Functional dissection of the transmembrane domains of the transporter associated with antigen processing (TAP)

The transporter associated with antigen processing (TAP1/2) translocates cytosolic peptides of proteasomal degradation into the endoplasmic reticulum (ER) lumen. A peptide-loading complex of tapasin, major histocompatibility complex class I, and several auxiliary factors is assembled at the transporter to optimize antigen display to cytotoxic T-lymphocytes at the cell surface. The heterodimeric TAP complex has unique N-terminal domains in addition to a 6 + 6-transmembrane segment core common to most ABC transporters. Here we provide direct evidence that this core TAP complex is sufficient for (i) ER targeting, (ii) heterodimeric assembly within the ER membrane, (iii) peptide binding, (iv) peptide transport, and (v) specific inhibition by the herpes simplex virus protein ICP47 and the human cytomegalovirus protein US6. We show for the first time that the translocation pore of the transporter is composed of the predicted TM-(5-10) of TAP1 and TM-(4-9) of TAP2. Moreover, we demonstrate that the N-terminal domains of TAP1 and TAP2 are essential for recruitment of tapasin, consequently mediating assembly of the macromolecular peptide-loading complex.     

The first N-terminal transmembrane helix of each subunit of the antigenic peptide transporter TAP is essential for independent tapasin binding

The heterodimeric ABC transporter TAP translocates proteasomal degradation products from the cytosol into the lumen of the endoplasmic reticulum, where these peptides are loaded onto MHC class I molecules by a macromolecular peptide-loading complex (PLC) and subsequently shuttled to the cell surface for inspection by cytotoxic T lymphocytes. Tapasin recruits, as a central adapter protein, other components of the PLC at the unique N-terminal domains of TAP. We found that the N-terminal domains of human TAP1 and TAP2 can independently bind to tapasin, thus providing two separate loading platforms for PLC assembly. Moreover, tapasin binding is dependent on the first N-terminal transmembrane helix of TAP1 and TAP2, demonstrating that these two helices contribute independently to the recruitment of tapasin and associated factors.     

Membrane topology and dimerization of the two subunits of the transporter associated with antigen processing reveal a three-domain structure

Presentation of peptides derived from cytosolic and nuclear proteins by MHC class I molecules requires their translocation across the membrane of the endoplasmic reticulum (ER) by a specialized ABC (ATP-binding cassette) transporter, TAP. To investigate the topology of the heterodimeric TAP complex, we constructed a set of C-terminal deletions for the TAP1 and TAP2 subunits. We identified eight and seven transmembrane (TM) segments for TAP1 and TAP2, respectively. TAP1 has both its N and C terminus in the cytoplasm, whereas TAP2 has its N terminus in the lumen of the ER. A putative TM pore consists of TM1-6 of TAP1 and, by analogy, TM1-5 of TAP2. Multiple ER-retention signals are present within this region, of which we positively identified TM1 of both TAP subunits. The N-terminal domain containing TM1-6 of TAP1 is sufficient for dimerization with TAP2. A second, independent dimerization domain, located between the putative pore and the nucleotide-binding cassette, lies within the cytoplasmic peptide-binding domains, which are anchored to the membrane via TM doublets 7/8 and 6/7 of TAP1 and TAP2, respectively. We present a model in which TAP is composed of three subdomains: a TM pore, a cytoplasmic peptide-binding pocket, and a nucleotide-binding domain.     

Modulation of the antigenic peptide transporter TAP by recombinant antibodies binding to the last five residues of TAP1

