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Exosome Biogenesis: ESCRT, Ceramide and the Multivesicular Body

Por Benn Bluestein-Veyra, M.Sc. en Química Orgánica · Actualizado el 20 de octubre de 2026

Exosome biogenesis is the process by which cells form small vesicles inside endosomes and release them into the extracellular space. Intraluminal vesicles bud into maturing endosomes, which become multivesicular bodies, and fusion of a multivesicular body with the plasma membrane releases those vesicles as exosomes.^[1]

Several machineries shape the budding membrane: the ESCRT complexes, the lipid ceramide and tetraspanin microdomains.^[2] This page describes each of them, the proteins that dock and fuse multivesicular bodies with the cell surface, and how cells divide these organelles between secretion and degradation.

Early endosomes mature into multivesicular bodies

Endocytosed membrane proteins first reach early endosomes. Huotari and Helenius described the maturation program that converts these organelles into late endosomes, or multivesicular bodies, and separates them from early endosomes in function and position.^[3]

During maturation the limiting membrane buds inward to form intraluminal vesicles, or ILVs (Fig. 1). Ubiquitin marks the membrane proteins that enter these invaginations, and the ESCRT machinery sorts them and releases each vesicle from the perimeter membrane.^[4]

Electron tomography resolves this geometry directly. Adell and colleagues combined light microscopy with tomography of yeast multivesicular bodies and captured ILVs in the lumen alongside buds still attached to the limiting membrane (Fig. 2).^[5]

A multivesicular body can fuse with a lysosome, which degrades its contents, or with the plasma membrane, which releases its ILVs as exosomes. Vesicles shed directly from the plasma membrane form a separate class called microvesicles.^[1]

ESCRT-0 and ESCRT-I recognize ubiquitinated cargo

The ESCRT pathway was defined in budding yeast. Katzmann and colleagues showed in 2001 that ubiquitination is a signal for sorting into multivesicular bodies, and they identified ESCRT-I, a complex of Vps23, Vps28 and Vps37 that recognizes ubiquitinated cargo through a UBC-like domain in Vps23.^[6]

In human cells ESCRT-0 pairs HRS, the product of the HGS gene, with STAM, and HRS sorts monoubiquitinated membrane proteins into the multivesicular body.^[7] Ren and colleagues solved the ESCRT-0 core at 2.3 Å, two domain-swapped GAT domains joined by an antiparallel coiled coil, within a flexible 1:1 complex (Fig. 3).^[8]

TSG101 is the human counterpart of yeast Vps23.^[9] Sundquist and colleagues solved its UEV domain bound to ubiquitin at 2.0 Å: the domain contacts the Ile44 surface of ubiquitin and an adjacent loop through a highly solvated interface, and mutations at that interface inhibited sorting into multivesicular bodies (Fig. 4).^[10]

Wollert and Hurley reconstituted the pathway on giant unilamellar vesicles carrying a membrane-tethered ubiquitin cargo. ESCRT-0 gathered the cargo into domains, and ESCRT-I and ESCRT-II together bent the membrane into buds that confined it, with both complexes positioned at the bud neck.^[11]

• ESCRT-0 (HRS and STAM) binds ubiquitinated cargo and clusters it on the endosomal membrane.^[11]
• ESCRT-I, which contains TSG101, and ESCRT-II shape the bud and sit at its neck.^[11]
• ESCRT-III subunits assemble at the neck and cut it to release the ILV.^[11]
• VPS4 binds ESCRT-III, drives the final constriction of the neck and recycles the subunits.^[12]

ESCRT-III and VPS4 cut the neck of the intraluminal vesicle

In the reconstituted system, ESCRT-III subunits localized to the bud neck and cleaved it. The released ILVs carried the cargo and were free of ESCRT proteins, so the machinery works from the cytoplasmic side of the bud without being consumed.^[11]

Wollert and colleagues had shown a year earlier that three yeast ESCRT-III subunits, Vps20, Snf7 and Vps24, suffice to detach ILVs. Vps2 recruits the ATPase Vps4, whose activity recycles ESCRT-III for further rounds of budding, and this minimal set is conserved from archaea to animals.^[13]

