Multiple residues in the transmembrane helix and connecting peptide of mouse tapasin stabilize the transporter associated with the antigen-processing TAP2 subunit

J Biol Chem. 2007 Mar 30;282(13):9401-9410. doi: 10.1074/jbc.M610429200. Epub 2007 Jan 23.

Abstract

The type I endoplasmic reticulum (ER) glycoprotein tapasin (Tpn) is essential for loading of major histocompatibility complex class I (MHC-I) molecules with an optimal spectrum of antigenic peptides and for stable expression of the heterodimeric, polytopic TAP peptide transporter. In a detailed mutational analysis, the transmembrane domain (TMD) and ER-luminal connecting peptide (CP) of mouse Tpn were analyzed for their capacity to stabilize the TAP2 subunit. Replacement of the TMD of Tpn by TMDs from calnexin or the Tpn-related protein, respectively, completely abolished TAP2 stabilization after transfection of Tpn-deficient cells, whereas TMDs derived from distantly related Tpn molecules (chicken and fish) were functional. A detailed mutational analysis of the TMD and adjacent residues in the ER-luminal CP of mouse Tpn was performed to elucidate amino acids that control the stabilization of TAP2. Single amino acid substitutions, including a conserved Lys residue in the center of the putative TMD, did not affect TAP2 expression levels. Mutation of this Lys plus four additional residues, predicted to be neighbors in an assumed alpha-helical TMD arrangement, abrogated the TAP2-stabilizing capacity of Tpn. In the presence of a wild-type TMD, also the substitution of a highly conserved Glu residue in the CP of Tpn strongly affected TAP2 stabilization. Defective TAP2 stabilization resulted in impaired cell surface expression of MHC-I molecules. This study thus defines a novel, spatially arranged motif in the TMD of Tpn essential for stable expression of the TAP2 protein and a novel protein interaction mode involving an ER-luminal Glu residue close to the membrane.

Publication types

  • Comparative Study

MeSH terms

  • ATP Binding Cassette Transporter, Subfamily B, Member 3
  • ATP-Binding Cassette Transporters / chemistry
  • ATP-Binding Cassette Transporters / metabolism*
  • Amino Acid Sequence
  • Amino Acids / genetics
  • Amino Acids / physiology*
  • Animals
  • Antigen Presentation / immunology*
  • Cattle
  • Cell Line
  • Conserved Sequence
  • Dogs
  • Humans
  • Membrane Proteins / chemistry
  • Membrane Proteins / metabolism*
  • Membrane Transport Proteins / chemistry
  • Membrane Transport Proteins / genetics
  • Membrane Transport Proteins / physiology*
  • Mice
  • Molecular Sequence Data
  • Peptide Fragments / chemistry
  • Peptide Fragments / genetics
  • Peptide Fragments / physiology*
  • Protein Structure, Tertiary
  • Protein Subunits / chemistry
  • Protein Subunits / metabolism*
  • Rats

Substances

  • ATP Binding Cassette Transporter, Subfamily B, Member 3
  • ATP-Binding Cassette Transporters
  • Amino Acids
  • Membrane Proteins
  • Membrane Transport Proteins
  • Peptide Fragments
  • Protein Subunits
  • Tap2 protein, mouse
  • tapasin