The transporter associated with antigen processing (TAP) plays a pivotal role in the major histocompatibility complex (MHC) class I mediated immune response against infected or malignantly transformed cells. It belongs to the ATP-binding cassette (ABC) superfamily and consists of TAP1 (ABCB2) and TAP2 (ABCB3), each of which possesses a transmembrane and a nucleotide-binding domain (NBD). Here we describe the generation of recombinant Fv and Fab antibody fragments to human TAP from a hybridoma cell line expressing the TAP1-specific monoclonal antibody mAb148.3. The epitope of the antibody was mapped to the very last five C-terminal amino acid residues of TAP1 on solid-supported peptide arrays. The recombinant antibody fragments were heterologously expressed in Escherichia coli and purified to homogeneity from periplasmic extracts by affinity chromatography. The monoclonal and recombinant antibodies bind with nanomolar affinity to the last five C-terminal amino acid residues of TAP1 as demonstrated by ELISA and surface plasmon resonance. Strikingly, the recombinant antibody fragments confer thermal stability to the heterodimeric TAP complex. At the same time TAP is arrested in a peptide transport incompetent conformation, although ATP and peptide binding to TAP are not affected. Based on our results we suggest that the C terminus of TAP1 modulates TAP function presumably as part of the dimer interface of the NBDs.     

Head-head/tail-tail relative orientation of the pore-forming domains of the heterodimeric ABC transporter TAP

BACKGROUND: The transporter associated with antigen processing (TAP) is a heterodimeric member of the large family of ABC transporters. The study of interactions between the subunits TAP1 and TAP2 can reveal the relative orientation of the transmembrane segments, which form a translocation pore for peptides. This is essential for understanding the architecture of TAP and other ABC transporters. RESULTS: The amino-terminal six transmembrane segments (TMs) of human TAP1, TAP1 (1-6), and the amino-terminal five TMs of TAP2, TAP2(1-5), are thought to constitute the pore of TAP. Two new approaches are used to define dimer interactions. We show that TM6 of TAP1 (1-6) is able to change topology post-translationally. This TM, along with a cytoplasmic tail, is translocated into the endoplasmic reticulum lumen, unless TAP2 is expressed. Coexpression of TM(4-5) of TAP2 stabilizes the topology of TAP1 (1-6), even when the TM1 of TAP1 is subsitituted with another sequence. This suggests that the carboxy-terminal TMs of the pore-forming domains TAP1 (1-6) and TAP2(1-5) interact. An alternative assay uses photobleaching in living cells using TAP1 (1-6) tagged with the green fluorescent protein (GFP). Coexpression with TAP2(1-5) results in reduced movement of the heterodimer within the endoplasmic reticulum membrane, as compared with the single TAP1 (1-6) molecule. In contrast, TAP2(1-4) has no effect on the mobility of TAP1 (1-6)-GFP, indicating the importance of TM5 of TAP2 for dimer formation. Also, TM1 of both TAP1 and TAP2 is essential for formation of a complex with low mobility. CONCLUSIONS: Dimerization of the pore-forming transmembrane domains of TAP1 (TM1-6) with its TAP2 counterpart (TM1-5) prevents the post-translational translocation of TM6 of TAP1 and results in a complex with reduced mobility within the endoplasmic reticulum membrane compared with the free subunit. These techniques are used to show that the pore-forming domains of TAP are aligned in a head-head/tail-tail orientation. This positions the following peptide-binding segments of the two TAP subunits to one side of the pore.     

Three-dimensional structure of transporter associated with antigen processing (TAP) obtained by single Particle image analysis

The transporter associated with antigen processing (TAP) is an ATP binding cassette transporter responsible for peptide translocation into the lumen of the endoplasmic reticulum for assembly with major histocompatibility complex class I molecules. Immunoaffinity-purified TAP particles comprising TAP1 and TAP2 polypeptides, and TAP2 particles alone were characterized after detergent solubilization and studied by electron microscopy. Projection structures of TAP1+2 particles reveal a molecule approximately 10 nm across with a deeply staining central region, whereas TAP2 molecules are smaller in projection. A three-dimensional structure of TAP reveals it is isolated as a single heterodimeric complex, with the TAP1 and TAP2 subunits combining to create a central 3-nm-diameter pocket on the predicted endoplasmic reticulum-lumenal side. Its structural similarity to other ABC transporters demonstrates a common tertiary structure for this diverse family of membrane proteins.     

