The CORVET complex: compositions, function, and impact on cellular behaviour

Abstract

The endolysosomal system is positioned on the crossroad of the intracellular and extracellular environment and is therefore crucial to regulate many cellular processes. Proper function of the endolysosomal system greatly depends on the concept of membrane identity; the controlled protein and lipid composition of all organellar membranes. Maintaining this identity requires exact timing and regulation of membrane fusion and is crucial for all transport inside the cell. Multi-subunit tethering complexes such as CORVET and HOPS are increasingly implicated as interaction hubs for many aspects of membrane fusion as well as for organelle motility and signaling. In this thesis we investigate the structure and function of the mammalian CORVET and HOPS complexes and in addition focus on the CORVET subunits Vps3 and Vps8. Compared to yeast, the composition of the mammalian CORVET and HOPS complexes are less well described. Therefore we investigated the mammalian CORVET and HOPS complexes and the mammalian specific VIPAS39/Vps33B complex and unravel their molecular architecture. Despite being highly similar protein complexes, we unraveled the mechanism that allows HOPS to bind RILP, while the same subunits in CORVET do not bind this protein. Interaction of the HOPS complex with regulating proteins such as RILP and Arl8b connects late endosomes and lysosomes to motor complexes mediating retrograde and anterograde movement. This illustrates the connection between membrane fusion and endosome motility. Further investigations on CORVET showed that this complex is dynamic in its composition and we identified a separate complex consisting of CORVET subunits. This complex is involved in endocytic recycling, a pathway different from the function of the CORVET complex in early endosome fusion. The identified recycling pathway is cargo specific and is conserved in various cell types. In the altered polarized recycling system of epithelial cells this pathway is also present, indicating a conserved role in endocytic recycling. Together our results show that the CORVET and HOPS complexes are far more complex, both in composition as well as function, than originally described. We contribute to the emerging view that multi-subunit tethering complexes are a hub for many processes at endosomes, not just membrane fusion