title: Differential use of two AP-3-mediated pathways by lysosomal membrane proteins.
abstract: The adaptor protein complex AP-3 is involved in the sorting of lysosomal membrane proteins to late endosomes/lysosomes. It is unclear whether AP-3-containing vesicles form at the trans-Golgi network (TGN) or early endosomes. We have compared the trafficking routes of endolyn/CD164 and 'typical' lysosomal membrane glycoproteins (lgp120/lamp-1 and CD63/lamp-3) containing cytosolic YXXPhi-targeting motifs preceded by asparagine and glycine, respectively. Endolyn, which has a NYHTL-motif, is concentrated in lysosomes, but also occurs in endosomes and at the cell surface. We observed predominant interaction of the NYHTL-motif with the mu-subunits of AP-3 in the yeast two-hybrid system. Endolyn was mislocalized to the cell surface in AP-3-deficient pearl cells, confirming a major role of AP-3 in endolyn traffic. However, lysosomal delivery of endolyn (or a NYHTL-reporter), but not GYXXPhi-containing proteins, was practically abolished when AP-2-mediated endocytosis or traffic from early to late endosomes was inhibited in NRK and 3T3 cells. This indicates that endolyn is mostly transported along the indirect lysosomal pathway (via the cell surface), rather than directly from the TGN to late endosomes/lysosomes. Our results suggest that AP-3 mediates lysosomal sorting of some membrane proteins in early endosomes in addition to sorting of proteins with intrinsically strong AP-3-interacting lysosomal targeting motifs at the TGN.
authors.affiliation: Cambridge Institute for Medical Research, Department of Clinical Biochemistry, University of Cambridge, Wellcome Trust/MRC Building, Cambridge CB2 2XY, UK. gi200@cam.ac.uk
authors.forename: Gudrun
authors.initials: G
authors.lastname: Ihrke
authors.forename: Aija
authors.initials: A
authors.lastname: Kyttälä
authors.forename: Matthew R G
authors.initials: MR
authors.lastname: Russell
authors.forename: Brian A
authors.initials: BA
authors.lastname: Rous
authors.forename: J Paul
authors.initials: JP
authors.lastname: Luzio
mesh_terms: D033963:Adaptor Protein Complex 3; D020816:Amino Acid Motifs; D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D051929:CD146 Antigen; D004268:DNA-Binding Proteins / Q000378:metabolism*; D051930:Endolyn; D005998:Glycine / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D018613:Microscopy, Confocal; D041681:NIH 3T3 Cells; D019006:Neural Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism; D021382:Protein Sorting Signals / Q000235:genetics / Q000502:physiology; D011506:Proteins / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*
chemical_list: D033963:Adaptor Protein Complex 3; D015703:Antigens, CD; D051929:CD146 Antigen; C497899:Cd164 protein, mouse; D004268:DNA-Binding Proteins; D051930:Endolyn; D008565:Membrane Proteins; D019006:Neural Cell Adhesion Molecules; D021382:Protein Sorting Signals; D011506:Proteins; D014157:Transcription Factors; C018589:lysosomal proteins; D005998:Glycine