title: 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.
authors.affiliation: Institute for Genetics, University of Cologne, Zuelpicher Strasse 47, D-50674 Cologne, Germany.
authors.forename: Kirstin
authors.initials: K
authors.lastname: Keusekotten
authors.forename: Ralf M
authors.initials: RM
authors.lastname: Leonhardt
authors.forename: Sarah
authors.initials: S
authors.lastname: Ehses
authors.forename: Michael R
authors.initials: MR
authors.lastname: Knittler
mesh_terms: D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D000071450:ATP Binding Cassette Transporter, Subfamily B, Member 3; D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D017951:Antigen Presentation; D000941:Antigens / Q000378:metabolism*; D001692:Biological Transport / Q000502:physiology; D002460:Cell Line; D019281:Dimerization; D004721:Endoplasmic Reticulum / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D066298:In Vitro Techniques; D008214:Lymphocytes / Q000166:cytology; D008861:Microsomes / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D013329:Structure-Activity Relationship
chemical_list: D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D000071450:ATP Binding Cassette Transporter, Subfamily B, Member 3; D018528:ATP-Binding Cassette Transporters; D000941:Antigens; C486172:Tap1 protein, rat; C489514:Tap2 protein, rat; D046988:Proteasome Endopeptidase Complex