Phenotypic Analysis of NCR1, the Yeast Niemann Pick Type-C Disease Gene

In humans, the deadly neurodegenerative Niemann Pick Type-C disease (NPC) is caused by loss of function mutations in the NPC1 gene, which chiefly affect Purkinje neurons of the brain. While the underlying cause of NPC is believed to be lysosomal cholesterol accumulation, recent studies suggest that other lipid, protein and calcium defects contribute to the cellular defects in NPC. In contrast, deletion of the yeast NPC1 homologue, NCR1, does not cause any obvious growth or sterol trafficking defects. However, ncr1Δ cells are resistant to edelfosine, a phosphatidylcholine (PC) analogue drug that disrupts PC synthesis and distribution. Examination of PC trafficking in edelfosine treated ncr1Δ cells suggested that NCR1 deletion suppressed edelfosine-induced vacuolar PC trafficking defects. Ybt1p, the vacuolar membrane PC importer was found to be important for vacuolar PC uptake in edelfosine treated ncr1Δ cells. Because Ybt1p affects vacuolar calcium levels and human NPC neurons show calcium defects, ncr1Δ cells were tested for calcium phenotypes, where we found that these mutants were sensitive to high calcium concentrations. Both the ncr1∆-associated edelfosine and calcium defects were exacerbated in cells expressing multicopy LEU2, which encodes an enzyme required for leucine biosynthesis. Because leucine activates the vacuolar Target Of Rapamycin Complex 1 (TORC1), we investigated a potential link between Ncr1p and TORC1. Like ncr1Δ cells, several TORC1-associated mutants were edelfosine resistant and a mutation causing constitutive TORC1 activation exacerbated ncr1Δ edelfosine resistance. This work uncovered novel PC- and calcium-related defects in ncr1Δ yeast cells, which might provide insights into conserved functions of Ncr1p/NPC1 that relate to defects in NPC-affected Purkinje neurons. This phenotypic analysis also implicates TORC1 in some aspect of Ncr1p function in yeast.