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. 2018 Nov 28;7:e40126. doi: 10.7554/eLife.40126

Figure 2. The TAPBPR K22-D35 loop is critical for peptide exchange.

(a) Typical peptide binding when cells gated for expressing high levels of surface TAPBPR were incubated with 10 nM YVVPFVAK*V peptide for 15 min at 37°C on IFNγ treated HeLaM-TAPBPRKO cells -/+expression of TAPBPRWT, TAPBPRØloop, TAPBPRL30G or TAPBPRØG30L. (b) Schematic representation of the experimental workflow used to compare the efficiency of peptide exchange by plasma membrane bound TAPBPRWT with the plasma membrane bound TAPBPR loop mutants. (c) Histograms show the level of dissociation of YVVPFVAK*V (YVV*) in the absence (blue line) and presence of 100 nM non-labelled competitor peptide YVVPFVAKV (YVV)(orange line) or EGVSKQSNG (ETVΔ2/9), a peptide in which the anchors which permit HLA-A*68:02 binding are mutated to produce a non-binding derivative (black line). Similar patterns of dissociation were found on cells incubated at 4°C demonstrating that the peptide exchange occurs directly on the cell surface (see Figure 2—figure supplement 1). (d-e) Graphs show the percentage of fluorescent peptide YVVPFVAK*V (YVV*) remaining in the presence of (d) 100 nM or (e) increasing concentrations of the non-labelled competitor peptide YVVPFVAKV as a percentage of the bound YVVPFVAK*V observed in the absence of competitor peptide from four independent experiments. Error bars show -/+SD.****p≤0.0001, ***p≤0.001 using unpaired two-tailed t-tests.

Figure 2.

Figure 2—figure supplement 1. Peptide Exchange at 4°C.

Figure 2—figure supplement 1.

To ensure peptide exchange was occurring directly on the surface MHC I rather than in intracellular vesicles, peptide exchange assays were performed on cells incubated at 4°C to inhibit membrane trafficking. HeLa-TAPBPRKO cells reconstituted with the panel of TAPBPR loop mutant were cooled to 4°C then incubated with 5 µM ETVSK*QSNV (ETV*) for 60 min at 4°C, followed by washing thoroughly in cold PBS to remove unbound peptide. Dissociation of the fluorescent peptide was subsequently monitored in the absence (blue line) or presence of 1 µM ETVSEQSNV (ETV) (orange line) for 60 min at 4°C.