Yes and no. We do know that they are made as ILVs; but first of all, not all ILVs finish up as exosomes, and second, the mechanism of their generation in endosomes is not fully understood. Most conventional membrane budding processes deform membrane from an organelle into the cytoplasm but in ILV formation the membrane buds away from the cytoplasm and into the endosome. This unconventional budding process is not limited to ILV generation but also takes place during enveloped virus budding from the cytosol and during cytokinesis [7], and it requires specialised machinery.

ILVs (and thus exosomes) can be generated at the endosomal limiting membrane by at least two mechanisms, one of which depends on the ESCRT machinery (ESCRT stands for endosomal sorting complexes required for transport) whereas the other is ESCRT-independent (Fig. 2).

Fig. 2 ILVs are generated by invagination of the endosomal membrane and have three possible fates. Inset: intraluminal vesicles (ILV) are formed by invagination of the endosomal membrane by either ESCRT-dependent or ESCRT-independent mechanisms. Matured endosomes accumulate ILVs within their lumen and have three distinct fates. They may deliver content that contributes to the biogenesis of specialized lysosome-related organelles (for example, melanosomes, Weibel-Palade bodies, azurophilic granules), they may fuse with lysosomes or they may fuse with the plasma membrane where released ILVs are now termed ‘exosomes’ Full size image

The ESCRT machinery consists of a set of cytosolic protein complexes that are recruited to endosomes by membrane proteins that have been tagged, usually with ubiquitin on their cytosolic domains. The ubiquitin tag is recognized by the first of the ESCRT complexes, ESCRT-0, which is thus recruited to the endosomal membrane and passes ubiquitinated cargos to ESCRT-I, one of whose components, Tsg101, also recognizes ubiquitin. The recruitment of the ESCRT machinery acts to both cluster the ubiquitinated cargo proteins on the endosome and induce curvature of the endosomal membrane to form ILVs.

But ILVs are still able to form in the absence of ESCRTs [8], so other means of generating ILVs must exist, although the mechanisms for their generation are less clear. Generation of these ESCRT-independent ILVs requires the tetraspanin CD63—a protein abundant on ILVs but with unclear function [9]—and may be facilitated by cone-shaped bending properties of lipids such as ceramide [10].