The dileucine motif within the tail of MPR46 is required for sorting of the receptor in endosomes

The cytoplasmic tail of MPR46 carries a leucine-based motif that is required for the sorting of lysosomal enzymes by the receptor. In addition, it is one of three independent, but functionally redundant, internalization signals present in the cytoplasmic tail of MPR46, We have analyzed a mutant of MPR46, in which the dileucine pair was replaced by alanines (MPR46 LL/AA) with respect to its intracellular distribution and trafficking. Ultrastructural analysis of cells expressing the MPR46 LL/AA mutant revealed that the substitution of the dileucine pair causes a shift of the receptor distribution from the TGN, where it is packaged into AP1-containing vesicles, to vesicular structures distributed throughout the cytoplasm. The vesicles could be identified as early endosomes with internalized BSA-gold and rab5 as markers. By analyzing the receptor trafficking biochemically, we found that return of the LL/AA mutant receptor from the plasma membrane/endosome pool back to the TGN was impaired, while recycling from endosomes to the plasma membrane was enhanced. In conclusion, our data indicate that the dileucine motif in the MPR46 tail is required for a sorting event in endosomes.