Biogenesis of Functional Antigenic Peptide Transporter TAP Requires Assembly of Pre-existing TAP1 with Newly Synthesized TAP2

Abstract

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.The antigen processing machinery plays an important role in the cellular immune response of vertebrates for the identification of infected or malignantly transformed cells. Peptides derived from proteasomal degradation of intracellular proteins are translocated via the transporter associated with antigen processing (TAP) 3 into the ER lumen and loaded onto major histocompatibility complex class I molecules (1). Presentation of "nonself " peptides at the cell surface to cytotoxic T-cells triggers the elimination of the infected or transformed cell. Lack of TAP expression e.g. in tumor tissue is associated with a dramatic loss of surface major histocompatibility complex class I and consequently promotes strongly the evasion of malignant cells from a proper cellular immune response (2). The ATP-binding cassette (ABC) transporter TAP is a heterodimer that comprises the two homologous subunits, TAP1 and TAP2, each of which consists of a hydrophobic transmembrane domain (TMD) and a hydrophilic, highly conserved cytoplasmic nucleotide-binding domain (NBD). The TMDs of both TAP subunits contribute to the formation of the peptide-binding site and the translocation pore (1). The binding and hydrolysis of ATP is believed to power the transport process by inducing conformational changes in the NBDs. These structural changes alter the conformation of the TMDs and cause the binding and movement of peptides across the ER membrane. The TMDs of TAP1 and TAP2 have unique N-terminal domains (N-domains with three to f...

Structural arrangement of the transmission interface in the antigen ABC transport complex TAP

“…Single-cysteine TAP subunits, expressed alone, yielded a single band of 174 and 150 kDa for TAP1 and TAP2, respectively, indicating the formation of homodimeric complexes (34,35). These homodimers, which form when one subunit is in excess (35), are most likely misfolded and thus inactive in peptide binding and translocation (17,25). This finding is in line with the observation that single cysteines placed in each X-loop can be cross-linked in the homodimer but not in the heterodimer (data not shown).…”

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

“…The data presented in this paper also suggest a somewhat unconventional model whereby TAP1 ubiquitination and degradation do not result in a similar change in TAP2 abundance. However, previous studies have demonstrated that newly synthesized TAP2 requires TAP1 for appropriate folding and stability (43). Moreover, TAP2 is targeted for proteasomal degradation when it is expressed in the absence of TAP1 in T2 lymphoblastoid cells or TAP1 abundance is reduced in vitro (43).…”

“…However, previous studies have demonstrated that newly synthesized TAP2 requires TAP1 for appropriate folding and stability (43). Moreover, TAP2 is targeted for proteasomal degradation when it is expressed in the absence of TAP1 in T2 lymphoblastoid cells or TAP1 abundance is reduced in vitro (43). Conversely, introduction of TAP1 into TAP1-deficient melanoma cells markedly enhances TAP2 protein levels (44).…”

Specific Lipids Modulate the Transporter Associated with Antigen Processing (TAP)

“…In the case of the ABC transporter TAP, this is even more challenging because (i) TAP functions as a heterodimeric complex, (ii) preformed TAP1 serves as a scaffold for the folding of TAP2 (44), and (iii) solubilized TAP is highly unstable and prone to aggregate or to dissociate into its subunits (21). Earlier attempts of TAP expression in human cells (45), Saccharomyces cerevisiae (46), and insect cells (20) allowed the investigation of some important aspects of the antigen translocation machinery.…”