title: COOH-terminal isoleucine of lysosome-associated membrane protein-1 is optimal for its efficient targeting to dense secondary lysosomes.
abstract: Lysosome-associated membrane protein-1 (LAMP-1) consists of a highly glycosylated luminal domain, a single-transmembrane domain and a short cytoplasmic tail that possesses a lysosome-targeting signal (GYQTI(382)) at the COOH terminus. It is hypothesized that the COOH-terminal isoleucine, I(382), could be substituted with any other bulky hydrophobic amino acid residue for LAMP-1 to exclusively localize in lysosomes. In order to test this hypothesis, we compared subcellular distribution of four substitution mutants with phenylalanine, leucine, methionine and valine at the COOH-terminus (termed I382F, I382L, I382M and I382V, respectively) with that of wild-type (WT)-LAMP-1. Double-labelled immunofluorescence analyses showed that these substitution mutants were localized as significantly to late endocytic organelles as WT-LAMP-1. However, the quantitative subcellular fractionation study revealed different distribution of WT-LAMP-1 and these four COOH-terminal mutants in late endosomes and dense secondary lysosomes. WT-LAMP-1 was accumulated three to six times more in the dense lysosomal fraction than the four mutants. The level of WT-LAMP-1 in late endosomal fraction was comparable to those of I382F, I382M and I382V. Conversely, I382L in the late endosomal fraction was approximately three times more abundant than WT-LAMP-1. These findings define the presence of isoleucine residue at the COOH-terminus of LAMP-1 as critical in governing its efficient delivery to secondary lysosomes and its ratio of lysosomes to late endosomes.
authors.affiliation: Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Fukuyama, Hiroshima, Japan. akasaki@fupharm.fukuyama-u.ac.jp
authors.forename: Kenji
authors.initials: K
authors.lastname: Akasaki
authors.forename: Michihisa
authors.initials: M
authors.lastname: Suenobu
authors.forename: Maki
authors.initials: M
authors.lastname: Mukaida
authors.forename: Akihiro
authors.initials: A
authors.lastname: Michihara
authors.forename: Ikuo
authors.initials: I
authors.lastname: Wada
mesh_terms: D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D000818:Animals; D002458:Cell Fractionation; D011992:Endosomes / Q000737:chemistry* / Q000378:metabolism; D000882:Haplorhini; D007532:Isoleucine / Q000737:chemistry*; D052118:Lysosomal-Associated Membrane Protein 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008247:Lysosomes / Q000737:chemistry* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary / Q000235:genetics; D021381:Protein Transport; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D052118:Lysosomal-Associated Membrane Protein 1; D007532:Isoleucine