Redox-regulated Export of the Major Histocompatibility Complex Class I-Peptide Complexes from the Endoplasmic Reticulum

In contrast to the fairly well-characterized mechanism of assembly of MHC class I-peptide complexes, the disassembly mechanism by which peptide-loaded MHC class I molecules are released from the peptide-loading complex and exit the endoplasmic reticulum (ER) is poorly understood. Optimal peptide binding by MHC class I molecules is assumed to be sufficient for triggering exit of peptide-filled MHC class I molecules from the ER. We now show that protein disulfide isomerase (PDI) controls MHC class I disassembly by regulating dissociation of the tapasin-ERp57 disulfide conjugate. PDI acts as a peptide-dependent molecular switch; in the peptide-bound state, it binds to tapasin and ERp57 and induces dissociation of the tapasin-ERp57 conjugate. In the peptide-free state, PDI is incompetent to bind to tapasin or ERp57 and fails to dissociate the tapasin-ERp57 conjugates, resulting in ER retention of MHC class I molecules. Thus, our results indicate that even after optimal peptide loading, MHC class I disassembly does not occur by default but, rather, is a regulated process involving PDI-mediated interactions within the peptide-loading complex.     

Cowpox virus exploits the endoplasmic reticulum retention pathway to inhibit MHC class I transport to the cell surface

Major histocompatibility complex (MHC) class I molecules assemble with peptides in the ER lumen and are transported via Golgi to the plasma membrane for recognition by T cells. Inhibiting MHC assembly, transport, and surface expression are common viral strategies of evading immune recognition. Cowpox virus, a clinically relevant orthopoxvirus, downregulates MHC class I expression on infected cells. However, the viral protein(s) and mechanisms responsible are unknown. We identify CPXV203 as a cowpox virus protein that associates with fully assembled MHC class I molecules and blocks their transport through the Golgi. A C-terminal KTEL motif in CPXV203 closely resembles the canonical ER retention motif KDEL and is required for CPXV203 function, indicating that a physiologic pathway is exploited to retain MHC class I in the ER. This viral mechanism for MHC class I downregulation may explain virulence differences between clinical isolates of orthopoxviruses.     

Human cytomegalovirus immediate early glycoprotein US3 retains MHC class I molecules by transient association

Human cytomegalovirus (HCMV) interferes with major histocompatibility complex (MHC) class I antigen presentation by a sequential multistep process to escape T cell surveillance. During the immediate early phase of infection, the glycoprotein US3 prevents intracellular transport of MHC class I molecules. Interestingly, US3 displays a significantly shorter half-life than US3-retained MHC class I molecules. Here we show that US3 associates only transiently with MHC class I molecules, exits the ER, and is inefficiently retrieved from the Golgi. US3 was degraded in a post-Golgi compartment, most likely lysosomes, because: i) Brefeldin A treatment prolonged the half-life of US3; and ii) US3 co-localized with the lysosomal marker protein LAMP in chloroquine-treated cells. In contrast, MHC class I molecules remained stable in the ER. Upon inhibition of protein synthesis MHC class I molecules were released suggesting that a continuous supply of newly synthesized US3 molecules is required for inhibition of transport. Thus, US3 does not seem to retain MHC class I molecules by a retrieval mechanism. Instead, our observations are consistent with US3 preventing MHC class I trafficking by blocking forward transport.     

Bap29/31 influences the intracellular traffic of MHC class I molecules

In this study, we examine the role of the putative cargo receptor B cell-associated protein (Bap)29/31 in the export of MHC class I molecules out of the endoplasmic reticulum (ER). We show that Bap31 binds to two allotypes of mouse class I molecules, with the interaction initiated at the time of H chain association with beta(2)-microglobulin and maintained until the class I molecule has left the ER. We also show that Bap31 is part of the peptide-loading complex, although is not required for its formation. Bap31 binds not only to class I molecules, but can bind to tapasin in the absence of class I. Consistent with an important role in recruiting class I molecules to transport vesicles, we show that in the absence of Bap29/31, there is a loss of class I colocalization with mSec31 (p137), a component of mammalian coat protein complex II coats. This observation is also associated with a delay in class I traffic from ER to Golgi. Our results are consistent with the view that class I molecules are largely recruited to ER exit sites by Bap29/31, and that Bap29/31 is a cargo receptor for MHC class I molecules.     

