Short communicationCytoplasmic domains of the transporter associated with antigen processing and P-glycoprotein interact with subunits of the proteasome
Introduction
The proteasome generates peptides for Class I antigen presentation within the cytoplasm, and these peptides are translocated into the lumen of the endoplasmic reticulum (ER) by TAP (the transporter associated with antigen processing) (Groll et al., 1997, Townsend, 1993). The form of the proteasome primarily involved in antigen presentation (the immunoproteasome) contains three γ-interferon-inducible subunits, one of which (β-5i; also termed Lmp7) is interchangeable with the β-5 subunit (also designated X, MB1, or ɛ Akiyama et al., 1994, Gaczynska et al., 1996).
TAP is a heterodimer of two subunits, TAP1 and TAP2, and is a member of the ABC superfamily of transporters. As free antigenic peptides generated by the proteasome are not present at measurable levels in the cytoplasm (Rotzschke et al., 1990), a direct interaction between TAP and the proteasome has been predicted (Brooks et al., 2000, Cerundolo et al., 1997, Deverson et al., 1990, Glynne et al., 1991, Rock et al., 1994, Rotzschke et al., 1990, Spies et al., 1990, Trowsdale et al., 1990). Such an interaction would not only efficiently channel peptides directly to the ER, but would also protect the peptides from degradation within the cytoplasm. We adopted a genetic approach to this question, using the yeast two-hybrid system to ask whether the cytoplasmic domains of TAP1 and TAP2 interact directly with the β-5and/or β-5i proteasome subunits. We also explored interactions of the proteasome with P-glycoprotein (P-gp), another member of the ABC transporter family that can confer multidrug resistance on tumors and has been linked to antigen presentation (Pendse et al., 2003).
Section snippets
Gene fusions
The polymerase chain reaction (PCR) was used to amplify, from plasmid DNA, sequences encoding the appropriate proteasome subunits and large intracellular domains (nucleotide-binding domains; NBDs) of each ABC transporter. The amino acid sequences comprising each domain are given in Table 1. The oligonucleotide primers used for the PCR were designed to add restriction endonuclease recognition sequences to facilitate subsequent cloning. The β-5 and β-5i proteasome subunits, the cytoplasmic
Results
To determine whether interactions between the proteasome and TAP could be detected, interaction trap fusions to the large cytoplasmic, nucleotide-binding domain (NBD) of each TAP subunit were constructed. In-frame, activation domain (AD) protein fusions were generated between the cytoplasmic domains of TAP1 and TAP2 (Table 1 and Fig. 1) and the ‘acid-blob’ transcriptional activation domain. Similarly, the β-5 and β-5i proteasome subunits were fused, separately, to the LexA DNA-binding domain (
Discussion and conclusions
We have demonstrated specific interactions between the cytoplasmic domains of the transporter associated with antigen processing and subunits of the proteasome, the large protease complex that generates peptides for antigen presentation. Antigenic peptides are transported into the lumen of the ER by TAP, and direct interaction between the proteasome and TAP would facilitate this process.
Significantly, TAP was found to interact with both the β-5 subunit of the proteasome and β-5i, the subunit
Acknowledgements
We are grateful to Enzo Cerundolo, Alain Townsend, Uzi Gileadi, Richard Callaghan, Liz Simpson, and Julian Dyson for helpful discussions. The two-hybrid interaction reagents were the generous gift of Lauren Ha and Roger Brent. Beatrice Py, Ronjon Chakraverty, Leonard Woo, and Yeun Shan Li kindly provided two-hybrid fusion plasmids used for specificity controls. This work was supported by the Medical Research Council, Cancer Research Campaign and the Imperial Cancer Research Fund.
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