ESCRT proteins and cell signalling.

Subunits of the endosomal sorting complex required for transport (ESCRT) were identified as components of a molecular machinery that sorts ubiquitinated membrane proteins into the intraluminal vesicles (ILVs) of multivesicular endosomes (MVEs) for subsequent delivery to the lumen of lysosomes or related organelles. As many of the membrane proteins that undergo ESCRT-mediated sorting are signalling receptors that are ubiquitinated in response to ligand binding, ESCRT subunits have been hypothesized to play a crucial role in attenuation of cell signalling by mediating ligand-induced receptor degradation. Here we discuss this concept based on the examples from loss-of-function studies in model organisms and cell lines. The emerging picture is that ESCRTs are indeed involved in downregulation of receptor signalling pathways associated with cell survival, proliferation and polarity. In addition, the recent discovery of a positive role for the ESCRT pathway in Wnt signalling through sequestration of an inhibitory cytosolic component into MVEs illustrates that ESCRTs may also control signalling in ways that are independent of degradative receptor sorting.

ESCRTs and Fab1 regulate distinct steps of autophagy.

Eukaryotes use autophagy to turn over organelles, protein aggregates, and cytoplasmic constituents. The impairment of autophagy causes developmental defects, starvation sensitivity, the accumulation of protein aggregates, neuronal degradation, and cell death [1, 2]. Double-membraned autophagosomes sequester cytoplasm and fuse with endosomes or lysosomes in higher eukaryotes [3], but the importance of the endocytic pathway for autophagy and associated disease is not known. Here, we show that regulators of endosomal biogenesis and functions play a critical role in autophagy in Drosophila melanogaster. Genetic and ultrastructural analysis showed that subunits of endosomal sorting complex required for transport (ESCRT)-I, -II and -III, as well as their regulatory ATPase Vps4 and the endosomal PtdIns(3)P 5-kinase Fab1, all are required for autophagy. Although the loss of ESCRT or Vps4 function caused the accumulation of autophagosomes, probably because of inhibited fusion with the endolysosomal system, Fab1 activity was necessary for the maturation of autolysosomes. Importantly, reduced ESCRT functions aggravated polyglutamine-induced neurotoxicity in a model for Huntington's disease. Thus, this study links ESCRT function with autophagy and aggregate-induced neurodegeneration, thereby providing a plausible explanation for the fact that ESCRT mutations are involved in inherited neurodegenerative disease in humans [4].

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.

Fab1 phosphatidylinositol 3-phosphate 5-kinase controls trafficking but not silencing of endocytosed receptors.

The trafficking of endocytosed receptors through phosphatidylinositol 3-phosphate [PtdIns(3)P]-containing endosomes is thought to attenuate their signaling. Here, we show that the PtdIns(3)P 5-kinase Fab1/PIKfyve controls trafficking but not silencing of endocytosed receptors. Drosophila fab1 mutants contain undetectable phosphatidylinositol 3,5-bisphosphate levels, show profound increases in cell and organ size, and die at the pupal stage. Mutant larvae contain highly enlarged multivesicular bodies and late endosomes that are inefficiently acidified. Clones of fab1 mutant cells accumulate Wingless and Notch, similarly to cells lacking Hrs, Vps25, and Tsg101, components of the endosomal sorting machinery for ubiquitinated membrane proteins. However, whereas hrs, vps25, and tsg101 mutant cell clones accumulate ubiquitinated cargo, this is not the case with fab1 mutants. Even though endocytic receptor trafficking is impaired in fab1 mutants, Notch, Wingless, and Dpp signaling is unaffected. We conclude that Fab1, despite its importance for endosomal functions, is not required for receptor silencing. This is consistent with the possibility that Fab1 functions at a late stage in endocytic receptor trafficking, at a point when signal termination has occurred.

Disruption of Vps4 and JNK function in Drosophila causes tumour growth.

Several regulators of endocytic trafficking have recently been identified as tumour suppressors in Drosophila. These include components of the endosomal sorting complex required for transport (ESCRT) machinery. Disruption of subunits of ESCRT-I and -II leads to cell-autonomous endosomal accumulation of ubiquitinated receptors, loss of apicobasal polarity and epithelial integrity, and increased cell death. Here we report that disruption of the ATPase dVps4, the most downstream component of the ESCRT machinery, causes the same array of cellular phenotypes. We find that loss of epithelial integrity and increased apoptosis, but not loss of cell polarity, require the activation of JNK signalling. Abrogation of JNK signalling prevents apoptosis in dVps4 deficient cells. Indeed double deficiency in dVps4 and JNK signalling leads to the formation of neoplastic tumours. We conclude that dvps4 is a tumour suppressor in Drosophila and that JNK is central to the cell-autonomous phenotypes of ESCRT-deficient cells.