Identification of domain boundaries within the N-termini of TAP1 and TAP2 and their importance in tapasin binding and tapasin-mediated increase in peptide loading of MHC class I

Before exit from the endoplasmic reticulum (ER), MHC class I molecules transiently associate with the transporter associated with antigen processing (TAP1/TAP2) in an interaction that is bridged by tapasin. TAP1 and TAP2 belong to the ATP-binding cassette (ABC) transporter family, and are necessary and sufficient for peptide translocation across the ER membrane during loading of MHC class I molecules. Most ABC transporters comprise a transmembrane region with six membrane-spanning helices. TAP1 and TAP2, however, contain additional N-terminal sequences whose functions may be linked to interactions with tapasin and MHC class I molecules. Upon expression and purification of human TAP1/TAP2 complexes from insect cells, proteolytic fragments were identified that result from cleavage at residues 131 and 88 of TAP1 and TAP2, respectively. N-Terminally truncated TAP variants lacking these segments retained the ability to bind peptide and nucleotide substrates at a level comparable to that of wild-type TAP. The truncated constructs were also capable of peptide translocation in vitro, although with reduced efficiency. In an insect cell-based assay that reconstituted the class I loading pathway, the truncated TAP variants promoted HLA-B*2705 processing to similar levels as wild-type TAP. However, correlating with the observed reduction in tapasin binding, the tapasin-mediated increase in processing of HLA-B*2705 and HLA-B*4402 was lower for the truncated TAP constructs relative to the wild type. Together, these studies indicate that N-terminal domains of TAP1 and TAP2 are important for tapasin binding and for optimal peptide loading onto MHC class I molecules.     

ABC transporters and immunity: mechanism of self-defense

The transporter associated with antigen processing (TAP) is a prototype of an asymmetric ATP-binding cassette (ABC) transporter, which uses ATP binding and hydrolysis to translocate peptides from the cytosol to the lumen of the endoplasmic reticulum (ER). Here, we review molecular details of peptide binding and ATP binding and hydrolysis as well as the resulting allosteric cross-talk between the nucleotide-binding domains and the transmembrane domains that drive translocation of the solute across the ER membrane. We also discuss the general molecular architecture of ABC transporters and demonstrate the importance of structural and functional studies for a better understanding of the role of the noncanonical site of asymmetric ABC transporters. Several aspects of peptide binding and specificity illustrate details of peptide translocation by TAP. Furthermore, this ABC transporter forms the central part of the major histocompatibility complex class I (MHC I) peptide-loading machinery. Hence, TAP is confronted with a number of viral factors, which prevent antigen translocation and MHC I loading in virally infected cells. We review how these viral factors have been used as molecular tools to decipher mechanistic aspects of solute translocation and discuss how they can help in the structural analysis of TAP.     

The human cytomegalovirus gene product US6 inhibits ATP binding by TAP

Human cytomegalovirus (HCMV) encodes several genes that disrupt the major histocompatibility complex (MHC) class I antigen presentation pathway. We recently described the HCMV-encoded US6 gene product, a 23 kDa endoplasmic reticulum (ER)-resident type I integral membrane protein that binds to the transporter associated with antigen processing (TAP), inhibits peptide translocation and prevents MHC class I assembly. The functional consequence of this inhibition is to prevent the cell surface expression of class I bound viral peptides and their recognition by HCMV-specific cytotoxic T cells. Here we describe a novel mechanism of action for US6. We demonstrate that US6 inhibits the binding of ATP by TAP1. This is a conformational effect, as the ER lumenal domain of US6 is sufficient to inhibit ATP binding by the cytosolic nucleotide binding domain of TAP1. US6 also stabilizes TAP at 37 degrees C and prevents conformational rearrangements induced by peptide binding. Our findings suggest that the association of US6 with TAP stabilizes a conformation in TAP1 that prevents ATP binding and subsequent peptide translocation.     

