STARD3 as a cellular switch determining organelle contact sites
Eukaryotic cells have established a sophisticated and highly coordinated system to maintain specific organellar lipid compositions. This is achieved by vesicular trafficking as well as by protein-mediated exchange of information and material at specialized sites of close membrane apposition termed organelle contact sites. By using the lysosomal storage disease Niemann-Pick disease type C (NPC) as a model, we have recently shown that the lysosomal protein NPC1 acts at lysosome-ER contact sites to facilitate lipid efflux from lysosomes. Down- regulation or loss of NPC1 reduced the number of lysosome-ER contact sites and led to lysosomal lipid accu- mulation which could be rescued by artificially tethering lysosomes to the ER. Surprisingly, loss in lysosome- ER contact sites was accompanied by an increase in lysosome-mitochondrial contact sites. Building on a previous report showing the endo/lysosomal integral membrane protein StAR-related lipid transfer domain containing protein (STARD) 3 to be required for cholesterol transport to mitochondria in NPC cells, we could demonstrate that indeed STARD3 is located at lysosome-mitochondria contacts and that depletion of STARD3 in NPC leads to loss of these lysosome-mitochondrial contacts. This finding, along with STARD3’s known role in sterol trafficking between ER and lysosomes leads us to suggest that STARD3 acts as a molecular switch which, on the one hand, interacts with ER-resident Vesicle-associate membrane proteins (VAPs) while on the other hand it is also required for the formation of lysosome-mitochondrial contact sites. The questions of how the STARD3 switch functions at the molecular level, how it is regulated and how it affects downstream cellular responses in lipid trafficking are the subjects of this proposal.
We aim to address these questions by a combination of biochemical and cell-based assays. In aim 1, we will identify STARD3 interacting partners at mitochondria by using proximity-based biotin labeling techniques and investigate whether STARD3 possesses ER- and mitochondria-specific interaction motifs. This will allow us to understand the molecular details how the STARD3 switch executes its function. In aim 2, we will identify signals that trigger STARD3 to switch between lysosome-ER and lysosome-mitochondria contact sites. We hypothe- size that the lipid content of lysosomes acts as a cue to modulate STARD3 interactions. We will test this by artificially raising lysosomal lipid levels using caged lipids as well as by creating mutants of STARD3 unable to bind sterols through its transmembrane MENTAL domain. In aim 3, we will investigate the effect of the STARD3 switch on lipid trafficking and test our hypothesis that lysosome-mitochondrial contacts serve as an alternate route for lysosomal lipids to reach the ER. To this end, we will use functionalized lipid probes and follow their metabolism as well as visualize their distribution in cells. By performing time-resolved experiments, we will measure the capacity of cells to export lipids from lysosomes and investigate whether manipulation of organelle contacts results in similarly altered lipid flows. In summary, by investigating the STARD3 switch we will gain insight into the composition and regulation of contact sites formed by lysosomes and their importance in maintaining cellular lipid homeostasis.