Recent findings suggest that Delta/Serrate/Lag2 (DSL) signals activate Notch by an unprecedented mechanism that requires the ligands to be endocytosed in signalsending cells to activate the receptor in signal-receiving cells. Here, we show that cells devoid of Epsin, a conserved adaptor protein for Clathrin-mediated endocytosis, behave normally except that they cannot send DSL signals. Surprisingly, we find that Epsin is not required for bulk endocytosis of DSL proteins. Instead, Epsin appears to be essential for targeting DSL proteins to a special endocytic pathway that they must enter to acquire signaling activity. We present evidence that DSL proteins must be monoubiquitinated to be targeted by Epsin to this pathway. Furthermore, we show that the requirements for both Epsin and mono-ubiquitination can be bypassed by introducing the internalization signal that mediates endocytosis and recycling of the Low Density Lipoprotein (LDL) receptor. We propose that Epsin is essential for DSL signaling because it targets mono-ubiquitinated DSL proteins to an endocytic recycling compartment that they must enter to be converted into active ligands. Alternatively Epsin may be required to target monoubiquitinated DSL proteins to a particular subclass of coated pits that have special properties essential for Notch activation. Wendland, 2002). Epsins were initially thought to be core components of the endocytic machinery because of the dominant-negative effects of truncated Epsin proteins on endocytosis in mammalian cells (Chen et al., 1998;Ford et al., 2002), their essential role in yeast endocytosis (Wendland et al., 1999), and their inherent capacity to induce membrane curvature (Ford et al., 2002) and bind other core components such as Clathrin and AP-2 (Chen et al., 1998;Owen et al., 1999;Rosenthal et al., 1999;Wendland et al., 1999;Drake et al., 2000). More recently, however, the identification of Ubiquitin-interacting motifs (UIMs) in Epsins, as well as in other proteins involved in membrane trafficking (Hofmann and Falquet, 2001), have led to the suggestion that Epsins belong to a family of cargoselective adapters that link mono-ubiquitinated cell-surface proteins with the endocytic machinery (Wendland, 2002).Here, we report that DSL ligands must normally be endocytosed in signal-sending cells via the action of Lqf to activate Notch on the surface of signal-receiving cells. Surprisingly, however, bulk endocytosis of DSL ligands appears normal in the absence of Lqf. We resolve this apparent paradox by providing evidence that Lqf is unique amongst adapters that target mono-ubiquitinated cargo proteins for internalization, in that it allows them to enter a special endocytic pathway that DSL ligands must enter to acquire signaling activity. We also show that this requirement can be bypassed by introducing the internalization signal that normally mediates internalization and recycling of the Low Density Lipoprotein (LDL) receptor. On the basis of these results, we hypothesize that Epsin-mediated endocytosis might be requi...
Ligands of the Delta/Serrate/Lag2 (DSL) family must normally be endocytosed in signal-sending cells to activate Notch in signal-receiving cells. DSL internalization and signaling are promoted in zebrafish and Drosophila, respectively, by the ubiquitin ligases Mind bomb (Mib)and Neuralized (Neur). DSL signaling activity also depends on Epsin, a conserved endocytic adaptor thought to target mono-ubiquitinated membrane proteins for internalization. Here, we present evidence that the Drosophila ortholog of Mib (Dmib) is required for ubiquitination and signaling activity of DSL ligands in cells that normally do not express Neur,and can be functionally replaced by ectopically expressed Neur. Furthermore,we show that both Dmib and Epsin are required in these cells for some of the endocytic events that internalize DSL ligands, and that the two Drosophila DSL ligands Delta and Serrate differ in their utilization of these Dmib- and Epsin-dependent pathways: most Serrate is endocytosed via the actions of Dmib and Epsin, whereas most Delta enters by other pathways. Nevertheless, only those Serrate and Delta proteins that are internalized via the action of Dmib and Epsin can signal. These results support and extend our previous proposal that mono-ubiquitination of DSL ligands allows them to gain access to a select, Epsin-dependent, endocytic pathway that they must normally enter to activate Notch.
The epidemic of obesity and diabetes is causing an increased incidence of dyslipidemia-related heart failure. While the primary etiology of lipotoxic cardiomyopathy is an elevation of lipid levels resulting from an imbalance in energy availability and expenditure, increasing evidence suggests a relationship between dysregulation of membrane phospholipid homeostasis and lipid-induced cardiomyopathy. In the present study, we report that the Drosophila easily shocked (eas) mutants that harbor a disturbance in phosphatidylethanolamine (PE) synthesis display tachycardia and defects in cardiac relaxation and are prone to developing cardiac arrest and fibrillation under stress. The eas mutant hearts exhibit elevated concentrations of triglycerides, suggestive of a metabolic, diabetic-like heart phenotype. Moreover, the low PE levels in eas flies mimic the effects of cholesterol deficiency in vertebrates by stimulating the Drosophila sterol regulatory element-binding protein (dSREBP) pathway. Significantly, cardiac-specific elevation of dSREBP signaling adversely affects heart function, reflecting the cardiac eas phenotype, whereas suppressing dSREBP or lipogenic target gene function in eas hearts rescues the cardiac hyperlipidemia and heart function disorders. These findings suggest that dysregulated phospholipid signaling that alters SREBP activity contributes to the progression of impaired heart function in flies and identifies a potential link to lipotoxic cardiac diseases in humans.
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