Lysosomal membrane glycoprotein termed LGP85 or LIMP 11 extends a COON-terminal cytoplasmic tail of R459GQGSMDEGTADERAPLIRT478, in which an L475 1476 sequence lies as a di-leucine-based motif for lysosomal targeting. In the present study, we explored the role of the 1476 residue in the localization of LGP85 to the endocytic organelles using two substitution mutants called 1476A and 1476L in which alanine and leucine are replaced at 1476, respectively, and 1476R477T478-deleted LG-P85 called Delta 476-478. Immunofluorescence analyses showed that 1476A and 1476L are largely colocalized in intracellular organelles with an endogenous late endosomal and lysosomal marker, LAMP-1, but there were some granules in which staining for the LGP85 mutants was prominent, while Delta 476-478 is detected in LAMP-1-positive and LAMP-1-negative intracellular organelles, and on the cell surface. The subcellular fractionation studies revealed that 1476A, 1476L, and Delta 476-478 are different from wild-type LGP85 in the distribution of early endosomes, late endosomes, and lysosomes. 1476A and 1476L are present more in late endosomes than in the densest lysosomes, whereas wild-type LGP85 is mainly lysosomal. Substitution of 1476 for A and L differentially modified the ratios of late endosomal to lysosomal LGP85. A major portion of Delta 476-478 resided in the light buoyant density fraction containing plasma membrane and early endosomes. Taken together, these results indicate that the existence of the 476th amino acid residue is essential for localization of LGP85 to late endocytic compartments. The fact that isoleucine but not leucine is in the 476th position is especially of importance in the proper distribution of LGP85 in late endosomes and lysosomes. (C) 2002 Elsevier Science (USA). All rights reserved.