into endocytic compartments in these transfectant cells.


Figure 6: Transport of the class IbulletIi p31 complex to endocytic compartments. A, fixed and permeabilized HeLaB27Iip31 cells were first stained with the anti-Ii PIN.1 antibody, followed by rhodamine B-conjugated goat anti-mouse Ig antibodies. After blocking free antigen binding sites of the goat antibodies, cells were then incubated with FITC-conjugated ME.1 antibody specific for HLA-B27. The background staining with mAb P3 (control) was negligible (not shown). B, after incubation of HeLaB27Iip31 cells with Texas red-conjugated ovalbumin, cells were fixed and permeabilized for staining with mAb ME.1 followed by FITC-conjugated goat anti-mouse Ig antibodies. Texas red was visualized through rhodamine filter sets.




DISCUSSION

Using direct biochemical analyses of human lymphocytes (Fig. 1Fig. 2Fig. 3) and reconstitution by transfection in HeLa cells (Fig. 4), we have demonstrated an association between class I molecules and Ii. The association of class II molecules with Ii is fairly strong and is maintained in detergents such as 1% Triton X-100(11) . In contrast, the association of class I molecules with Ii was too weak at least in vitro to be maintained in 0.5% Triton X-100 (data not shown). We observed that a small amount of Ii was coimmunoprecipitated with class I molecules in milder detergents such as 0.3% CHAPS (Fig. 1) and 1% digitonin (not shown), and we succeeded in visualizing the association more clearly in a reproducible way by the use of low concentrations of the chemical cross-linker DSP (Fig. 1). Previously, mouse class I heavy chains were expressed artificially in human cells, and an association with human Ii was reported(53) . Here, we demonstrate the presence of class IbulletIi complexes under physiological conditions in untransfected human cell lines. Moreover, we show that the class IbulletIi complex remained assembled in vivo during transport from the ER, through the Golgi, to endocytic compartments, which was evidenced by pulse-chase experiments (Fig. 5) and immunofluorescence microscopy analysis (Fig. 6). Thus, the association of Ii with class I molecules is stable in vivo and results in sorting of this pool of class I molecules out of the direct secretory pathway.

What is the biological significance of this association? The colocalization of class IbulletIi complex with endocytosed ovalbumin leads one to consider the possible involvement of this complex in presentation of exogenous antigens by class I molecules. Several reports have shown that exogenous antigens can be presented by class I molecules to T cells both in vivo and in vitro(31, 32, 33, 34) . Recent evidence has shown that cells capable of this presentation are phagocytic macrophages, and indeed phagocytosis by these cells of exogenous antigens, such as bacteria or ovalbumin linked to beads, and transport of these antigens to endosomal compartments are critical for antigen presentation(33, 34) . We speculate that Ii may transport a subset of class I molecules, as well as class II molecules, to endocytic compartments, where Ii is cleaved and class I molecules acquire peptides derived from endocytosed exogenous antigens. Although a recently identified compartment where peptide loading onto class II molecules takes place does not contain class I molecules, a small amount of class I molecules are found in endosomes in a human B cell line, JY(15) , in which no internalization from the cell surface is detected for class I molecules(24) . Further studies are required to determine definitively the extent to which class I molecules (colocalized with Ii and with class II molecules) are present in endocytic compartments.

Another possible function of class IbulletIi complexes is to provide the source of self class I-derived peptides for binding to class II molecules. Analysis of naturally processed peptides bound to HLA-DR1 shows that self HLA-A2 derived peptides as well as those derived from Ii are found in association with DR1 molecules(54) , and indeed MHC class II molecules have been shown to present endogenous class I-derived peptides to cytotoxic T cells in a Brefeldin A-sensitive manner(29) . Given the limited internalization of class I molecules from the cell surface, Ii-mediated transport of class I molecules to endocytic compartments might be crucial to the supply of certain self antigens (such as MHC class I proteins) for presentation by class II molecules.

Finally, based on these results and previous studies from our laboratory(22) , we believe that calnexin may act as a molecular chaperone in the assembly of class IbulletIi complexes. Calnexin has been shown to play an important role in the assembly of both class I and class II molecules(11, 21, 22, 55) . In the case of class II molecules, calnexin binds newly synthesized alpha, beta and Ii chains and remains associated with assembling intermediate complexes until the complete nonamer, containing three alphabeta dimers and the Ii trimer, is formed (11) . In the case of class I assembly, we recently showed that newly synthesized class I heavy chains bind to calnexin and that association with beta(2)m triggers heavy chains to dissociate from calnexin(22) . Since the interaction between class I heavy chains and Ii was also observed in beta(2)m deficient Daudi cells (data not shown), we speculate that calnexin may mediate association of a subset of class I heavy chains with Ii, and that, following association with beta(2)m, class I heavy chainbulletbeta(2)mbulletIi complex may dissociate from calnexin. These findings potentially indicate stronger parallels between the assembly of class II molecules and a subset of class I molecules than previously have been recognized.


FOOTNOTES

*
This work was supported in part by grants from the National Institutes of Health (to M. B. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Partly supported by the Uehara Memorial Foundation and by the Japan Rheumatism Foundation. To whom all correspondence should be addressed: Seeley G. Mudd Bldg., Rm. 507, 250 Longwood Ave., Boston, MA 02115. Tel.: 617-432-0948; Fax: 617-432-2799.

Scholar of the Leukemia Society of America.

(^1)
The abbreviations used are: MHC, major histocompatibility complex; Ii, invariant chain; ER, endoplasmic reticulum; beta(2)m, beta(2)-microglobulin; mAb, monoclonal antibody; HLA, human leukocyte antigen; DSP, dithiobis[succinimidylpropionate]; PAGE, polyacrylamide gel electrophoresis; endo H, endoglycosidase H; FITC, fluorescein isothiocyanate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.


ACKNOWLEDGEMENTS

We thank Dr. Per Peterson for Ii p31 cDNA; Dr. William Biddison for HLA-B27 cDNA; Dr. Peter Cresswell for mAb PIN.1, T1, and T2 cells; and Dr. Hidde Ploegh for mAb HC10 and concanamycin B. We also thank Dr. Steven Porcelli for critically reading the manuscript.


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