Role of the interaction of AP-3 and the HOPS tethering complex in intracellular protein transport

• Applicant: Dr. Alexander Stockhammer
• Subject Area: Biochemistry ; Cell Biology
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 564459937

Although studied for many years, the role and mechanism of action of AP-3 in intracellular protein transport in metazoan cells remains poorly understood. In yeast, an AP-3 pathway that directly connects the Golgi with the vacuole is well established, and interaction of the Vps41 subunit of the HOPS tethering complex and AP-3 has been shown to be crucial for the AP-3 pathway to function efficiently. Nevertheless, it is still unclear how AP-3 vesicles are recognized, how their interaction with HOPS is regulated and when and how they shed the AP-3 coat to allow fusion with the vacuolar membrane. The designed project aims to address the pressing question of the role of the AP-3 coat in intracellular transport and how the interaction with HOPS regulates AP-3 trafficking. Therefore, I will work in two different model organisms, yeast and mammalian cells, to benefit from advantages of each model organism and overcome inherant limitiations. The ultimate goal is to understand to understand whether and to which degree the AP-3 function and the AP-3 pathway is conserved across species. The work program includes the generation of different CRISPR-knock-in cell lines, to visualize AP-3, HOPS as well as the underlying membranes simultaneously in metazoan cells using multi-color live-cell imaging. This will clarify if and where there the interaction of AP-3 with HOPS takes place. In addition, fast-time lapse imaging will resolve the dynamic nature of the interaction and the fate of the AP-3 coat after the encounter with the HOPS complex. Furthermore, I will employ interactome mapping in yeast and mammalian cells to reveal which proteins are recruited specifically to sites of AP-3/HOPS interaction. Candidate proteins will then be probed for their role regarding AP-3 trafficking. By doing so, I will be able to understand the underlying mechanism triggered once AP-3 interacts with the HOPS complex. This project will answer the long-standing question of to which degree the AP-3 pathway is conserved from yeast to mammals and will also shed light on the role of HOPS in the context of AP-3. Until now, the adaptor protein complexes were thought to exclusivly act in cargo enrichment and vesicle formation, an additional role in uncoating and membrane fusion would transform the view we have on the role of adaptor proteins in intracellular transport.

• DFG Programme: WBP Position