Fig. 7. The type of tyrosine motif determines the lysosomal delivery of LAP and Lamp 1. BHK cells expressing the same two LAP reporter proteins, which were described in Fig. 6, containing the two different variants of the tyrosine motifs of LAP and Lamp 1 (Fig. 1) were incubated for 15 minutes (A and B) or one hour (C and D) at 37°C in the presence of antibodies against the lumenal domain of LAP. Subsequently, the cells were fixed and stained for LAP (green colour in all images) and the early endosomal marker EEA1 (red colour in A and B) or endogenous Lamp 1 (red colour in C and D). Changing the residues +2 and +3 in the tyrosine motif of LAP to those of Lamp 1 (LAP
7/TI) results in the efficient lysosomal delivery of the mutant within one hour (C) as indicated by the almost complete colocalisation with endogenous Lamp 1. Within 15 minutes of endocytosis, only a small fraction of the mutant can be detected in early endosomes (colocalization with EEA1 in A). Conversely, the substitution of the last two residues in the tyrosine motif of Lamp 1 for those of LAP results in endosomal retention of the mutant (colocalisation with EEA1 in B), which is also reflected by the small degree of colocalisation with endogenous Lamp 1 after one hour of endocytosis.