Biogenesis of functional antigenic peptide transporter TAP requires assembly of pre-existing TAP1 with newly synthesized TAP2

The transporter associated with antigen processing (TAP) is essential for the delivery of antigenic peptides from the cytosol into the endoplasmic reticulum (ER), where they are loaded onto major histocompatibility complex class I molecules. TAP is a heterodimeric transmembrane protein that comprises the homologous subunits TAP1 and TAP2. As for many other oligomeric protein complexes, which are synthesized in the ER, the process of subunit assembly is essential for TAP to attain a native functional state. Here, we have analyzed the individual requirements of TAP1 and TAP2 for the formation of a functional TAP complex. Unlike TAP1, TAP2 is very unstable when expressed in isolation. We show that heterodimerization of TAP subunits is required for maintaining a stable level of TAP2. By using an in vitro expression system we demonstrate that the biogenesis of functional TAP depends on the assembly of preexisting TAP1 with newly synthesized TAP2, but not vice versa. The pore forming core transmembrane domain (core TMD) of in vitro expressed TAP2 is necessary and sufficient to allow functional complex formation with pre-existing TAP1. We propose that the observed assembly mechanism of TAP protects newly synthesized TAP2 from rapid degradation and controls the number of transport active transporter molecules. Our findings open up new possibilities to investigate functional and structural properties of TAP and provide a powerful model system to address the biosynthetic assembly of oligomeric transmembrane proteins in the ER.     

Tapasin is required for efficient peptide binding to transporter associated with antigen processing

The transporter associated with antigen processing (TAP) binds peptides in its cytosolic part and subsequently translocates the peptides into the lumen of the endoplasmic reticulum (ER), where assembly of major histocompatibility complex (MHC) class I and peptide takes place. Tapasin is a subunit of the TAP complex and binds both to TAP1 and MHC class I. In the absence of tapasin, the assembly of MHC class I in the ER is impaired, and the surface expression is reduced. To clarify the function of tapasin in the processing of antigenic peptides, we studied the interaction of peptide and TAP, peptide transport across the membrane of the ER, and association of peptides with MHC class I molecules in the microsomes derived from tapasin mutant cell line 721.220, its sister cell line 721.221 expressing tapasin, and their HLA-A2 transfectants. The binding of peptides to TAP in tapasin mutant 721.220 cells was significantly diminished in comparison with 721.221 cells. Impaired peptide-TAP interaction resulted in a defective peptide transport in tapasin mutant 721.220 cells. Interestingly, despite the diminished peptide binding to TAP, the transport rate of TAP-associated peptides was not significantly altered in 721.220 cells. After transfection of tapasin cDNA into 721.220 cells, efficient peptide-TAP interaction was restored. Thus, we conclude that tapasin is required for efficient peptide-TAP interaction.     

An Annular Lipid Belt Is Essential for Allosteric Coupling and Viral Inhibition of the Antigen Translocation Complex TAP (Transporter Associated with Antigen Processing)

The transporter associated with antigen processing (TAP) constitutes a focal element in the adaptive immune response against infected or malignantly transformed cells. TAP shuttles proteasomal degradation products into the lumen of the endoplasmic reticulum for loading of major histocompatibility complex (MHC) class I molecules. Here, the heterodimeric TAP complex was purified and reconstituted in nanodiscs in defined stoichiometry. We demonstrate that a single heterodimeric core-TAP complex is active in peptide binding, which is tightly coupled to ATP hydrolysis. Notably, with increasing peptide length, the ATP turnover was gradually decreased, revealing that ATP hydrolysis is coupled to the movement of peptide through the ATP-binding cassette transporter. In addition, all-atom molecular dynamics simulations show that the observed 22 lipids are sufficient to form an annular belt surrounding the TAP complex. This lipid belt is essential for high affinity inhibition by the herpesvirus immune evasin ICP47. In conclusion, nanodiscs are a powerful approach to study the important role of lipids as well as the function, interaction, and modulation of the antigen translocation machinery.     

