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(459)GQGSMDEGTADERAPLIRT(478). Two acidic amino acid residues, D(470) and E(471), in the cytoplasmic tail of LGP85 are crucial for its binding to adaptor-like complex AP-3. In the present study we investigated their role(s) in intracellular distributions of LGP85 using two alanine substitution mutants at D(470) and E(471) (defined as D470A and E471A, respectively). Immunofluorescence analysis showed that D470A and E471A are localized to endocytic organelles as well as wild-type LGP85. However, the subcellular fractionation study revealed that D470A and E471A are different from wild-type LGP85 in the distribution among early endosomes, late endosomes, and lysosomes. A major portion of wild-type LGP85 existed in the densest lysosomal fraction. In contrast, a significant amount of D470A existed in the early endosomal fraction with a light buoyant density, while less D470A resided in the lysosomal fraction. E471A broadened 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 within the endocytic pathway, which finally makes the highest concentration of LGP85 in the dense secondary lysosomes.
Tabuchi
N
N
Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Fukuyama, Hiroshima, 729-0292, Japan.
Akasaki
K
K
Tsuji
H
H
D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism*; D001483:Base Sequence; D018955:CD36 Antigens / Q000737:chemistry / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D017931:DNA Primers; D018698:Glutamic Acid / Q000378:metabolism*; D006801:Humans; D051907:Lysosomal Membrane Proteins; D008247:Lysosomes / Q000378:metabolism*; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D051116:Receptors, Scavenger; D012795:Sialoglycoproteins; D013347:Subcellular Fractions / Q000378:metabolism; D014407:Tumor Cells, Cultured
D018955:CD36 Antigens; D017931:DNA Primers; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D051116:Receptors, Scavenger; C074299:SCARB2 protein, human; C489452:Scarb2 protein, mouse; D012795:Sialoglycoproteins; D001224:Aspartic Acid; D018698:Glutamic Acid