Time-resolved ultrastructural detection of phosphatidylinositol 3-phosphate.

Phosphatidylinositol 3-phosphate [PtdIns(3)P] plays an important role in recruitment of various effector proteins in the endocytic and autophagic pathways. In an attempt to follow the distribution of PtdIns(3)P at the ultrastructural level, we are using the Fab1, YOTB, Vac1, and EEA1 (FYVE) domain, which is a zinc finger motif specifically binding to PtdIns(3)P. To follow PtdIns(3)P trafficking during a defined time window, here we have used a monomeric dimerizable FYVE probe, which binds with high avidity to PtdIns(3)P only after rapalog-induced dimerization.

Multivesicular endosome biogenesis in the absence of ESCRTs.

The endosomal sorting complex required for transport (ESCRT) protein machinery comprises four complexes, ESCRT-0, ESCRT-I, ESCRT-II and ESCRT-III, that facilitate receptor sorting into the lumen of multivesicular endosomes (MVEs) in order to terminate signalling receptors for final degradation within the lysosomes. Even though ESCRT proteins appear to be essential for the biogenesis of MVEs in Saccharomyces cerevisae, it is not clear whether ESCRT-independent pathways for MVE biogenesis exist in higher organisms. In this study we maximized inhibition of ESCRT-dependent pathway by depleting cells of key subunits of all four ESCRTs and followed MVE formation and epidermal growth factor (EGF) receptor (EGFR) traffic using electron and confocal microscopy.

The role of ESCRT proteins in attenuation of cell signalling.

The ESCRT (endosomal sorting complex required for transport) machinery consists of four protein complexes that mediate sorting of ubiquitinated membrane proteins into the intraluminal vesicles of multivesicular endosomes, thereby targeting them for degradation in lysosomes. In the present paper, we review how ESCRT-mediated receptor down-regulation affects signalling downstream of Notch and growth factor receptors, and how ESCRTs may control cell proliferation, survival and cytoskeletal functions and contribute to tumour suppression.

ESCRT proteins in physiology and disease.

As a mechanism of signal attenuation, receptors for growth factors, peptide hormones and cytokines are internalized in response to ligand binding, followed by degradation in lysosomes. Receptor ubiquitination is a key signal for such downregulation, and four protein complexes known as endosomal sorting complex required for transport (ESCRT)-0, -I, -II and -III have been identified as the machinery required for degradative endosomal sorting of ubiquitinated membrane proteins in yeast and metazoans. Three of these complexes contain ubiquitin-binding domains whereas ESCRT-III instead recruits deubiquitinating enzymes.

Functional multivesicular bodies are required for autophagic clearance of protein aggregates associated with neurodegenerative disease.

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.

RILP is required for the proper morphology and function of late endosomes.

Lysosomal degradation of signalling receptors such as the epidermal growth factor (EGF) receptor (EGFR) is an important mechanism for termination of cell signalling. Such degradation involves the endosomal sorting of ubiquitylated receptors into intralumenal vesicles (ILVs) of multivesicular endosomes (MVEs) that move along microtubules to fuse with perinuclear lysosomes. The Rab7-interacting lysosomal protein RILP is interesting in this context as it interacts with Vps22 (also known as EAP30) and Vps36 (also known as EAP45), subunits of the endosomal sorting complex required for transport II (ESCRT-II), as well as with the dynein-dynactin motor complex.

Vps22/EAP30 in ESCRT-II mediates endosomal sorting of growth factor and chemokine receptors destined for lysosomal degradation.

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.

The ESCRT-III subunit hVps24 is required for degradation but not silencing of the epidermal growth factor receptor.

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.