Lethal (2) giant discs (Lgd)/CC2D1 is required for the full activity of the ESCRT machinery

A major task of the endosomal sorting complex required for transport (ESCRT) machinery is the pinching off of cargo-loaded intraluminal vesicles (ILVs) into the lumen of maturing endosomes (MEs), which is essential for the complete degradation of transmembrane proteins in the lysosome. The ESCRT machinery is also required for the termination of signalling through activated signalling receptors, as it separates their intracellular domains from the cytosol. At the heart of the machinery lies the ESCRT-III complex, which is required for an increasing number of processes where membrane regions are abscised away from the cytosol. The core of ESCRT-III, comprising four members of the CHMP protein family, organises the assembly of a homopolymer of CHMP4, Shrub in Drosophila, that is essential for abscission. We and others identified the tumour-suppressor lethal (2) giant discs (Lgd)/CC2D1 as a physical interactor of Shrub/CHMP4 in Drosophila and mammals, respectively.

Here, we show that the loss of function of lgd constitutes a state of reduced activity of Shrub/CHMP4/ESCRT-III. This hypomorphic shrub mutant situation causes a slight decrease in the rate of ILV formation that appears to result in incomplete incorporation of Notch into ILVs. We found that the forced incorporation in ILVs of lgd mutant MEs suppresses the uncontrolled and ligand-independent activation of Notch. Moreover, the analysis of Su(dx) lgd double mutants clarifies their relationship and suggests that they are not operating in a linear pathway. We could show that, despite prolonged lifetime, the MEs of lgd mutants have a similar ILV density as wild-type but less than rab7 mutant MEs, suggesting the rate in lgd mutants is slightly reduced. The analysis of the MEs of wild-type and mutant cells in the electron microscope revealed that the ESCRT-containing electron-dense microdomains of ILV formation at the limiting membrane are elongated, indicating a change in ESCRT activity. Since lgd mutants can be rescued to normal adult flies if extra copies of shrub (or its mammalian ortholog CHMP4B) are added into the genome, we conclude that the net activity of Shrub is reduced upon loss of lgd function. Finally, we show that, in solution, CHMP4B/Shrub exists in two conformations. LGD1/Lgd binding does not affect the conformational state of Shrub, suggesting that Lgd is not a chaperone for Shrub/CHMP4B.

Our results suggest that Lgd is required for the full activity of Shrub/ESCRT-III. In its absence, the activity of the ESCRT machinery is reduced. This reduction causes the escape of a fraction of cargo, among it Notch, from incorporation into ILVs, which in turn leads to an activation of this fraction of Notch after fusion of the ME with the lysosome. Our results highlight the importance of the incorporation of Notch into ILV not only to assure complete degradation, but also to avoid uncontrolled activation of the pathway.