Unravelling HOPS dependent and independent mechanisms of VPS41 function in neuronal lysosome biogenesis

• Applicant: Professorin Judith Klumperman, Ph.D.
• Subject Area: Cell Biology
• Term: since 2017
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 323732846

Lysosomal resident proteins are synthesized in the endoplasmic reticulum, transferred to the trans-Golgi network (TGN), and from here to lysosomes. The best understood TGN-to-lysosome pathway is mannose 6-phosphate receptor (MPR) dependent transport of lysosomal hydrolases. Relatively little is known on MPR-independent TGN-to-lysosome transport pathways. Recently, we identified a MPR-independent pathway, which we refer to as LAMP (lysosome associated membrane proteins) pathway. The central vehicles in this pathway are 200 nm sized, non-coated carriers, which form at the TGN and fuse with late endosomes. In human cells they transport LAMP-1 and LAMP-2 and our preliminary data suggest that they also carry lysosomal integral membrane protein-2 (LIMP-2) and its ligand glucocerebrosidase (GBA). In Drosophila, the LAMP pathway is furthermore required for transport of the V0-ATPase and the cholesterol transporter protein Niemann Pick C1 (NPC1). Fusion of LAMP carriers with late endosomes requires the tethering protein VPS41. Recently, 3 patients with mutations in VPS41 were identified which show symptoms similar to Parkinsons disease. Interestingly, GBA (causative gene for Gaucher disease) and NPC1 (causative gene for NPC1 disease) are also implicated in Parkinsons disease. These data imply an important role for the LAMP pathway in transport of lysosomal proteins and suggest that mutations in GBA, NPC1 and VPS41 may lead to overlapping pathomechanisms. The objectives of this project are to characterize the molecular machinery of the LAMP pathway, identify its cargo proteins and reveal its role in lysosome and cellular physiology. Our studies will be in collaboration with various partners of the consortium and especially make use of the proteomics expertise of the Gieselmann/Winter group. Moreover, we will make our own extensive expertise in electron microscopy (EM) available to this consortium. To identify the molecular machinery of the LAMP pathway we will test known interactors of VPS41 for their role in LAMP trafficking and identify novel VPS41 interactors by co-immuno-precipitation and mass spectrometry. We will establish the role of the LAMP pathway in transport of putative cargo molecules, like GBA and NPC1, by live cell transport assays and immuno-EM. Moreover, we will perform proteomics of enriched LAMP carriers to detect additional cargo proteins. Finally, we will analyze lysosomal protein contents of control cells and VPS41 mutant or depleted cells by proteomics and of the cellular metabolome by metabolomics. Addressing these questions will reveal fundamental insights in lysosomal protein transport and may yield essential information to understand the phenotype of VPS41 patients and related pathologies.

• DFG Programme: Research Units
• Subproject of: FOR 2625: Mechanisms of Lysosomal Homeostasis
• International Connection: Netherlands