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Browsing by Author "Bradshaw, Amy R."

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    Two specific ESCRT components rescue the sphingolipid-mediated intracellular accumulation of ergosterol
    (Te Herenga Waka—Victoria University of Wellington, 2015) Bradshaw, Amy R.; Munkacsi, Andrew
    Niemann-Pick type C (NP-C) disease is a lysosomal storage disorder characterised by the accumulation of cholesterol and sphingolipids in the late endosome/lysosome. The primary cause of NP-C disease is mutations in NPC1, a lysosomal transmembrane protein. As NPC1 is functionally conserved from yeast to humans, here I investigated the conditional synthetic lethal interaction between Ncr1p (the yeast ortholog of human NPC1) and the F-box recycling protein Rcy1p in the presence of the sphingolipid inhibitor myriocin. To determine if this lethality could be rescued by the deletion of another gene, a Synthetic Genetic Array (SGA) analysis was utilized to identify two endosomal sorting complexes required for transport (ESCRT) proteins, Vps20p and Vps22p that are each required for this lethality. Fluorescent microscopy analysis of unesterified ergosterol in ncr1Δ rcy1Δ treated with a sub-lethal dose of myriocin resulted in the intracellular accumulation of unesterified ergosterol, while the additional deletion of Vps20p or Vps22p restored the normal transport of ergosterol in myriocin-treated ncr1Δ rcy1Δ cells. Vps20p and Vps22p are members of the ESCRT-II and ESCRT-III complexes, respectively. Fifteen additional gene deletions representing ESCRT-0, ESCRT-I, ESCRT-II and ESCRT-III were evaluated as genetic suppressors, and not one of these gene deletions suppressed the conditional synthetic lethal NCR1-RCY1 interaction. The exact mechanisms underlying this sphingolipid-mediated sterol accumulation regulated by specific components of the ESCRT machinery remain unresolved and are critical to developing myriocin-treated ncr1Δ rcy1Δ as the first yeast model to phenocopy the intracellular accumulation of cholesterol that is a tool in the diagnosis of human NP-C disease.