Comparative study of the steady-state subcellular distribution of lysosome-associated membrane glycoprotein-2 (LAMP-2) isoforms with GYXXΦ-type tyrosine-based motifs that interact differently with four adaptor protein (AP) complexes

• Published in: Journal of biochemistry (Tokyo) Vol. 175; no. 3; pp. 275 - 287
• Main Authors: Yamaguchi, Fumiaki, Sakane, Hiroshi, Akasaki, Kenji
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 04-03-2024
• Subjects: Adapters; Adaptor Proteins, Signal Transducing; Amino Acids; Comparative studies; Density; Endosomes; Fractionation; Glycoproteins; Hybrid systems; Hydrophobicity; Immunofluorescence; Isoforms; LAMP-1 protein; LAMP-2 protein; Localization; Lysosomes; Membranes; Organelles; Protein Isoforms - genetics; Protein transport; Proteins; Transcription Factors; Tyrosine; Yeasts; adaptor protein complex; tyrosine-based motif; lysosome targeting; subcellular distribution; isoform
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/jb/mvad096

Abstract
Lysosome-associated membrane protein-1 and -2 (LAMP-1 and LAMP-2, respectively) are type I transmembrane proteins. LAMP-2 comprises three splice isoforms (LAMP-2A, -B and-C) with different cytoplasmic tails (CTs). These three CTs possess different tyrosine-based motifs (GYXXΦ, where Φ is a bulky hydrophobic amino acid) at their C-termini. Interactions between tyrosine-based motifs and μ-subunits of four tetrameric adaptor protein (AP) complexes are necessary for their vesicular transport to lysosomes. Little is known about how the interaction strengths of these tyrosine motifs with μ-subunits affect the localization of isoforms to lysosomes. The interactions were first investigated using a yeast two-hybrid system to address this question. LAMP-2A-CT interacted with all four μ-subunits (μ1, μ2, μ3A and μ4 of AP-1, AP-2, AP-3 and AP-4, respectively). The interaction with μ3A was more robust than that with other μ-subunits. LAMP-2B-CT interacted exclusively and moderately with μ3A. LAMP-2C-CT did not detectably interact with any of the four μ-subunits. Immunofluorescence microscopy showed that all isoforms were localized in late endosomes and lysosomes. LAMP-2C was present in the plasma membrane and early endosomes; however, LAMP-2A and -2B were barely detectable in these organelles. In cell fractionation, LAMP-2A was the most abundant in the dense lysosomes, whereas LAMP-2C was significantly present in the low-density fraction containing the plasma membrane and early endosomes, in addition to the dense lysosomes. LAMP-2B considerably existed in the low-density late endosomal fraction. These data strongly suggest that the LAMP-2 isoforms are distributed differently in endocytic organelles depending on their interaction strengths with AP-3.
Graphical Abstract
Graphical Abstract