**2. ESCRT dependent and independent multivesicular body biogenesis**

Endosomal sorting complex required for transport (ESCRT) proteins and ESCRT associated proteins including TSG101, ALG-2 interacting protein X (ALIX), SKD1, and Chmp4 are required for ESCRT dependent MVB biogenesis. ESCRT is a ubiquitin-dependent mechanism that contributes to the sorting of ubiquitinated proteins into exosomes. ESCRT consist of four complexes that are numbered according to their sequential action. For example, ESCRT-0 aka vacuolar protein sorting (VPS) associated protein 27/heat shock element 1 complex VPS27/HSE1 recruits ESCRT-1, which plays an essential role in MVB cargo sorting and bud formation. VPS36 associates with ESCRT-1 via a ubiquitin moiety while VPS25 is required for the assembly of the ESCRT-II complex [3]. ESCRT-II also plays a role in cargo sorting and regulates

#### *Mechanisms of Extracellular Vesicle Biogenesis, Cargo Loading, and Release DOI: http://dx.doi.org/10.5772/intechopen.100458*

ESCRT-III formation. VPS20 is involved in the assembly of the ESCRT-III complex and VPS24 completes the scission of the budding membrane [4].

In addition to sorting of cargo within multivesicular bodies, ESCRT-III participates in ESCRT recycling and binds ALIX to allow for cargo sorting [5]. ALIX also stimulates exosome secretion containing the tetraspanins CD63, CD9 and CD81 [5].

Alternatively, there are multiple ESCRT independent MVB biogenesis pathways that utilize various other proteins. This particular mechanism may be dependent on alternate mechanisms including the Rab associated proteins, ceramides, sphingomyelins, cholesterols, and tetraspanins. Members of the Rab family of small GTPases regulate various steps in the formation and transition of the endosomal transport network. Rab5 is thought to regulate the formation and fusion of early endosomes [6, 7]. Rab7 is thought to regulate the fusion of late endosomes/multivesicular endosomes with lysosomes resulting in degradation of the intraluminal vesicles [8, 9]. Rab27 is thought to regulate multivesicular endosome docking and fusion with the plasma membrane to allow for release of the intraluminal vesicles as exosomes [10]. One report provided evidence that Rab31 engages flotillin proteins from lipid rafts to orchestrate epidermal growth factor receptor entry into multivesicular endosomes to form intraluminal vesicles (ILV) and the release of exosomes [11].
