Investigation of the Lipid Transfer ProteinVPS13D at the Golgi Apparatus - Project Summary/Abstract The formation and maintenance of membranes is essential for the health of the nervous system. Consistent with the unique sensitivity of the nervous system to lipid dynamics, each of the four mammalian paralogues in the VPS13 family of proteins, which were recently proposed to function as bridges that connect lipid synthesis in the ER to recipient membranes for the nonvesicular transfer of lipids (Leonzino et al., 2021), are associated with neurodegenerative and neurodevelopmental disease. Of the four VPS13 paralogues, VPS13D is the only one essential for life. While full loss of function is not compatible even with cellular life, partial loss of function of VPS13D leads to neurodegenerative diseases, such as spastic ataxia, which motivates this proposal to investigate mechanisms of VPS13D-dependent neurodegenerative disease. Previous studies showed that exogenous, and thus overexpressed, VPS13D resides at the Golgi apparatus and also at contacts between the ER, via VAP, and both mitochondria and peroxisomes via an interaction with Miro (Guillen-Samander et al., 2021). This proposal seeks to understand VPS13D’s still-unknown role at the Golgi complex, in spite of its prominent localization at this organelle using cellular models. The first aim is to elucidate the precise localization of VPS13D within the Golgi complex, where preliminary data suggests an association of overexpressed VPS13D with the trans Golgi/TGN. I established an endogenously tagged VPS13D^V5 line that I find localizes at the Golgi apparatus, wherein I propose to investigate its precise localization using nocodazole ministacks, superresolution, and immune-electron microscopy. The second aim focuses on how VPS13D is recruited to the Golgi complex. Proteomics screens based on proximity labeling will be used to complement co-immunoprecipitation of endoVPS13D^V5 towards the identification of binding partners on membranes of this organelle. In preliminary proximity labelling mass spectrometry experiments, I identified candidates at the TGN, which I propose methods to test. In the final aim, my preliminary data show that VPS13D depletion leads to a selective dispersion of TGN46 and impaired trafficking of its associated cargo, Lysozyme C, implicating VPS13D, and thus its putative lipid transport function, in the maintenance of the TGN. Furthermore, I find that VPS13D loss leads to decreased ability of membranes to form ordered raft- like domains, which are critical for sorting at the TGN. Proposed experiments include investigating the effect of VPS13D loss on purified Golgi with lipidomics, and how my findings from cell lines translate to conditional knockout mice and cells derived from them.
