Lipid raft-associated protein sorting in exosomes.
Exosomes are small membrane vesicles secreted by cells upon fusion of multivesicular endosomes with the cell surface. The mechanisms underlying the specific sorting of proteins in exosomal membranes are far from being unraveled. We demonstrate here, using different cells, that some molecules are released in the extracellular medium via their association with lipid raft domains of the exosomal membrane. Various typical raft-associated molecules could be detected by immunoblot in exosomes and Triton X-100-insoluble fractions isolated from exosomes of different origins. Partial localization of major histocompatibility complex (MHC) class II molecules with detergent-resistant fractions isolated from Daudi-secreted exosomes was demonstrated by immunoblot and confirmed by electron microscopy colocalization of MHC class II molecules and ganglioside GM1. Moreover, we found that exosome-associated Lyn (1) had a lower molecular weight compared with Lyn detected in cell-isolated detergent-resistant domains, (2) was absent from the Triton X-100-insoluble fraction isolated from exosomes, and (3) had lost its partitioning capacity in Triton X-114. Exosomal Lyn is probably cleaved by a caspase-3-like activity contained in secreted vesicles. All together, the data highlight the presence of lipid microdomains in exosomal membranes and suggest their participation in vesicle formation and structure, as well as the direct implication of exosomes in regulatory mechanisms.
de Gassart
Aude
A
UMR 5539, Univ Montpellier II-cc107, Montpellier 34095, France.
Geminard
Charles
C
Fevrier
Benoit
B
Raposo
Graca
G
Vidal
Michel
M
D000818:Animals; D001692:Biological Transport; D001798:Blood Proteins / Q000378:metabolism; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D003902:Detergents / Q000494:pharmacology; D011992:Endosomes / Q000502:physiology; D005677:G(M1) Ganglioside / Q000378:metabolism; D000949:Histocompatibility Antigens Class II / Q000378:metabolism; D006801:Humans; D020014:K562 Cells / Q000166:cytology / Q000378:metabolism; D008561:Membrane Fusion; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D017830:Octoxynol / Q000494:pharmacology; D011092:Polyethylene Glycols / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012156:Reticulocytes / Q000166:cytology / Q000378:metabolism*; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism
D001798:Blood Proteins; D003902:Detergents; D000949:Histocompatibility Antigens Class II; D008565:Membrane Proteins; C036860:STOM protein, human; C106354:flotillins; D005677:G(M1) Ganglioside; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases; C505407:CASP3 protein, human; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases