Fuhrmann Fonden - Molecular dissection of sterol transport through the vacuole by yeast homolog of Niemann-Pick type C proteins 1 and 2

In mammalian cells, the Niemann-Pick type C proteins, NPC1 and NPC2, are required for redistribution of sterols from lysosomes to other membranes by binding and transferring cholesterol derived from endocytosed low-density lipoproteins. Absence of one of these proteins leads to accumulation of cholesterol and other lipids in lysosomes. This disease is known as the neurodegenerative disorder, Niemann-Pick disease (Kwon, Abi-Mosleh et al. 2009, Wang, Motamed et al. 2010, Tsuji, Fujimoto et al. 2017). NPC2 is a small soluble protein localized in the lysosomal lumen (Wang, Motamed et al. 2010), whereas NPC1 is a large integral protein in the limiting membrane of lysosomes. Its N-terminal domain (NTD) projects into the lumen of the lysosome, where NPC1 likely interacts with NPC2 for sterol transfer according to the so-called ‘hydrophobic hand-off’ model (Kwon, Abi-Mosleh et al. 2009). In this model, NPC1 subsequently transfers the sterol across the glycocalyx layer and into the lysosomal membrane. Parts of the molecular mechanism for sterol transfer through this partway remains to be elucidated. This can be done using yeast proteins and cells due to the relatively high similarity between the yeast proteins and human proteins and the fact that yeast is more easily amenable for genetic and experimental manipulation (Berger, Hanson et al. 2005, Berger, Vanderford et al. 2005).
The experiments for this project will be conducted in the laboratory of Assoc. Prof. Daniel Wüstner at the Department of Biochemistry and Molecular Biology and under his guidance and supervision.
The results of my ISA indicate that the yeast vacuole engulfs lipids droplets containing sterols during starvation (in my experiments tested by imaging the fluorescent ergosterol analog, DHE) in vivo (Winkler, Kidmose et al. 2019). During my master thesis, the dynamic between lipid droplets, vacuole and NCR1 and perhaps NPC2 will be studied in molecular detail. To support the data obtained in my ISA, lipid autophagy facilitated by the vacuole will be investigated with highresolution microscopy and in cells with no/inhibited lipases in the cytosol and/or in the vacuole. This will help address the location of the lipid droplets during the hydrolysis. Besides that, isolation of vacuoles will enable many interesting experiments. Isolating vacuoles at different timepoints after introducing a starving cell to DHE will also reveal when and if the DHE is inside the vacuole. The interaction between NCR1 and NPC2 in isolated vacuoles will reveal more about the sterol transferring mechanism. The third part of the experiments will be to examine the dynamics of NCR1 including the distribution and movement of the protein, and the lateral organization of functional NCR1 and whether it changes under starving conditions.