Figure 1. ERAAP Is Essential for Generating pMHC I on the Cell Surface from ER-Targeted N-Terminally Extended Precursors.

(A) The DNA constructs encoding the vector alone or the SHL8 (SIINFEHL) antigenic peptide downstream of an ER-targeting signal sequence (ES) were transiently transfected into fibroblasts derived from either TAP-deficient or ERAAP-TAP double-deficient mice. The constructs included either no (ES-[SHL8]) or five (ES-X5[SHL8], X5 = AIVMK) additional amino acids between the signal peptidase cleavage site of the ES-signal sequence and the SHL8 peptide. 2 days later, indicated number of transfected cells were incubated overnight with the lacZ-inducible, SHL8-K^b-specific B3Z T cell hybridoma. The lacZ activity was measured by the conversion of the substrate chlorophenolred-β-D-galacto-pyrannoside into chlorophenol red, which absorbs light at 595 nm.

(B) ER-targeted, N-terminally extended versions of the antigenic peptides SHL8, SVL9 (SSVVGVWYL), WI9 (WMHHNMDLI), or QL9 (QLSPFPFDL) were cotransfected with either vector alone or mouse ERAAP into ERAAP-TAP double-deficient fibroblasts. The cDNA encoding L^d was also included with the ES-X6[QL9] construct. 2 days later, varying number of transfected cells were used as APCs for the indicated T cells, and their lacZ response was measured as in (A). The MHC molecule presenting the peptides is shown in parentheses. Data are representative of at least three independent experiments.