Human ESCRT-III is built from the charged multivesicular body proteins, and the CHMP4 subfamily assembles into membrane-attached filaments.^[14] Azad and colleagues solved membrane-coated CHMP2A and CHMP3 filaments by cryo-EM at 3.3 and 3.6 Å, showing heterodimers in the open conformation that present a positively charged surface to the membrane (Fig. 5).^[15]

In those filaments the C-terminal sequence that VPS4 recognizes points toward the tube interior. VPS4 constricted and cleaved CHMP2A and CHMP3 membrane tubes, and the authors concluded that the three proteins form a minimal fission machinery.^[15]

Humans carry two paralogs of yeast Vps4, VPS4A and VPS4B, and both associate with endosomal compartments.^[16] Adell and colleagues found by electron tomography in yeast that ESCRT-III assembly induces or stabilizes the necks of growing ILVs, and that Vps4 binding, coordinated through Vps2 and Snf7, is coupled to neck constriction.^[12]

Lattice light-sheet microscopy then measured the assemblies on yeast endosomes: each lasted 3 to 45 s and gathered 75 to 200 Snf7 and 15 to 50 Vps24 molecules. Productive budding required at least four Vps4 hexamers and continuous exchange of subunits.^[5]

Pfitzner and colleagues traced a sequence of subunit exchanges powered by Vps4. Replacing Vps24 with Did2 tilted the polymer and converted flat spirals into helical filaments, and a highly constricted Did2 and Ist1 copolymer promoted fission from inside membrane necks.^[17]

The same machinery severs membrane necks from their inner face in cytokinesis, nuclear envelope reformation and autophagy.^[18][19] McCullough and colleagues review the structures of ESCRT-III filaments and of Vps4 engaged with an ESCRT-III substrate that underlie this shared mechanism.^[20]

ESCRT components shape exosome secretion in mammalian cells

Colombo and colleagues screened 23 ESCRT components and associated proteins by RNA interference in HeLa-CIITA cells. Silencing HRS, STAM1 or TSG101 reduced secretion of vesicle-associated CD63 and MHC class II, and each changed the size or protein composition of the secreted vesicles in a different way.^[21]

Depletion of VPS4B increased secretion and left vesicle features unchanged. Silencing ALIX raised MHC class II in vesicles secreted by HeLa-CIITA cells. In primary dendritic cells it raised MHC class II in the cells and left the CD63-positive vesicles unchanged, a difference the authors attributed to vesicle heterogeneity.^[21]

Stuffers and colleagues depleted key subunits of all four ESCRT complexes in mammalian cells. EGF-induced multivesicular bodies were highly sensitive to the depletion, yet EGF-independent multivesicular bodies still formed, with enlarged ILVs that excluded the EGF receptor.^[22]

The authors concluded that mammalian cells use both ESCRT-dependent and ESCRT-independent mechanisms of multivesicular body biogenesis.^[22] Henne and colleagues describe the ESCRT pathway itself as a cargo recognition and membrane sculpting machine.^[9]

Ceramide drives ESCRT-independent inward budding

Trajkovic and colleagues found in 2008 that cargo segregates into distinct subdomains on the endosomal membrane. Transfer of the exosome-associated domains into the endosome lumen proceeded independently of ESCRT function and required the sphingolipid ceramide.^[23]

Purified exosomes were enriched in ceramide, and inhibiting neutral sphingomyelinases reduced exosome release.^[23] The authors proposed that the cone shape of ceramide, with its small head group, and its tendency to coalesce into domains favor the negative curvature of an inward bud.^[23]

Neutral sphingomyelinase 2 is encoded by SMPD3 and carries sphingomyelin phosphodiesterase activity.^[24] Airola and colleagues solved its catalytic domain at 1.85 Å, a DNase-I-type fold whose hydrophobic track to the active site is gated by a conserved element they named the DK switch (Fig. 6).^[25]

A membrane-embedded N-terminal domain anchors the enzyme and binds phosphatidylserine. The juxtamembrane region linking it to the catalytic domain was necessary and sufficient for allosteric activation, which suggests that ceramide production follows the lipid composition of the membrane the enzyme sits in.^[25]

