Spatiotemporal dynamics and function of multisubunit trafficking complexes with dual functionality within the endolysosomal pathway

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Several lines of evidence indicate that the lysosomal/vacuolar HOPS and endosomal CORVET complexes are far more dynamic than anticipated from the profile of the purified hexameric complexes on gels. In yeast, and possibly in higher eukaryots, endosomes seem to accumulate at distinct sites proximal to the vacuole, which can be enhanced by overexpression of the CORVET subunit Vps8. Likewise, Vps39 has at least two additional functions outside of HOPS, both in vCLAMP formation and in regulating the TORC1. How these Rab-specific subunits disassemble from their hexamers and how this is regulated and controlled remains largely unexplored. Within this project, we will dissect how HOPS and CORVET assemble and disassemble to release individual subunits that function in other trafficking-related processes beyond tethering and fusion, both in yeast, and later in metazoan cells. In Aim 1, we will reveal the dynamics of the Rab-specific subunits of CORVET and HOPS with a focus on their distribution, regulation of complex assembly and disassembly, and functions beyond the tethering complexes. In particular, we will address the regulation of the Vps39 function at TORC1. Aim 2 will focus on the role of the hybrid complex of HOPS Vps41 and CORVET Vps3, where its formation, localization, function, and physiology will be clarified. In Aim 3, insights on HOPS (and later CORVET) dynamics will be transferred into metazoan cells by monitoring dynamics and regulation of Vps41 and Vps39 relative to known Rabs and coat proteins such as AP-3. We will thus reveal novel insights into the largely unexplored assembly and disassembly of HOPS and CORVET and its role in endolysosomal function.