Figure 5. Isoleucine at position 337 (I₃₃₇) is required for accelerated internalization and lysosomal targeting.

(A and B) Expression of A2/C cytoplasmic tail point mutations. CEM T cells (negative control) or CEM T cells expressing the indicated MHC-I were pulse-labeled as described in Figure C, and analyzed by SDS PAGE. Bands were quantified using a phosphorimager and ImageQuant software. The results are displayed in part B as the means +/- SD from three experiments. (C and D) I₃₃₇ is necessary for reduced steady state cell surface expression of HA-A2/C. CEM T cell lines were generated expressing the indicated MHC-I. (C) CEM T cells expressing the indicated mutant were stained with an antibody directed against the HA-tag and analyzed by flow cytometry. The filled gray curve represents the expression of the indicated molecule while the filled black curve represents untagged cells stained with the HA antibody. (D) Quantitation of the HA-A2/C cytoplasmic tail mutant surface expression. The results are depicted as MFI +/- SD from three experiments. (E) I₃₃₇ is not required for slow export of HA-A2/C. A transport assay was performed as in Figure 3A, except cells were chased in biotin for only 1hr. The transport assay was quantified as in Figure 3B, except that percent of HA-A2 transported to the cell surface was set to 100%. The mean +/- standard deviation is shown for two experiments. (F) I₃₃₇ is necessary for accelerated internalization of HA-A2/C. A flow cytometric internalization assay was used as in Figure 3C to determine the percentage of each molecule remaining on the cell surface over time. The mean +/- SD for an experiment performed in triplicate is shown. (G and H) I₃₃₇ is necessary for lysosomal targeting of HA-A2/C. A pulse chase was performed as in Figure 4E and was quantified as in Figure 4F. The mean +/- SD is shown for two experiments.