The transporter associated with antigen processing, TAP

After the demonstration that proteins lacking signals for access to the secretory compartment could contribute peptide epitopes for presentation via class I molecules at the cell surface, the existence of an auxiliary molecule that could be involved in the transport of cytosolic peptides into the ER was proposed.

A first suggestion that transporter proteins existed was provided by studying the class I-deficient mutant cell line, RMA-S, which could present exogenous peptides but not cytosolic ones. A similar phenotype was observed for several other mutants with defects in class I assembly. It was proposed that they could be deficient in a specific mechanism for translocation of peptides from the

cytosol to the ER (Townsend et a l 1989). Furthermore, the majority of class I molecules expressed in the mutants .174 and T2 contained either no peptide, or a limited set of peptides derived from signal sequences that are cleaved in the ER (Wei

e t a l 1992, Arnold gf a/. 1992).

Analysis of the .174 hybrid with normal cells established that the deletion in the MHC on chromosome 6 of .174 was responsible for the defect in antigen presentation and the class I assembly defect (Salter et a l 1986, Erlich et a l 1986, Cerundolo et a l 1990). It was suggested that gene or genes encoding the peptide transport should exist within this deletion (Cerundolo et a l 1990).

In 1990 four laboratories localized genes in the MHC encoding proteins very similar to the ABC family of transporters (Spies et a l 1990, Trowsdale et a l

1990, Monaco et a l 1990, Deverson et a l 1990), The transporter genes were then called TAPI and TAP2, for transporters associated with antigen processing. The human genes were cloned by analysing the mutant .174 cell line which has a large deletion in the class II region of the MHC.

Immunoelectron microscopy analysis showed that TAPI is localized in the ER membrane with the ATP binding domain most probably oriented to the cytosol (Kleijmeer et a l 1992). The TAPI and TAP2 genes in humans are not highly polymorphic, and until now, 3 alleles of TAPI and 5 alleles of TAP2, have been identified (Powis et a l 1992a)^ However, the rat TAP2 locus is highly polymorphic, probably to compensate the low number of class I alleles in this species. In rat the TAP genes have probably evolved in a way to increase the diversity of peptides presented by the limited range of class I available.

In order to study the function of the TAP genes, mutant cell lines that lacked class I at the cell surface were transfected with either TAPI or TAP2 genes or both. The mutant line .134 was transfected with the TAPI gene, RMA-S was transfected with the TAP2 gene and .174 with both TAPI and TAP2 (Spies and DeMars 1991, Spies et a l 1992, Powis et a l 1991, Attaya et a l 1992, Kelly er a l

1992, Arnold et a l 1992, Momburg et a l 1992). In each case class I expression was restored to 50-100% of wild type, and the mutant phenotype was a result of mutations of either TA PI or TAP2. In addition, it was possible to reconstitute antigen presentation in the mutant cell with TAP genes from different species (Momburg et a l 1992, Powis et a l 1991). In the murine mutant RMA-S the TAP2 deficiency could be restored for presentation of influenza antigens to murine T cells by transfection with the rat TAP2 sequence (Powis et a l 1991).

The role of TAP in peptide transport was questioned by experiments showing the existence of ATP- and TAP-independent peptide transport into microsomal vesicles (Koppelman et al. 1992, Levy et al. 1991). Later, a number of studies described the development of in vitro peptide transport assays that showed that peptide translocation was ATP- and TAP- dependent (Dobberstein 1992, Shepherd erfl/. 1993, Neffjes etal. 1993, Androlewicz e r <2/. 1993)

Two further independent assays using lymphoblastoid cells permeabilized by streptolysin O showed accumulation of test peptides in the ER in an ATP- and TAP-dependent manner (Neefjes et al. 1993, Androlewicz et al. 1993, Shepperd et al.

1993). In the one assay the retention of peptides in the ER was achieved by including a consensus N-glycosylation site in the test peptide, enabling ER-translocated peptides to be recovered later on a ConA Sepharose column (Neefjes et al. 1993). In the second assay, peptide accumulation in the ER was observed by measuring peptides bound and retained by the nascent class I molecule of different cells (Androlewicz et al. 1993, Shepperd et al. 1993).

To analyse in detail the conditions for transport of peptides by TAP in

vitro assays were developed. Because class I molecules bind preferentially peptides

8-10 amino acids in length the possibility of TAP having any additional function by modifying peptides to be loaded in the ER, or if any additional proteolytic activity in the ER lumen would cleave peptides from longer precursors derived from the cytosol. In most assays the TAPs showed preference for the length of the peptide transported, which corresponded to the peptide length preferred by the class I molecule (Shepherd

et al. 1993, Androlewicz etal. 1993, Momburg etal. 1994b 1994c, Androlewicz and

Cresswell 1994a). Cells permeabilized with streptolysin O could transport iodinated 16-40 mer peptides, which ones were first proteolytically processed into shorter peptides prior their transportation (Momburg et al. 1994b).

It was recently proposed that peptides longer than 8-10 amino acids long could also be transported into the ER, and be loaded onto class I molecules more frequently than previously supposed. Loaded longer peptides can have the amino -or boxy-terminal amino acids located in their complementary pockets in the class I groove with the extra length accomwcdated by bulging (Collins et al. 1994). It is also possible that longer peptides do not have an ideal binding site and they have one or both termini left hanging loose (Collins et al. 1994, Urban et al. 1994). It has been shown that the identity of the peptide C-terminal residue essentially governs the species-specific substrate specificity of TAP (Neefjes et al 1995). Rat and Mouse u TAP alleles preferentially transport peptides with hydrophobic C-terminal residues.

No such selection was reported for human TAP or for rat TAP a allele (Momburg et al 1996).

To identify the peptide binding site in TAP proteins, photoactivable peptide cross-linkers were used, revealing that TAP has a combinatorial binding site that consists of both TAP chains (Androlewicz et al 1994b). It was also observed that peptides bind to TAPs in an ATP independent manner. ATP hydrolysis is necessary for peptide translocation. Expression of TAPI and TAP2 in insect cells has shown that they are the minimal components necessary for peptide translocation across the ER membrane, in conjunction with ATP binding to the COOH-terminal of murine TAPI and TAP2 (Meyer et al. 1994, van Endert et a l 1994, Wang et a l 1994).

It was shown that the TAP complex interacts with MHC molecules, and dissociation of the transporter from class I molecules coincides with exit of the complexes from the ER (Suh et a l 1994, Ortmann et a l 1994). However, it was not known if this association was direct or mediated by another protein. By studying a new mutant cell line 721.220, it was observed that the expressed functional class I and TAP molecules did not associate in the ER (Greenwood et a l 1994). This cell line showed a 80% reduction of class I surface expression that could be restored by introducing an unidentified 48kd MHC-linked gene, suggesting that acfeional proteins may be involved in the assembly of class I molecules (Grandea et a l 1995).