The Identification and Characterisation of Protein-Protein Interactions with the Yeast Orthologue of Niemann-Pick Type C Disease Protein-1
Niemann-Pick type C (NPC) disease is a rare lysosomal-storage disease caused by mutations in NPC1 (95% cases) or NPC2 (5% cases). It is widely accepted that NPC1 and NPC2 function together in cholesterol egress from the lysosome, whereby upon mutation, cholesterol and other lipids accumulate and cause major pathologies. Interestingly, it is not fully understood (even in healthy individuals) how cholesterol is transported from NPC1 residing at the lysosomal membrane to the endoplasmic reticulum and plasma membrane. Niemann-Pick type C related protein-1 (Ncr1), the yeast orthologue of NPC1, functions similarly to NPC1; when transfected into a mammalian cell lacking NPC1, Ncr1 rescues cholesterol and sphingolipid accumulation typically seen in diseased cells. My thesis aims to identify and characterise protein-protein interactions (PPIs) with the yeast Ncr1 protein.
In Chapter 2, I present results from a split-ubiquitin membrane yeast two-hybrid (MYTH) screen that identified six proteins interacting with Ncr1 (Cyb5, Lip1, Pga3, Phs1, Sss1 and Ysy6).Interestingly, several proteins that function in protein complexes also interact with Ncr1; Lip1 and Lag1 (components of the ceramide synthase complex) and Sss1, Sec61, Sbh1, Ssh1 and Sbh2 (components of the Sec61/Ssh1 protein translocation complex). Protein interactions with Cyb5, Lip1 and Sss1 were confirmed using co-immunoprecipitation (Co-IP) analysis, while in silico analyses supported interactions with Cyb5 and Pga3. Lastly, network analysis was conducted to highlight biological processes (protein translocation, folding/maturation of membrane proteins, and lipid metabolism) enriched in the network and thus important to the PPIs with Ncr1.
In Chapter 3, I aimed to characterise the PPIs occurring with Ncr1. To map the site of interaction on the Ncr1 protein, fragments of Ncr1 were tagged with a yellow-fluorescent protein and protein localisation assessed. Unfortunately, no fragment localised to the vacuolar membrane and subsequent MYTH analysis was not conducted on the mislocalised constructs. To map the site of interaction on the prey proteins, two complementary experiments were conducted. MYTH analyses on prey protein fragments with full-length Ncr1 identified possible regions required for the PPI (Cyb5, 68-120 aa; Lip1, 11-20 aa; Pga3, 1-175 aa; and Ysy6, 15-29 aa). Alanine mutagenesis of cytosolic protruding sequences identified regions required for the PPIs (Cyb5, 2-78 aa; Pga3, 105-215 aa; Phs1, 119-142 aa; and Sss1, 30-44 aa). To further characterise the PPIs, drugs/conditions involved in different aspects of lipid metabolism were incorporated into the MYTH analysis; Ncr1-Cyb5, Ncr1-Lip1 and Ncr1-Pga3 were observed to be important in ergosterol, fatty acid and sphingolipid metabolism.
In Chapter 4, I investigated the biological significance of the PPIs with Ncr1 via functional analyses on single and double mutant strains so to support the proposed physical interactions identified in Chapter 2 and 3. Cells lacking NCR1 and CYB5 (ncr1Δcyb5Δ) displayed stunted growth; sensitivity to the sphingolipid biosynthesis inhibitor (myriocin) and fatty acid biosynthesis inhibitor (cerulenin); increased number of lipid droplets; and dysregulated ergosterol intermediates. In addition to lipid analysis, intracellular transport via GFP-Atg8 (autophagy marker) and Mup1-GFP (endocytosis marker) analysis was investigated. Cells lacking NCR1 displayed reduced fluorophore expression in the vacuole, therefore suggesting a component of uptake at the vacuolar membrane is dysregulated. Together, this chapter highlighted several intriguing lipid metabolism phenotypes for the NCR1-CYB5 interaction.
To conclude, in this thesis I set out to identify proteins that physically interact with Ncr1. I identified 11 proteins physically interacting with Ncr1, some of which may provide further molecular insight into previously described functions of Ncr1 in ergosterol metabolism. Additionally, I predict new functions of Ncr1 (and possibly NPC1) in their function in the Cyb5/Cbr1 electron transfer system (via the Cyb5 and Pga3 interactions), the ceramide synthase complex (via the Lip1 and Lag1 interactions); fatty acid elongation (via the Phs1 interaction), and their interaction with the Sec61/Sbh1 protein translocation complex (via the Sss1, Sec61, Sbh1, Ssh1 and Sbh2 interactions). Further work is necessary to gain a more complete understanding of these proposed interactions.
