Membrane recruitment and removal of the ESCRT-III regulator Lethal (2) giant discs/CC2D1A by the AAA-ATPase Vps4 during ESCRT mediated formation of intraluminal vesicles

• Applicant: Professor Dr. Thomas Klein
• Subject Area: Developmental Biology
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 558314618

The ESCRT machinery is the only known device in cells that can abscise membranes away from the cytosol and is used in many fundamental processes. One prime example is the formation of intraluminal vesicles (ILVs), which are abscised from the limiting membrane (LM) of maturing endosomes (ME). The machinery consists of four in sequence acting complexes with ESCRT-III at its core. ESCRT-III forms a polymer, consisting of Shrub protomers, at the LM that abscise the membrane in concert with the AAA-ATPase Vps4 in a manner that is not understood. During abscission, the polymer is disassembled into its Shrub monomers that reside in the cytosol in an inactive closed form until recruitment to the LM for the next round of ILV formation. The disassembly of the ESCRT-III polymer requires the interaction of MIM domains of the ESCRT members with the MIT domain of Vps4. The activity of Shrub/ESCRT-III is regulated by the evolutionary conserved tumoursuppressor Lethal (2) giant discs (Lgd). Lgd directly interact with Shrub and, together with Shrub, it cycles between the cytosol and the LM of the ME in a Vps4-dependent manner. Our preliminary data suggests that Lgd enhances the disassembly of the Shrub/ESCRT-III polymer by Vps4. Moreover, the sequence analysis combined with Alphafold2 predictions revealed so far unrecognised domains in Lgd family members and a proline-rich sequence (PR) in each of the family-specific DM14 domains. The PR resembles the consensus sequence of MIM2 domains. Using the plethora of genetic and molecular techniques of Drosophila, we will analyse the membrane recruitment and removal of Lgd/CC2D1A by Vps4. To do so, we will perform a structure/function analysis of the previously unrecognised domains of Lgd and will clarify how Lgd is recruited to the LM. We focus on the hypothesis that the PRs of the DM14s are functional MIM2 domains that are required for the Vps4-dependent removal of Lgd from the LM of the ME, together with attached Shrub oligomers. The results will reveal the function of LGD family proteins in the regulation of one of a fundamental machinery of cells.

• DFG Programme: Research Grants