[This web page was produced as an assignment for an undergraduate course at Davidson College.]

Transporter associated with Antigen Processing
(TAP1 and TAP2)


Fig. 1:
TAP function in the bigger picture. Used by permission.
Link to original site.

Introduction: TAP is a protein which plays a major role in the immune system by providing suitable peptides to assembling MHC-I molecules in the ER of eukaryotic cells. In an uninfected cell, cytosolic peptides are tagged with ubiquitin and degraded by in the Proteasome. Short peptide sequences (around 10 amino acids in length) are translocated by TAP into the ER where they bind to an MHC-I molecule for transport to the cell surface. All cells need to display MHC-I on their surface lest they be destroyed by the immune system. When a virus infects a cell, interferon gamma causes changes in proteasome and TAP expression leading to the binding of foreign peptides of viral origin to the MHC-I. Cytotoxic T-Cells recognize foreign peptide bound to the MHC-I on the cell surface and destroy infected cells to prevent the spread of this infection (Janeway et al., 1999).
To see how the TAP complex relates to overall MHC-I loading, check out Dr. Campbell's Molecular Movie of MHC-I loading.

Figure 2 shows schematically the role of TAP in immune function.


Fig. 2:
Courtesy of Stephen Man. 1998.
Human cellular immune responses against human papillomaviruses in cervical neoplasia. Expert Reviews in Molecular Medicine © Cambridge University Press
Used by permission, though the editor encourages you to
visit their site, and to see the animation on the same subject.

Specifics: TAP is a heterodimeric protein complex consisting of the 70kD TAP1 and 72kD TAP2 subunits, though other sizes have been reported (Ortmann et al., 1997). TAP1 and 2 have formerly borne the monikers HAM1/2 (in mice), PSF1/2 or RING4/11 (in humans) and MTP1/2 (in rats) (Van Kaer et al., 1992).
TAP is a member of the "ATP-binding cassette (ABC)" family of membrane translocators, a group which also includes:

(Tampé, R., Shepherd et al., 1993). Proteins in the ABC superfamily have a hydrophobic domain which spans the membrane six to eight times as well as a cytosolic nucleotide binding domain, while both domains are repeated twice, either in a single polypeptide or through dimerization (Shepherd et al., 1993). Additionally, the ABC transporters actively transport their substrates across a membrane(Shepherd et al., 1993).


Fig. 3:
Black arrows represent sites of translocation by ABC transporters in Eukaryotic/Prokaryotic cells.
Used with permission from Robert Tampé, Universitaet Marburg.
Link to original posting.

Location and structure: Investigators have found TAP to be present in the ER and cis-golgi organelles, though the localization mechanism is unknown. Interestingly, location does not seem to be dependant upon association of TAP1 with TAP2 because T2 cells expressing no TAP2 show the same expression of TAP1 as wt cells (Kleijmeer et al., 1992). Our current picture of TAP is based on the understanding that the cytosolic portion of TAP contains the ATP binding domain (Kleijmeer et al., 1992).


Fig. 4:
Distribution of GFP-tagged TAP1 in living cells. CLSM analysis shows a typical ER distribution 
with a large reticular membranous network in the cytoplasm and a pronounced nuclear envelope. 
(Permission has been requested to use this figure and its legend.
Link to original posting.)

Function: Since TAP is the primary cellular component which which delivers candidate pathogen antigen peptides from the site of their hydrolysis in the cytosol to the site of binding with the MHC-I molecule in the ER and cis-golgi, it would stand to reason that TAP would exercise selectivity in peptide translocation. This was indeed the finding of Shepherd et al. in 1992. They found ATP is required for peptide translocation, and that the optimal substrates were between 7 and 13 amino acids in length. Additionally, certain sequences were translocated much poorer when reversed, proving that translocation is sequence specific, and not just dependant on overall charge or hydrophobicity. A great deal of research has been performed more recently to elucidate the specific binding preferences for TAP, resulting in the finding that the N-terminal and C-terminal residues of short peptides are the most important determinants (Daniel et al., 1998). See http://www.bioinfo.de/isb/1998/01/0010/, (Table 1), for the peptide binding motif for human TAP, published in Brusic et al. 1999. One research team also found a correllation between sequence affinity for TAP and specific HLA proteins, suggesting that TAP co-evolved with HLA-B27, -A3 and -A24 (Brusic et al., 1999). Researchers may also be interested to know that a serachable database now exists, called MHCPEP, containg over 13,000 MHC binding peptide sequences.
The mechanism of peptide translocation is currently thought to be a two step process of peptide binding and ATP dependant translocation (Tampé, 1999).


Fig. 5: Peptide translocation is thought to occur in two two closely coupled stages, with only the second being ATP dependant.
Courtesy of Robert Tampé, Universitaet Marburg.
Link to original posting.

Anti-TAP1 affinity purification of cell contents preciptates a complex of proteins including Calnexin, TAP1, TAP2, Calreticulin, Tapasin and Class I HC, in equal quantities (Ortmann et al., 1997). Association of these molecules is required for proper peptide loading and cells defective in MHC-I association with TAP1 show greatly reduced surface expression of MHC-I. This has lead investigators to propose that interaction between MHC-I and TAP1 may allow peptides to load more or less directly into the binding cleft, before they are exposed to further degradtion or dilution in the ER (Grandea et al., 1995).