Structural and functional dissection of human cytomegalovirus US3 in binding major histocompatibility complex class I molecules

The human cytomegalovirus US3, an endoplasmic reticulum (ER)-resident transmembrane glycoprotein, forms a complex with major histocompatibility complex (MHC) class I molecules and retains them in the ER, thereby preventing cytolysis by cytotoxic T lymphocytes. To identify which parts of US3 confine the protein to the ER and which parts are responsible for the association with MHC class I molecules, we constructed truncated mutant and chimeric forms in which US3 domains were exchanged with corresponding domains of CD4 and analyzed them for their intracellular localization and the ability to associate with MHC class I molecules. All of the truncated mutant and chimeric proteins containing the luminal domain of US3 were retained in the ER, while replacement of the US3 luminal domain with that of CD4 led to cell surface expression of the chimera. Thus, the luminal domain of US3 was sufficient for ER retention. Immunolocalization of the US3 glycoprotein after nocodazole treatment and the observation that the carbohydrate moiety of the US3 glycoprotein was not modified by Golgi enzymes indicated that the ER localization of US3 involved true retention, without recycling through the Golgi. Unlike the ER retention signal, the ability to associate with MHC class I molecules required the transmembrane domain in addition to the luminal domain of US3. Direct interaction between US3 and MHC class I molecules could be demonstrated after in vitro translation by coimmunoprecipitation. Together, the present data indicate that the properties that allow US3 to be localized in the ER and bind MHC class I molecules are located in different parts of the molecule.     

The group II chaperonin TRiC protects proteolytic intermediates from degradation in the MHC class I antigen processing pathway

MHC class I molecules present precisely cleaved peptides of intracellular proteins on the cell surface. For most antigenic precursors, presentation requires transport of peptide fragments into the ER, but the nature of the cytoplasmic peptides and their chaperones is obscure. By tracking proteolytic intermediates in living cells, we show that intracellular proteolysis yields a mixture of antigenic peptides containing only N-terminal flanking residues for ER transport. Some of these peptides were bound to the group II chaperonin TRiC and were protected from degradation. Destabilization of TRiC by RNA interference inhibited the expression of peptide-loaded MHC I molecules on the cell surface. Thus, the TRiC chaperonin serves a function in protecting proteolytic intermediates in the MHC I antigen processing pathway.     

Prediction of intracellular targets of a small compound by analyzing peptides presented on MHC class I

In chemical biology, the elucidation of chemical target is crucial for successful drug development. Because MHC class I molecules present peptides from intracellular damaged proteins, it might be possible to identify targets of a chemical by analyzing peptide sequences on MHC class I. Therefore, we treated cells with the autophagy-inducing chemical TMD-457 and identified the peptides presented on MHC class I. Many of the peptides were derived from molecules involved in ER trafficking and ER stress, which were confirmed by morphological and biochemical analyses. Therefore, our results demonstrate that analyzing MHC class I peptides is useful for the detection of chemical targets.     

Traffic of proteins and peptides across membranes for immunosurveillance by CD8(+) T lymphocytes: a topological challenge

Cytotoxic CD8(+) T lymphocytes kill infected cells that display major histocompatibility complex (MHC) class I molecules presenting peptides processed from pathogen proteins. In general, the peptides are proteolytically processed from newly made endogenous antigens in the cytosol and require translocation to the endoplasmic reticulum (ER) for MHC class I loading. This last task is performed by the transporters associated with antigen processing (TAP). Sampling of suspicious pathogen-derived proteins reaches beyond the cytosol, and MHC class I loading can occur in other secretory or endosomal compartments besides the ER. Peptides processed from exogenous antigens can also be presented by MHC class I molecules to CD8(+) T lymphocytes, in this case requiring delivery from the extracellular medium to the processing and MHC class I loading compartments. The endogenous or exogenous antigen can be processed before or after its transport to the site of MHC class I loading. Therefore, mechanisms that allow the full-length protein or processed peptides to cross several subcellular membranes are essential. This review deals with the different intracellular pathways that allow the traffic of antigens to compartments proficient in processing and loading of MHC class I molecules for presentation to CD8(+) T lymphocytes and highlights the need to molecularly identify the transporters involved.     

