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J. Biol. Chem., Vol. 281, Issue 26, 17545-17551, June 30, 2006

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

Kirstin Keusekotten, Ralf M. Leonhardt¹, Sarah Ehses, and Michael R. Knittler²

From the University of Cologne, Institute for Genetics, Zuelpicher Strasse 47, D-50674 Cologne, Germany and the Institute of Immunology, Friedrich-Loeffler-Institute, Federal Research Institute for Animal Health, Paul-Ehrlich-Strasse 28, D-72076 Tubingen, Germany

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.

Received for publication, March 13, 2006

^* This work was supported by Land Nordrhein-Westfalen through the University of Cologne and by the Deutsche Forschungsgemeinschaft (DFG Grants KN541-1 and SFB635/Project P7). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Section of Immunobiology, Howard Hughes Medical Inst., Yale University School of Medicine, New Haven, CT 06520-8011.

² To whom correspondence should be addressed: Inst. of Immunology, Friedrich-Loeffler-Inst., Federal Research Inst. for Animal Health, Paul Ehrlich Str. 28, D-72076 Tubingen, Germany. Tel.: 49-7071-967-210; Fax: 49-7071-967-303; E-mail: michael.knittler{at}fli.bund.de.

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