ALIX- and ESCRT-III–dependent sorting of tetraspanins to exosomes

Abstract

The intraluminal vesicles (ILVs) of endosomes mediate the delivery of activated signaling receptors and other proteins to lysosomes for degradation, but they also modulate intercellular communication when secreted as exosomes. The formation of ILVs requires four complexes, ESCRT-0, -I, -II, and -III, with ESCRT-0, -I, and -II presumably involved in cargo sorting and ESCRT-III in membrane deformation and fission. Here, we report that an active form of the ESCRT-associated protein ALIX efficiently recruits ESCRT-III proteins to endosomes. This recruitment occurs independently of other ESCRTs but requires lysobisphosphatidic acid (LBPA) in vivo, and can be reconstituted on supported bilayers in vitro. Our data indicate that this ALIX- and ESCRT-III–dependent pathway promotes the sorting and delivery of tetraspanins to exosomes. We conclude that ALIX provides an additional pathway of ILV formation, secondary to the canonical pathway, and that this pathway controls the targeting of exosomal proteins.

Extracellular Vesicle Membrane-Associated Proteins: Emerging Roles in Tumor Angiogenesis and Anti-Angiogenesis Therapy Resistance

“…Protease-activated receptor 1 (PAR1) bypasses the requirement for ubiquitination and the ubiquitin-binding early ESCRT complexes by binding the ESCRT accessory protein ALG-2-interacting protein X (ALIX) that recruits the ESCRT-III complex to sort PAR1 into ILVs [34]. Tetraspanins are also sorted to exosomes via ALIX-dependent ESCRT-III recruitment [35]. Furthermore, ALIX mediates sorting of syndecans to exosomes via its interaction with the syndecan adaptor syntenin [36].…”

Transmissible Endosomal Intoxication: A Balance between Exosomes and Lysosomes at the Basis of Intercellular Amyloid Propagation

“…Here, syntenin and Alix generate a bridge between the sorted cargo and ESCRT-III components via the recruitment of charged multivesicular body protein 4 CHMP4 (ESCRT-III) and the ATPase Vsp4 [65]. Other ESCRT-independent pathways for ILV biogenesis involve specific proteins and lipids, such as tetraspanins [66] and sphingolipid ceramides [67], but these pathways could act synergistically as tetraspanins sorting to ILVs would depend on semi-ESCRT-dependent pathways [65,68]. The tetraspanin TSPAN6, which is found to be increased in the prefrontal cortex of AD patients, interacts with syntenin and regulates the sorting of APP-CTF into ILVs [69,70].…”

Isolation and characterization of exosomes for cancer research

“…Evidence has proven that the endosomal sorting complex required for transport (ESCRT) participates in ILV formation. Four separate ESCRT subunits (0 through III) work cooperatively to promote MVB formation, vesicle budding, and protein cargo sorting [ 75 – 77 ]. It has been demonstrated that the ESCRT-0 subunit of the complex recruits proteins for internalization, including ubiquitinated proteins and clathrin; that ESCRT-I and ESCRT-II initiate the beginning of the budding process and facilitate enzymatic de-ubiquitination of cargo proteins; and that ESCRT-III is involved in the final stage of membrane invagination and separation [ 78 , 79 ].…”