Catalytic site modifications of TAP1 and TAP2 and their functional consequences

The transporter associated with antigen processing (TAP), a member of the ATP binding cassette (ABC) family of transmembrane transporters, transports peptides across the endoplasmic reticulum membrane for assembly of major histocompatibility complex class I molecules. Two subunits, TAP1 and TAP2, are required for peptide transport, and ATP hydrolysis by TAP1.TAP2 complexes is important for transport activity. Two nucleotide binding sites are present in TAP1.TAP2 complexes. Compared with other ABC transporters, the first nucleotide binding site contains non-consensus catalytic site residues, including Asp(668) in the Walker B region of TAP1 (in place of a highly conserved glutamic acid), and Gln(701) in the switch region of TAP1 (in place of a highly conserved histidine). At the second nucleotide binding site, a glutamic acid (TAP2 Glu(632)) follows the Walker B motif, and the switch region contains a histidine (TAP2 His(661)). We found that alterations at Glu(632) and His(661) of TAP2 significantly reduced peptide translocation and/or TAP-induced major histocompatibility complex class I surface expression. Alterations of TAP1 Asp(668) alone or in combination with TAP1 Gln(701) had only small effects on TAP activity. Thus, the naturally occurring Asp(668) and Gln(701) alterations of TAP1 are likely to contribute to attenuated catalytic activity at the first nucleotide binding site (the TAP1 site) of TAP complexes. Due to its enhanced catalytic activity, the second nucleotide binding site (the TAP2 site) appears to be the main site driving peptide transport. A mechanistic model involving one main active site is likely to apply to other ABC transporters that have an asymmetric distribution of catalytic site residues within the two nucleotide binding sites.     

Function of the transport complex TAP in cellular immune recognition

The transporter associated with antigen processing (TAP) is essential for peptide loading onto major histocompatibility complex (MHC) class I molecules by translocating peptides into the endoplasmic reticulum. The MHC-encoded ABC transporter works in concert with the proteasome and MHC class I molecules for the antigen presentation on the cell surface for T cell recognition. TAP forms a heterodimer where each subunit consists of a hydrophilic nucleotide binding domain and a hydrophobic transmembrane domain. The transport mechanism is a multistep process composed of an ATP-independent peptide association step which induces a structural reorganization of the transport complex that may trigger the ATP-driven transport of the peptide into the endoplasmic reticulum lumen. By using combinatorial peptide libraries, the substrate selectivity and the recognition principle of TAP have been elucidated. TAP maximizes the degree of substrate diversity in combination with high substrate affinity. This ABC transporter is also unique as it is closely associated with chaperone-like proteins involved in bonding of the substrate onto MHC molecules. Most interestingly, virus-infected and malignant cells have developed strategies to escape immune surveillance by affecting TAP expression or function.     

Loss of a glycine in the alpha2 domain affects MHC peptide binding but not chaperone binding.

Prior to the binding of peptide in the endoplasmic reticulum (ER), the major histocompatibility complex (MHC) class I heavy chain associates with an assembly complex that includes the transporter associated with antigen processing (TAP). The proximity of a part of the MHC class I alpha2 domain alpha-helix to areas previously shown to influence assembly complex binding suggests that this region might also be involved in chaperone association. Position 151, found in this part of the alpha2 domain alpha-helix, has a side chain that points up, away from direct contact with peptide, and is occupied by a glycine in all murine MHC class I heavy chains. We found that substitution of this glycine in H-2L(d) with a histidine substantially increased the proportion of peptide-free forms, although TAP binding was not abrogated. Thus, interaction of the heavy chain with peptides, but not with the assembly complex, is influenced by this glycine.     

Structure of the ABC ATPase domain of human TAP1, the transporter associated with antigen processing

The transporter associated with antigen processing (TAP) is an ABC transporter formed of two subunits, TAP1 and TAP2, each of which has an N-terminal membrane-spanning domain and a C-terminal ABC ATPase domain. We report the structure of the C-terminal ABC ATPase domain of TAP1 (cTAP1) bound to ADP. cTAP1 forms an L-shaped molecule with two domains, a RecA-like domain and a small alpha-helical domain. The diphosphate group of ADP interacts with the P-loop as expected. Residues thought to be involved in gamma-phosphate binding and hydrolysis show flexibility in the ADP-bound state as evidenced by their high B-factors. Comparisons of cTAP1 with other ABC ATPases from the ABC transporter family as well as ABC ATPases involved in DNA maintenance and repair reveal key regions and residues specific to each family. Three ATPase subfamilies are identified which have distinct adenosine recognition motifs, as well as distinct subdomains that may be specific to the different functions of each subfamily. Differences between TAP1 and TAP2 in the nucleotide-binding site may be related to the observed asymmetry during peptide transport.     

