Functional cysteine-less subunits of the transporter associated with antigen processing (TAP1 and TAP2) by de novo gene assembly

Heintke, Susanne; Chen, Min; Ritz, Ulrike; Lankat-Buttgereit, Brigitte; Koch, Joachim; Abele, Rupert; Seliger, Barbara; Tampé, Robert

« Previous Next »FEBS Letters
Volume 533, Complete , Pages 42-46, 2 January 2003

Functional cysteine-less subunits of the transporter associated with antigen processing (TAP1 and TAP2) by de novo gene assembly

Edited by Irmgard Sinnig

Susanne Heintke
- Institut für Biochemie, Biozentrum, Johann Wolfgang Goethe-Universität Frankfurt, Marie Curie Str. 9, D-60439 Frankfurt/M, Germany
Min Chen
- Institut für Biochemie, Biozentrum, Johann Wolfgang Goethe-Universität Frankfurt, Marie Curie Str. 9, D-60439 Frankfurt/M, Germany
Ulrike Ritz
- III. Med. Klinik and Poliklinik, Johannes Gutenberg-Universität Klinikum Mainz, Langenbeckstr. 1, D-55131 Mainz, Germany
Brigitte Lankat-Buttgereit
- Institut für Physiologische Chemie, Philipps-Universität Marburg, Karl-von-Frisch-Str. 1, D-35043 Marburg, Germany
Joachim Koch
- Institut für Biochemie, Biozentrum, Johann Wolfgang Goethe-Universität Frankfurt, Marie Curie Str. 9, D-60439 Frankfurt/M, Germany
Rupert Abele
- Institut für Biochemie, Biozentrum, Johann Wolfgang Goethe-Universität Frankfurt, Marie Curie Str. 9, D-60439 Frankfurt/M, Germany
Barbara Seliger
- III. Med. Klinik and Poliklinik, Johannes Gutenberg-Universität Klinikum Mainz, Langenbeckstr. 1, D-55131 Mainz, Germany
Robert Tampé
- Institut für Biochemie, Biozentrum, Johann Wolfgang Goethe-Universität Frankfurt, Marie Curie Str. 9, D-60439 Frankfurt/M, Germany
- Corresponding author. Fax: (49)-69-798 29495

Received 17 October 2002; accepted 21 November 2002.

Abstract

Within the adaptive immune system the transporter associated with antigen processing (TAP) plays a pivotal role in loading of peptides onto major histocompatibility (MHC) class I molecules. As a central tool to investigate the structure and function of the TAP complex, we created cysteine-less human TAP subunits by de novo gene synthesis, replacing all 19 cysteines in TAP1 and TAP2. After expression in TAP-deficient human fibroblasts, cysteine-less TAP1 and TAP2 are functional with respect to adenosine triphosphate (ATP)-dependent peptide transport and inhibition by ICP47 from herpes simplex virus. Cysteine-less TAP1 and TAP2 restore maturation and intracellular trafficking of MHC class I molecules to the cell surface.

Keywords: Adenosine triphosphate-binding cassette transporter, Antigen processing, Cysteine-scanning mutagenesis, Membrane protein

Abbreviations: ECL, enhanced chemiluminescence, FACS, fluorescence-activated cell sorting, MHC, major histocompatibility complex, NBD, nucleotide-binding domain, TAP, transporter associated with antigen processing, TMD, transmembrane domain