A mouse cytomegalovirus glycoprotein, gp34, forms a complex with folded class I MHC molecules in the ER which is not retained but is transported to the cell surface.

Murine cytomegalovirus (MCMV) interferes with antigen presentation by means of retaining major histocompatibility complex (MHC) class I molecules in the endoplasmic reticulum (ER). Here we identify and characterize an MCMV-encoded glycoprotein, gp34, which tightly associates with properly conformed MHC class I molecules in the ER. Gp34 is synthesized in large quantities during MCMV infection and it leaves the ER only in association with MHC class I complexes. Many but not all class I molecules are retained in the ER during the early phase of MCMV infection, and we observe an inverse correlation between amounts of gp34 synthesized during the course of infection and class I retention. An MCMV deletion mutant lacking several genes, including the gene encoding gp34, shows increased class I retention. Thus, MCMV gp34 may counteract class I retention, perhaps to decrease susceptibility of infected cells to recognition by natural killer cells.     

Release from Endoplasmic Reticulum Matrix Proteins Controls Cell Surface Transport of MHC Class I Molecules

The anterograde transport of secretory proteins from the endoplasmic reticulum (ER) to the plasma membrane is a multi‐step process. Secretory proteins differ greatly in their transport rates to the cell surface, but the contribution of each individual step to this difference is poorly understood. Transport rates may be determined by protein folding, chaperone association in the ER, access to ER exit sites (ERES) and retrieval from the ER‐Golgi intermediate compartment or the cis‐Golgi to the ER. We have used a combination of folding and trafficking assays to identify the differential step in the cell surface transport of two natural allotypes of the murine major histocompatibility complex (MHC) class I peptide receptor, H‐2D^b and H‐2K^b. We find that a novel pre‐ER exit process that acts on the folded lumenal part of MHC class I molecules and that drastically limits their access to ERES accounts for the transport difference of the two allotypes. Our observations support a model in which the cell surface transport of MHC class I molecules and other type I transmembrane proteins is governed by the affinity of all their folding and maturation states to the proteins of the ER matrix.      

Anti-peptide antibody blocks peptide binding to MHC class I molecules in the endoplasmic reticulum

The finding that MHC class I molecules are physically associated with the TAP transporter has suggested that peptides may be directly transported into the binding groove of the class I molecules rather than into the lumen of the endoplasmic reticulum (ER) where they subsequently would encounter class I molecules by diffusion. Such a mechanism would protect peptides from peptidases in the ER and/or escaping back into the cytoplasm. However, we find that an anti-peptide Ab that is cotranslationally transported into the ER prevents TAP-transported peptides from being presented on class I molecules. The Ab only blocks the binding of its cognate peptide (SIINFEKL) but not other peptides (KVVRFKDL, ASNENMETM, and FAPGNYPAL). Therefore, most TAP-transported peptides must diffuse through the lumen of the ER before binding stably to MHC class I molecules.     

Functional dissection of HCMV US11 in mediating the degradation of MHC class I molecules

The human cytomegalovirus (HCMV) gene product US11 dislocates MHC I heavy chains from the endoplasmic reticulum (ER) and targets them for proteasomal degradation in the cytosol. To identify the structural and functional domains of US11 that mediate MHC class I molecule degradation, we constructed truncated mutants and chimeric proteins, and analyzed these to determine their intracellular localization and their ability to degrade MHC class I molecules. We found that only the luminal domain of US11 was essential to confer ER localization to the protein but that the ability to degrade MHC class I molecules required both the transmembrane domain and the luminal domain of US11. By analyzing a series of point mutants of the transmembrane domain, we were also able to identify Gln(192) and Gly(196) as being crucial for the functioning of US11, suggesting that these residues may play a critical role in interacting with the components of the protein degradation machinery.     