Pseudomonas aeruginosa Cif Protein Enhances the Ubiquitination and Proteasomal Degradation of the Transporter Associated with Antigen Processing (TAP) and Reduces Major Histocompatibility Complex (MHC) Class I Antigen Presentation

Cif (PA2934), a bacterial virulence factor secreted in outer membrane vesicles by Pseudomonas aeruginosa, increases the ubiquitination and lysosomal degradation of some, but not all, plasma membrane ATP-binding cassette transporters (ABC), including the cystic fibrosis transmembrane conductance regulator and P-glycoprotein. The goal of this study was to determine whether Cif enhances the ubiquitination and degradation of the transporter associated with antigen processing (TAP1 and TAP2), members of the ABC transporter family that play an essential role in antigen presentation and intracellular pathogen clearance. Cif selectively increased the amount of ubiquitinated TAP1 and increased its degradation in the proteasome of human airway epithelial cells. This effect of Cif was mediated by reducing USP10 deubiquitinating activity, resulting in increased polyubiquitination and proteasomal degradation of TAP1. The reduction in TAP1 abundance decreased peptide antigen translocation into the endoplasmic reticulum, an effect that resulted in reduced antigen available to MHC class I molecules for presentation at the plasma membrane of airway epithelial cells and recognition by CD8⁺ T cells. Cif is the first bacterial factor identified that inhibits TAP function and MHC class I antigen presentation.     

Functional asymmetry of the ATP-binding-cassettes of the ABC transporter TAP is determined by intrinsic properties of the nucleotide binding domains.

The ATP-binding-cassette (ABC) transporter associated with antigen processing (TAP) delivers peptides into the ER. TAP consists of two polypeptides (TAP1 and TAP2) each with an N-terminal transmembrane (TMD) and a C-terminal nucleotide binding domain (NBD). The two highly homologous NBDs of TAP show different nucleotide binding specificites, and identical mutations in the domains can have different effects on peptide transport. We asked whether this functional asymmetry of the NBDs is an intrinsic property or is imposed by the TMDs to which they are linked. To investigate the functional interdependence of the TAP domains, we created various TAP variants in which TMDs and/or NBDs were exchanged. All TAP variants except those with two TMDs of TAP1 could assemble. The TMDs did not affect the different nucleotide binding properties of the NBDs. The TAP variant with switched NBDs showed active peptide transport while the variants with pairs of identical NBDs or TMDs were inactive. Although both types of TMDs and NBDs have to be present for peptide transport they do not have to be assorted as in wild-type TAP. Thus, TAP domains seem to preserve functional autonomy despite their fusion into single polypeptide chains. We propose that the two NBDs act as nonequivalent 'modules' that directly determine the functional asymmetry of the included ATP-binding-cassettes. This provides a new insight into the function of NBDs and opens up new possibilities to investigate the molecular mechanism of the 'NBD engine' in ABC transporters.     

Role of the carboxyl terminal stop transfer sequence of UGT1A6 membrane protein in ER targeting and translocation of upstream lumenal domain

We investigated the role of the stop transfer sequence of human UGT1A6 in ER assembly and enzyme activity. We found that this sequence was able to address and translocate the upstream lumenal domain into microsomal membranes in vitro co- and posttranslationally. The signal activity of this sequence was further demonstrated in HeLa cells by its ability to target and maintain the CD4 protein deleted from both the N-terminal signal peptide and C-terminal transmembrane domain into the ER. We showed that total or partial deletion of the stop transfer sequence of UGT1A6 severely impaired enzyme activity highlighting its importance in both membrane assembly and function.