Two acidic amino acid residues, Asp(470) and Glu(471), contained in the carboxyl cytoplasmic tail of a major lysosomal membrane protein, LGP85/LIMP II, are important for its accumulation in secondary lysosomes

Lysosomal membrane glycoprotein termed LGP85 or LIMP II has a COOH-terminal cytoplasmic tail whose amino acid sequence is R(469)GQGSMDEGTA-DERAPLIRT(4 78). Two acidic amino acid residues, D-470, and E-471, in the cytoplasmic t ail of LGP85 are crucial for its binding to adaptor-like complex AP-3. In t he present study we investigated their role(s) in intracellular distributio ns of LGP85 using two alanine substitution mutants at D-470 and E-471 (defi ned as D470A and E471A, respectively). Immunofluorescence analysis showed t hat D470A and E471A are localized to endocytic organelles as well as wild-t ype LGP85. However, the subcellular fractionation study revealed that D470A and E471A are different from wild-type LGP85 in the distribution among ear ly endosomes, late endosomes, and lysosomes. A major portion of wild-type L GP85 existed in the densest lysosomal fraction. In contrast, a significant amount of D470A existed in the early endosomal fraction with a light buoyan t density, while less D470A resided in the lysosomal fraction. E471A broade ned from the early endosomal fraction to the lysosomal fraction without the high lysosomal peak. These findings indicate that the two acidic residues, D-470 and E-471, play an important role in regulation of LGP85 movement wi thin the endocytic pathway, which finally makes the highest concentration o f LGP85 in the dense secondary lysosomes. (C) 2000 Academic Press.