Selective export of HLA-F by its cytoplasmic tail

MHC class I molecules exit the endoplasmic reticulum (ER) by an unknown mechanism. Although a selective export mechanism has been proposed for the anterograde transport of class I, a motif responsible for export has never been identified. Although classical class I molecules lacking their cytoplasmic tail are expressed on the cell surface, we found that HLA-F was entirely dependent on its cytoplasmic tail for export from the ER. Two known export motifs were recognizable in HLA-F. A C-terminal valine residue functioned in ER export and interacted with coat complex (COP)II, while an RxR motif also played an important role in anterograde transport and bound to 14-3-3 proteins. This divergent trafficking of HLA-F implicates an alternative function for HLA-F, independent of loading with peptides in the ER.     

A cytomegalovirus glycoprotein re-routes MHC class I complexes to lysosomes for degradation

Mouse cytomegalovirus (MCMV) early gene expression interferes with the major histocompatibility complex class I (MHC class I) pathway of antigen presentation. Here we identify a 48 kDa type I transmembrane glycoprotein encoded by the MCMV early gene m06, which tightly binds to properly folded beta2-microglobulin (beta2m)-associated MHC class I molecules in the endoplasmic reticulum (ER). This association is mediated by the lumenal/transmembrane part of the protein. gp48-MHC class I complexes are transported out of the ER, pass the Golgi, but instead of being expressed on the cell surface, they are redirected to the endocytic route and rapidly degraded in a Lamp-1(+) compartment. As a result, m06-expressing cells are impaired in presenting antigenic peptides to CD8(+) T cells. The cytoplasmic tail of gp48 contains two di-leucine motifs. Mutation of the membrane-proximal di-leucine motif of gp48 restored surface expression of MHC class I, while mutation of the distal one had no effect. The results establish a novel viral mechanism for downregulation of MHC class I molecules by directly binding surface-destined MHC complexes and exploiting the cellular di-leucine sorting machinery for lysosomal degradation.     

MHC class II-restricted presentation of intracellular antigen.

An endogenously produced immunoglobulin light chain (lambda 2(315] is processed and presented to T cells in association with major histocompatibility complex (MHC) class II molecules. Using transfectants producing variant forms of lambda 2(315) that are neither expressed on the cell surface nor secreted, we demonstrate that intracellular lambda 2(315), which has never been exported outside of the cell, is the source of processed lambda 2(315) idiotype. This challenges the currently accepted paradigm that endogenous antigens are only presented by MHC class I molecules. Variants of lambda 2(315) protein that are retained in the endoplasmic recticulum (ER) are also presented. Variants that are expressed in the cytosol as well as those that are transported into the nucleus rather than the ER are not presented. Thus, the ER is likely to be the processing compartment.     

Calreticulin‐dependent recycling in the early secretory pathway mediates optimal peptide loading of MHC class I molecules

Calreticulin is a lectin chaperone of the endoplasmic reticulum (ER). In calreticulin‐deficient cells, major histocompatibility complex (MHC) class I molecules travel to the cell surface in association with a sub‐optimal peptide load. Here, we show that calreticulin exits the ER to accumulate in the ER–Golgi intermediate compartment (ERGIC) and the cis‐Golgi, together with sub‐optimally loaded class I molecules. Calreticulin that lacks its C‐terminal KDEL retrieval sequence assembles with the peptide‐loading complex but neither retrieves sub‐optimally loaded class I molecules from the cis‐Golgi to the ER, nor supports optimal peptide loading. Our study, to the best of our knowledge, demonstrates for the first time a functional role of intracellular transport in the optimal loading of MHC class I molecules with antigenic peptide.     

Association with BiP and aggregation of class II MHC molecules synthesized in the absence of invariant chain.