Tetraspanins and ALIX sort cargo into intraluminal vesicles

Tetraspanin-enriched microdomains offer a third sorting route. In melanocytes, van Niel and colleagues showed that CD63 directly participates in ESCRT-independent sorting of the PMEL luminal domain into ILVs, where it is processed into the amyloid fibrils of melanosomes.^[26]

Inactivating CD63 in cultured cells or in mice impaired fibril formation and melanosome morphogenesis. The remaining PMEL transmembrane fragment required functional ESCRTs, and in the absence of CD63 the luminal domain followed it to ESCRT-dependent degradation, one cargo handled by two ILV sorting processes.^[26]

ALIX is one of the early-acting factors that nucleate ESCRT-III assembly (Fig. 7).^[20] Baietti and colleagues found that the adaptor syntenin binds ALIX through LYPXnL motifs and links syndecan heparan sulfate proteoglycans to intraluminal budding.^[27]

Larios and colleagues showed that an active form of ALIX recruits ESCRT-III to endosomes independently of the other ESCRTs, in a step that requires lysobisphosphatidic acid (LBPA). This ALIX and ESCRT-III pathway promoted the sorting of tetraspanins to exosomes.^[28]

Matsuo and colleagues had earlier shown that LBPA can drive the formation of multivesicular liposomes. The process depended on a pH gradient like the one across endosome membranes and was controlled by ALIX.^[29]

RAB27 and SNARE proteins deliver multivesicular bodies to the plasma membrane

Ostrowski and colleagues screened Rab GTPases by RNA interference in HeLa cells and identified five that promote exosome secretion. Two of them, Rab27a and Rab27b, act in docking of multivesicular bodies at the plasma membrane.^[30]

The two isoforms act at different steps. Rab27a silencing strongly enlarged multivesicular bodies, Rab27b silencing redistributed them toward the perinuclear region, and silencing the effectors Slp4 and Slac2b reproduced the two phenotypes in turn.^[30]

Verweij and colleagues imaged fusion directly with tetraspanin-based pH-sensitive reporters and total internal reflection microscopy. Depleting the target membrane SNAREs SNAP23 and syntaxin-4 reduced fusion frequency, and histamine H1 receptor signaling raised it through phosphorylation of SNAP23 at Ser110.^[31]

Gross and colleagues found that Wnt proteins travel with their cargo receptor Evi through endosomal compartments onto exosomes in fruit flies and human cells. The exosomes carried Wnts on their surface and induced Wnt signaling in target cells, and the route requires the R-SNARE YKT6.^[32]

Multivesicular bodies balance secretion and lysosomal degradation

ILVs are either sorted for degradation in lysosomes or secreted as exosomes. Trajkovic and colleagues found that cargo segregates into distinct subdomains of the endosomal membrane, which places one point of that choice at the moment of budding.^[23]

Villarroya-Beltri and colleagues identified ISGylation, a ubiquitin-like modification, as a regulator of this balance. Conjugation of ISG15 decreased the number of multivesicular bodies, moved them toward lysosomes and impaired exosome secretion in cells and in mice.^[33]

ISGylation of TSG101 by itself triggered the aggregation and degradation of the protein and was sufficient to impair secretion. Blocking lysosomal function or autophagy restored exosome release, consistent with degradation and secretion drawing on a shared pool of multivesicular bodies.^[33]

The fate of a multivesicular body therefore reflects how its ILVs were formed and the docking and degradation signals it receives. These choices help explain why vesicle preparations from different cells differ in size and composition.^[1][21]

Where to go from here

The cargo sorting reference follows the signals that load proteins and RNA into ILVs, including the syndecan, syntenin and ALIX route. The lipidome reference describes the ceramide and cholesterol content of the exosome membrane, and the CD63 reference covers the tetraspanin at the center of ESCRT-independent sorting.

For measurement, the quality references on MISEV 2023 and on tetraspanin marker panels explain how vesicle preparations are characterized. Verified practices can request the certificate of analysis for lot-specific data, and our team can explain how each result was measured.