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.

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)

“…WT and mutant LAMP-1 pcDNA3.1 constructs were prepared previously. 11) Plasmid Construction cDNA fragments corresponding to the cytoplasmic tails (CTs) (amino acids 372-382) of WT and mutant LAMP-1 were amplified by PCR using WT and mutant LAMP-1 pcDNA3.1 aliquots as templates and introduced into the EcoRI and BamHI sites of a pGBKT7 vector to produce pGBKT7-WT and mutants ( Fig. 1).…”

“…15) As described above, WT and various COOH-terminal mutants of LAMP-1 are different in the late endosomal and lysosomal abundance. 11) The most efficient trafficking was observed for WT (GYQTI) and the second most was I382L (GYQTL). Lower levels of I382F (GYQTF), I382M (GYQM), and I382V (GYQTV) were present in the late endosomal and lysosomal fraction.…”

“…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.…”

“…[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.…”