The endosomal sorting complexes required for transport (ESCRTs) are required to sort integral membrane proteins into intralumenal vesicles of the multivesicular body (MVB). Mutations in the ESCRT-III subunit CHMP2B were recently associated with frontotemporal dementia and amyotrophic lateral sclerosis (ALS), neurodegenerative diseases characterized by abnormal ubiquitin-positive protein deposits in affected neurons. We show here that autophagic degradation is inhibited in cells depleted of ESCRT subunits and in cells expressing CHMP2B mutants, leading to accumulation of protein aggregates containing ubiquitinated proteins, p62 and Alfy. Moreover, we find that functional MVBs are required for clearance of TDP-43 (identified as the major ubiquitinated protein in ALS and frontotemporal lobar degeneration with ubiquitin deposits), and of expanded polyglutamine aggregates associated with Huntington's disease. Together, our data indicate that efficient autophagic degradation requires functional MVBs and provide a possible explanation to the observed neurodegenerative phenotype seen in patients with CHMP2B mutations.

The process by which clathrin-coated vesicles are produced involves interactions of multifunctional adaptor proteins with the plasma membrane, as well as with clathrin and several accessory proteins and phosphoinositides. Here we review recent findings highlighting new insights into mechanisms underlying clathrin-dependent endocytosis.

Cell migration requires endocytosis and recycling of integrins, but it is not known whether degradation of these membrane proteins is involved. Here we demonstrate that in migrating cells, a fraction of the endocytosed fibronectin receptor, alpha 5 beta 1 integrin, is sorted into multivesicular endosomes together with fibronectin and degraded in lysosomes. This sorting requires fibronectin-induced ubiquitination of the alpha 5 subunit, and the activity of the endosomal sorting complex required for transport (ESCRT) machinery, which interacts with alpha 5 beta 1 integrin. Importantly, we demonstrate that both alpha 5 ubiquitination and ESCRT functions are required for proper migration of fibroblasts. We propose that ligand-mediated degradation of alpha 5 beta 1 integrin via the ESCRT pathway is required in order to prevent endosomal accumulation of ligand-bound integrins that might otherwise form nonproductive adhesion sites. Fibronectin and alpha 5 beta 1 integrin therefore are trafficked to lysosomes in a similar way to growth factors and their receptors.

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The endosomal sorting complexes required for transport, ESCRT-I, -II, and -III, are thought to mediate the biogenesis of multivesicular endosomes (MVEs) and endosomal sorting of ubiquitinated membrane proteins. Here, we have compared the importance of the ESCRT-I subunit tumor susceptibility gene 101 (Tsg101) and the ESCRT-III subunit hVps24/CHMP3 for endosomal functions and receptor signaling. Like Tsg101, endogenous hVps24 localized mainly to late endosomes. Depletion of hVps24 by siRNA showed that this ESCRT subunit, like Tsg101, is important for degradation of the epidermal growth factor (EGF) receptor (EGFR) and for transport of the receptor from early endosomes to lysosomes. Surprisingly, however, whereas depletion of Tsg101 caused sustained EGF activation of the mitogen-activated protein kinase pathway, depletion of hVps24 had no such effect. Moreover, depletion of Tsg101 but not of hVps24 caused a major fraction of internalized EGF to accumulate in nonacidified endosomes. Electron microscopy of hVps24-depleted cells showed an accumulation of EGFRs in MVEs that were significantly smaller than those in control cells, probably because of an impaired fusion with lyso-bisphosphatidic acid-positive late endosomes/lysosomes. Together, our results reveal functional differences between ESCRT-I and ESCRT-III in degradative protein trafficking and indicate that degradation of the EGFR is not required for termination of its signaling.

The ubiquitin-binding protein Hrs and endosomal sorting complex required for transport (ESCRT)-I and ESCRT-III are involved in sorting endocytosed and ubiquitinated receptors to lysosomes for degradation and efficient termination of signaling. In this study, we have investigated the role of the ESCRT-II subunit Vps22/EAP30 in degradative protein sorting of ubiquitinated receptors. Vps22 transiently expressed in HeLa cells was detected in endosomes containing endocytosed epidermal growth factor receptors (EGFRs) as well as Hrs and ESCRT-I and ESCRT-III. Depletion of Vps22 by small interfering RNA, which was accompanied by decreased levels of other ESCRT-II subunits, greatly reduced degradation of EGFR and its ligand EGF as well as the chemokine receptor CXCR4. EGFR accumulated on the limiting membranes of early endosomes and aberrantly small multivesicular bodies in Vps22-depleted cells. Phosphorylation and nuclear translocation of extracellular-signal-regulated kinase1/2 downstream of the EGF-activated receptor were sustained by depletion of Hrs or the ESCRT-I subunit Tsg101. In contrast, this was not the case when Vps22 was depleted. These results indicate an important role for Vps22 in ligand-induced EGFR and CXCR4 turnover and suggest that termination of EGF signaling occurs prior to ESCRT-II engagement.

Endocytosed membrane proteins that are destined for degradation in lysosomes are ubiquitylated and recognised by sorting complexes on endosome membranes. The ubiquitin-binding sorting component Hrs as well as ubiquitylated cargo are enriched in a characteristic flat clathrin coat on the endosome membrane. The function of clathrin within this coat has not been investigated. Here, we show that both clathrin and the clathrin-box motif of Hrs are required for the clustering of Hrs into restricted microdomains. The C-terminus of Hrs, which contains the clathrin-box, is sufficient to redirect a phosphatidylinositol(3)-phosphate-binding protein into the Hrs- and clathrin-containing microdomains. Although these microdomains show little lateral diffusion in the membrane, they are dynamic structures that exchange Hrs and clathrin with similar kinetics, and acquire the downstream sorting component Tsg101. The clathrin-mediated clustering is essential for the function of Hrs in degradative protein sorting. We conclude that clathrin is responsible for concentrating Hrs in endosomal microdomains specialised for recognition of ubiquitylated membrane proteins, thus enabling efficient sorting of cargo into the degradative pathway.

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