Constitutive expression of a COOH-terminal leucine mutant of lysosome-associated membrane protein-1 causes its exclusive localization in low density intracellular vesicles

Abstract

Lysosome-associated membrane protein-1 (LAMP-1) is a type I transmembrane protein with a short cytoplasmic tail that possesses a lysosome-targeting signal of GYQTI(382)-COOH. Wild-type (WT)-LAMP-1 was exclusively localized in high density lysosomes, and efficiency of LAMP-1's transport to lysosomes depends on its COOH-terminal amino acid residue. Among many different COOH-terminal amino acid substitution mutants of LAMP-1, a leucine-substituted mutant (I382L) displays the most efficient targeting to late endosomes and lysosomes [Akasaki et al. (2010) J. Biochem. 148: , 669-679]. In this study, we generated two human hepatoma cell lines (HepG2 cell lines) that stably express WT-LAMP-1 and I382L, and compared their intracellular distributions. The subcellular fractionation study using Percoll density gradient centrifugation revealed that WT-LAMP-1 had preferential localization in the high density secondary lysosomes where endogenous human LAMP-1 was enriched. In contrast, a major portion of I382L was located in a low density fraction. The low density fraction also contained approximately 80% of endogenous human LAMP-1 and significant amounts of endogenous β-glucuronidase and LAMP-2, which probably represents occurrence of low density lysosomes in the I382L-expressing cells. Double immunofluorescence microscopic analyses distinguished I382L-containing intracellular vesicles from endogenous LAMP-1-containing lysosomes and early endosomes. Altogether, constitutive expression of I382L causes its aberrant intracellular localization and generation of low density lysosomes, indicating that the COOH-terminal isoleucine is critical for normal localization of LAMP-1 in the dense lysosomes.

Targeting of Wild-Type and Mutated Forms of Lysosome-Associated Membrane Protein-1 (LAMP-1) to Late Endosomes/Lysosomes Depends on Affinities of Their Cytoplasmic Tail Peptides with a Medium Subunit of Adaptor Protein Complex-3 (AP-3)

“…Lysosome-associated membrane proteins (LAMP)-1 and LAMP-2 represent a major portion of the lysosomal membrane proteins 4,5) and may have an important role in lysosomal integrity. 6) These two proteins share some structural similarities; a large and highly glycosylated luminal domain, a single transmembrane domain and a short cytoplasmic tail (CT) at the COOH-terminus. 7) For targeting of LAMP-1 and LAMP-2 to lysosomes, they have tyrosine-based motifs in their CTs, which conform to GYXXΦ, where Φ is a hydrophobic amino acid residue.…”

“…[7][8][9][10][11]18) Lysosomal localization of LAMP-1 varies by changing hydrophobic amino acid residues in the Φ position. 6,8,11) The COOH-terminal isoleucine intrinsic to wild-type (WT)-LAMP-1 is optimal for its efficient targeting to dense lysosomes. 6,11) The tyrosine-based motifs are well known to interact with medium subunits μ1, μ2, μ3 (A or B), and μ4 of adaptor protein (AP) complexes AP-1, AP-2, AP-3, and AP-4, respectively (μ3A and μ3B are ubiquitous and neuron-specific forms, respectively).…”

“…6,8,11) The COOH-terminal isoleucine intrinsic to wild-type (WT)-LAMP-1 is optimal for its efficient targeting to dense lysosomes. 6,11) The tyrosine-based motifs are well known to interact with medium subunits μ1, μ2, μ3 (A or B), and μ4 of adaptor protein (AP) complexes AP-1, AP-2, AP-3, and AP-4, respectively (μ3A and μ3B are ubiquitous and neuron-specific forms, respectively). 10,[12][13][14][15] The interactions of GYXXΦ to AP complexes cause selective incorporation of the integral membrane proteins such as LAMP-1 into clathrin-coated vesicles that carry these cargo proteins to their own cellular destinations.…”