Organelle specific rules for the self-assembly of ESCRT-III

Membrane remodeling is essential for the biogenesis and function of organelles in eukaryotic cells. The endosomal sorting complexes required for transport (ESCRT) form one of the most fascinating and versatile membrane remodeling machineries. The inventory of the ESCRT machinery is well characterized. Its components transiently assemble on membranes of endosomes, the plasma membrane (PM) and at the inner nuclear membrane (INM) and, by processes that are incompletely understood, mediate task-specific membrane remodeling. Common to these different processes is the assembly of ESCRT-III filaments and their interaction with Vps4, a AAA (ATPase associated with a variety of cellular activities) ATPase. Despite the wealth of structural and biophysical information on ESCRT-III, the rules that govern the self-assembly of ESCRT-III subunits into functional filaments are only partially understood in vivo. This knowledge gap prevents us from understanding how ESCRT-III / Vps4 assemblies form and function on different organelles. We do not know if the molecular architecture and organization of ESCRT-III filaments is organelle-specific and adapted to specific biological tasks or if the formation of ESCRT-III assemblies follows a set of general rules. In particular, we lack information on how the ESCRT-III subunits Vps20, Vps24 and Vps2 interact with each other and with Snf7 protofilaments to assemble into functional ESCRT-III heteropolymers on endosomes, at the PM and at the INM. To fill this knowledge gap, we will generate new tools to probe the connectivity of ESCRT-III subunits in the assembling of the filaments in vivo. Therefore, we will combine detailed genetic interaction mapping in budding yeast (S. cerevisiae) and fission yeast (S. japonicus) with live cell imaging and biochemical approaches including different cross-linking approaches. We expect to define for the first time how ESCRT-III subunits interact with each other to self-assemble into functional ESCRT-III hetero-filaments that recruit Vps4 on endosomes, at the PM and at the INM. These results will pave the way to establish organelle specific rules for the assembly of ESCRT-III filaments. In a wider context the results of this research project will help to understand how cells shapes their membranes to maintain organelle function and cellular integrity.

Eukaryotic cells rely on molecular machines for the biogenesis of their organelles. One of the most fascinating of these machines is the endosomal sorting complex required for transport (ESCRT), which can remodel membranes and thereby plays a central role in determining organelle shape and function. The components of the ESCRT machinery are now well characterized. It has also been established that ESCRT assembles transiently at diverse membranes, including the plasma membrane, endo-lysosomal compartments, and the inner nuclear membrane. Intriguingly, at each of these sites ESCRT fulfills organelle-specific functions, all of which are associated with membrane remodeling. How the ESCRT machinery catalyzes these distinct processes, however, is not fully understood. In this project, we address this question by characterizing the assembly of ESCRT at the plasma membrane and at endosomes in molecular detail. To achieve this in living cells, we have developed new molecular tools and apply a combination of genetic, biochemical, and imaging approaches in Saccharomyces cerevisiae, which represents the best-suited model system for this purpose. Our findings show that while ESCRT complexes at endosomes and at the stressed plasma membrane rely on the same intermolecular interactions, they nevertheless operate differently: at endosomes, ESCRT assemblies are smaller and less dynamic, driving the formation of intraluminal vesicles. At the plasma membrane, by contrast, ESCRT assemblies are larger and more dynamic, where they function to respond to fluctuations in sphingolipid homeostasis and prevent membrane damage. Together, these results define a molecular rule set for ESCRT assembly at different organelles. More broadly, they provide new insight into the processes underlying organelle formation, function, and repair, thereby advancing our understanding of the molecular mechanisms that shape the architecture of eukaryotic cells.