ABSTRACT
E-cadherin, a transmembrane protein involved in epithelial cell-cell adhesion and signaling, is found in exosomal fractions isolated from human body fluids. A cellular mechanism for recruitment of E-cadherin into extracellular vesicles (EVs) has not yet been defined. Here, we show that E-cadherin is incorporated into the membrane of EVs with the extracellular domain exposed at the vesicle surface. This recruitment depends on the endosomal sorting complex required for transport I (ESCRT-I) component Tsg101 and a highly conserved tetrapeptide P(S/T)AP late domain motif in the cytoplasmic tail of E-cadherin that mediates interaction with Tsg101. Mutation of this motif results in a loss of interaction and a dramatic decrease in exosomal E-cadherin secretion. We conclude, that the process of late domain mediated exosomal recruitment is exerted by this endogenous non-ESCRT transmembrane protein.
ABSTRACT
Cells use unconventional secretion to deliver the ß-galactoside binding lectin galectin-3 from the cell interior into the extracellular milieu. This process starts with galectin-3 recruitment into intraluminal vesicles (ILVs), which are later released at the plasma membrane as exosomes. Electron microscopy is utilized to determine the location of GFP-tagged galectin-3 in pelleted exosomes. We also describe how these vesicles are harvested from cell culture media to determine their composition. The fluorescent protein GFP was fused with the exosomal sorting motif of galectin-3 to direct GFP into exosomes. Recruitment of this fusion construct into the lumen of exosomes can be assessed by proteinase K accessibility analysis.
