Competing sorting signals guide endolyn along a novel route to lysosomes in MDCK cells.
We have examined the trafficking of the mucin-like protein endolyn in transfected, polarized MDCK cells using biochemical approaches and immunofluorescence microscopy. Although endolyn contains a lysosomal targeting motif of the type YXXPhi and was localized primarily to lysosomes at steady state, significant amounts of newly synthesized endolyn were delivered to the apical cell surface. Antibodies to endolyn, but not lamp-2, were preferentially internalized from the apical plasma membrane and efficiently transported to lysosomes. Analysis of endolyn-CD8 chimeras showed that the lumenal domain of endolyn contains apical targeting information that predominates over basolateral information in its cytoplasmic tail. Interestingly, surface polarity of endolyn was independent of O-glycosylation processing, but was reversed by disruption of N-glycosylation using tunicamycin. At all times, endolyn was soluble in cold Triton X-100, suggesting that apical sorting was independent of sphingolipid rafts. Our data indicate that a strong, N-glycan-dependent apical targeting signal in the lumenal domain directs endolyn into a novel biosynthetic pathway to lysosomes, which occurs via the apical surface of polarized epithelial cells.
Ihrke
G
G
Department of Clinical Biochemistry and Cambridge Institute for Medical Research, University of Cambridge, Wellcome Trust/MRC Building, Cambridge CB2 2XY, UK. gi200@cus.cam.ac.uk
Bruns
J R
JR
Luzio
J P
JP
Weisz
O A
OA
D020816:Amino Acid Motifs; D000818:Animals; D015703:Antigens, CD / Q000737:chemistry / Q000276:immunology; D019921:Biotinylation; D051929:CD146 Antigen; D016827:CD8 Antigens / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003902:Detergents / Q000378:metabolism / Q000494:pharmacology; D004285:Dogs; D051930:Endolyn; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism; D051907:Lysosomal Membrane Proteins; D008247:Lysosomes / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000276:immunology / Q000502:physiology*; D021962:Membrane Microdomains / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D017830:Octoxynol / Q000494:pharmacology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011817:Rabbits; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection
D015703:Antigens, CD; D051929:CD146 Antigen; D016827:CD8 Antigens; D003902:Detergents; D051930:Endolyn; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins; D017830:Octoxynol