Class II molecules of the major histocompatibility complex (MHC) are composed of two polymorphic glycoprotein chains (alpha and beta), that associate in the ER with a third, non-polymorphic glycoprotein known as the invariant chain (Ii). We have examined the relationship between the intracellular transport and physico-chemical characteristics of various combinations of murine alpha, beta and Ii chains. Biochemical and morphological analyses of transfected fibroblasts expressing class II MHC chains show that both unassembled alpha and beta chains, as well as a large fraction of alpha+beta complexes synthesized in the absence of Ii chain, are retained in the ER in association with the immunoglobulin heavy chain binding protein, BiP. Analyses by sedimentation velocity on sucrose gradients show that most incompletely assembled class II MHC species exist as high molecular weight aggregates in both transfected fibroblasts and spleen cells from mice carrying a disruption of the Ii chain gene. This is in contrast to the sedimentation properties of alpha beta Ii complexes from normal mice, which migrate as discrete, stoichiometric complexes of M(r) approximately 200,000-300,000. These observations suggest that assembly with the Ii chain prevents accumulation of aggregated alpha and beta chains in the ER, which might relate to the known ability of the Ii chain to promote exit of class II MHC molecules from the ER.     

Association of tapasin and COPI provides a mechanism for the retrograde transport of major histocompatibility complex (MHC) class I molecules from the Golgi complex to the endoplasmic reticulum

Tapasin is a subunit of the transporter associated with antigen processing (TAP). It associates with the major histocompatibility complex (MHC) class I. We show that tapasin interacts with beta- and gamma-subunits of COPI coatomer. COPI retrieves membrane proteins from the Golgi network back to the endoplasmic reticulum (ER). The COPI subunit-associated tapasin also interacts with MHC class I molecules suggesting that tapasin acts as the cargo receptor for packing MHC class I molecules as cargo proteins into COPI-coated vesicles. In tapasin mutant cells, neither TAP nor MHC class I are detected in association with the COPI coatomer. Interestingly, tapasin-associated MHC class I molecules are antigenic peptide-receptive and detected in both the ER and the Golgi. Our data suggest that tapasin is required for the COPI vesicle-mediated retrograde transport of immature MHC class I molecules from the Golgi network to the ER.     

MHC class I molecules are incorporated into human herpesvirus‐6 viral particles and released into the extracellular environment

Human herpesvirus‐6 (HHV‐6), which belongs to the betaherpesvirus subfamily, mainly replicates in T lymphocytes. Here, we show that MHC class I molecules are incorporated into HHV‐6 viral particles and released into the extracellular environment. In addition, HHV‐6A/B‐infected T cells showed reduced surface and intracellular expression of MHC class I molecules. The cellular machinery responsible for molecular transport appears to be modified upon HHV‐6 infection, causing MHC class I molecules to be transported to virion assembly sites.     

Assembly and cell surface expression of TAP-independent,chloroquine-sensitive and interferon-gamma-inducible class I MHC complexes in transformed fibroblast cell lines are regulated by tapasin

Antigen processing and presentation by class I MHC molecules generally require assembly with peptide epitopes generated by the proteasome and transported into the ER by the transporters associated with antigen presentation (TAP). Recently, TAP-independent pathways supporting class I MHC-mediated presentation of exogenous antigens, as well as of endogenously synthesized viral antigens, were described. We now characterize a TAP-independent pathway that is operative in both TAP1- and TAP2-deficient Adenovirus (Ad)-transformed fibroblast cell lines. To the best of our knowledge, this is the first time that the existence of such a pathway has been described in non-infected cells that do not belong to the hematopoietic lineage. We show that this pathway is proteasome-independent and chloroquine-sensitive. Cell surface expression of these TAP-independent class I complexes is modulated by tapasin levels and is enhanced by IFN-gamma. The data imply that IFN-gamma increases the relative level of TAP-independent high affinity class I complexes that exit the ER on their way to the cell surface and to vacuolar compartments where peptide cleavage/exchange might take place before recycling to the cell surface. Since both TAP and tapasin expression are altered in numerous tumors and in virus-infected cells, TAP-independent class I complexes may be a valuable target source for immune responses.