Drugs: The only drug known to directly inhibit the action of TAP is the Herpes-Simplex-Virus Protein ICP47 (Neumann, 1996). Such a viral adaptation is to be expected as there is selective pressure for any tactic which might prevent antigen presentation to cytotoxic T-cells. For details on the mechanism of ICP47/TAP interaction, see the doctoral thesis of L. Neumann (in German). Application of the cytokine interferon-gamma to cells in vitro is known to significantly upregulate the expression of TAP1 and TAP2, hence the assumption that this is a part of the IFN-gamma response in vivo (Fisk et al., 1994).

Knock out: Several cell lines have been developed which exhibit impaired TAP1 or TAP2 function, and have proved very useful in the investigation of TAP function. TAP disfunction removes the major source of peptide fragments for loading onto MHC-I molecules and thus greatly reduces surface levels of MHC-I (Van Kaer et al., 1992). Some research indicates that in TAP mutants at lower temperatures, MHC-I molecules can assemble without associated peptides and even appear at the surface, but are labile at 37° C, leading to low measured surface levels of MHC-I in TAP mutants (Ljunggren et al., 1990). In fact, TAP1 and TAP2 genes were originally identified by their ability to rescue MHC-I expression on the surface of mutant cell lines (Powis et al., 1991, Attaya et al., 1992).

 

Works Cited:
Attaya, M., Jameson, S., Martinez, C.K., Hermel, E., Aldrich, C., Forman, J., Lindahl, K.F., Bevan, M.J., Monaco, J. 1992. HAM-2 corrects the class I antigen-processing defect in RMA-S cells. Nature 355:647-649.

Brusic V., van Endert P., Zeleznikow J., Daniel S., Hammer J. and Petrovsky N. 1999. A neural network model approach to the study of human TAP transporter. In Silico Biology, 1(2):109-121. (also on web at: http://www.bioinfo.de/isb/1998/01/0010/).

Daniel S., Brusic V., Caillat-Zucman S., Petrovsky N., Harrison L., Riganelli D., Sinigaglia F., Gallazzi F., Hammer J. and van Endert P.M. 1998. Relationship between peptide selectivities of human transporters associated with antigen processing and HLA class I molecules. Journal of Immunology, 161(2):617-624.

Fisk, B., Ioannides, C., Aggarwal, S., Wharton, JT., O'Brian, C.A., Restifo, N., Glisson, B. 1994. Enhanced expression of HLA-A, B, C and inducibility of TAP-1, TAP-2 and HLA-A, B, C by interferon-gamma in a multidrug-resistant small cell lung cancer line. Lymphokine and Cytokine research 13(2):125-131.

Grandea, AG., Androlewicz, MJ., Athwal, RS, Geraghty, DE., Spies, T. 1995. Dependance of Peptide Binding by MHC class I molecules on their interaction with TAP. Science 270:105-107.

Janeway, Charles A., Travers, Paul, Walport, Mark, Capra, J. Donald. 1999. Immunobiology: the immune system in health and disease, 4th Edition. London: Current Biology. 116-135

Kleijmeer, MJ, Kelly, A., Geuze, HJ., Slot, JW., Townsend, A., Trowsdale, J. 1992. Location of MHC-encoded transporters in the endoplasmic reticulum and cis-golgi. Nature 357:342-357.

Ljunggren, H., Stam, N.J., Ohlen, C., Jeefjes, J.J., Hoglund, P., Heemels, M., Bastin, J., Schumacher, T.N.M., Townsend, A., Karre, K., Ploegh, H.l. 1990 Empty MHC class I molecules come out in the cold. Nature 346:476-480.

Neumann, L. Charakterisierung des viralen TAP-Inhibitors ICP47 und Entwicklung nicht-radioaktiver Untersuchungsmethoden <http://www.o4y.com/Katalog/Chemie/Biochemie/seite76.htm> Acessed 2000 Mar 3.

Ortmann, B., Copeman, J., Lehner, PJ, Sadasivan, B., Herberg, JA, Grandea, AG, Riddell, SR, Tampe, R., Spies, T., Trowsdale, J., Cresswell, P. 1997. A critical role for Tapasin in the assembly and function of multimeric MHC Class I-TAP complexes. Science 277: 1306-1309.

Powis, S.J., Townsend, A.R.M., Deverson, EV., Bastin, J., Butcher, G.W., Howard, JC. 1991 Restoration of antigen presentation to the mutant cell line RMA-S by an MHC-linked transporter. Nature 354:528-531.

Shepherd, J.C., Schumacher, T.N.M., Ashton-Rickardt, P.G., Imaeda, S. Ploegh, H.L., Janeway, C.A. 1993. TAP1-Dependant peptide translocation in vitro is ATP dependant and peptide selective. Cell 74:577-587.

Tampé, R. Overview of the Transporter associated with antigen presentation (TAP) <http://www.uni-marburg.de/biochem/tampe.htm> Accessed 2000 Mar 3.

Van Kaer, L., Ashton-Rickardt, P.G., Ploegh, H.L., Tonegawa, S. 1992 TAP1 mutant mice are deficient in antigen presentation, surface class I molecules and CD4-8+ T cells. Cell 71:1205-1214.

 

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