Lethal (2) giant discs (Lgd)/CC2D1 is required for the full activity of the ESCRT machinery
Authors: Baeumers Miriam , Ruhnau Kristina , Breuer Thomas , Pannen Hendrik , Goerlich Bastian , Kniebel Anna , Haensch Sebastian , Weidtkamp-Peters Stefanie , Schmitt Lutz , Klein Thomas 
Published: December 2020

Background

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 inDrosophila, 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 inDrosophilaand mammals, respectively.

Results

Here, we show that the loss of function oflgdconstitutes a state of reduced activity of Shrub/CHMP4/ESCRT-III. This hypomorphicshrubmutant 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 oflgdmutant MEs suppresses the uncontrolled and ligand-independent activation of Notch. Moreover, the analysis ofSu(dx) lgddouble mutants clarifies their relationship and suggests that they are not operating in a linear pathway. We could show that, despite prolonged lifetime, the MEs oflgdmutants have a similar ILV density as wild-type but less thanrab7mutant MEs, suggesting the rate inlgdmutants 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. Sincelgdmutants can be rescued to normal adult flies if extra copies ofshrub(or its mammalian orthologCHMP4B) are added into the genome, we conclude that the net activity of Shrub is reduced upon loss oflgdfunction. 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.

Conclusion

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.