Three Tapasin Docking Sites in TAP Cooperate To Facilitate Transporter Stabilization and Heterodimerization

Abstract

The transporter associated with antigen processing (TAP) translocates peptide antigens into the lumen of the endoplasmic reticulum (ER) for loading onto major histocompatibility complex (MHC) class I molecules. MHC class I acquires its peptide cargo in the peptide loading complex (PLC), an oligomeric complex that the chaperone tapasin organizes by bridging TAP to MHC class I and recruiting accessory molecules such as ERp57 and calreticulin. Three tapasin binding sites on TAP have been described, two of which are located in the N-terminal domains (N domains) of TAP1 and TAP2. The third binding site is present in the core transmembrane domain (coreTMD) of TAP1 and is only used by the unassembled subunits. Tapasin is required to promote TAP stability, but through which binding site(s) it is acting is unknown. In particular the role of tapasin binding to the coreTMD of TAP1 single chains is mysterious as this interaction is lost upon TAP2 association. In this study, we map the respective binding site in TAP1 to the polar face of the amphipathic transmembrane helix TM9 and identify key residues that are essential to establish the interaction. We find that this interaction is dispensable for the peptide transport function but essential to achieve full stability of human TAP1. The interaction is also required for proper heterodimerization of the transporter. Based on similar results obtained using TAP mutants lacking tapasin binding to either N domain, we conclude that all three tapasin-binding sites in TAP cooperate to achieve high transporter stability and efficient heterodimerization.

Multiple Structural and Epigenetic Defects in the Human Leukocyte Antigen Class I Antigen Presentation Pathway in a Recurrent Metastatic Melanoma Following Immunotherapy

“…This result is different from those of previous studies that established the TAP-stabilizing function of tapasin using other cell line systems (12,(45)(46)(47). Several possibilities may explain this discrepancy.…”

Hydrophobic Interactions Are Key To Drive the Association of Tapasin with Peptide Transporter Subunit TAP2

“…7, only TAP2/ TPN complexes were highly sensitive to benzene, whereas TAP1/ TPN and 1DN/TPN complexes were not affected by the cyclic hydrocarbon (see TPN/TAP chain signal ratios for the different protein loads). Thus, consistent with a recent study (52), the transient complex formation between TPN and the core TMD of single TAP1 chains does not appear to be driven by hydrophobic interactions. We note that these results are also an excellent internal control for all of our above experiments, as they demonstrate that benzene selectively disrupts only hydrophobic interactions, even among identical binding partners (i.e., TPN binding to the N-domain of TAP1 is fully benzene sensitive; Fig.…”

“…The three TPN-docking sites in TAP have distinct biochemical requirements for TPN binding Single as well as assembled TAP2 chains interact with TPN exclusively via the N-domain (7), whereas single TAP1 subunits use a TPN-docking site in the core TMD (7, 52), which is lost upon TAP2 association (52). The respective TPN-binding site of core TAP1 is located in the polar face of the amphipathic TM9 and depends on charged and polar but not hydrophobic residues (52).…”

“…However, mutations of K408 do cause a failure to stabilize heterodimeric TAP and, as a consequence, abrogate stable PLC formation (61). A possible explanation is that K408 of TPN is required for the transient interaction with the core TMD (not the N-domain) of TAP1 during the biogenesis of functional TAP heterodimers (52). This particular interaction was recently investigated and shown to require polar residues, and is thus probably very different in nature than the TPN association with the TAP N-domains examined in the present study (52).…”

“…A possible explanation is that K408 of TPN is required for the transient interaction with the core TMD (not the N-domain) of TAP1 during the biogenesis of functional TAP heterodimers (52). This particular interaction was recently investigated and shown to require polar residues, and is thus probably very different in nature than the TPN association with the TAP N-domains examined in the present study (52). A scenario in which TPN binding to the N-domains and to the core TMD of single TAP1 has different biochemical requirements is further supported by their differential sensitivity to benzene (Figs.…”