Exploration of role of Ncrlp, yeast homologue of NPC-1, in membrane trafficking

Abstract

Niemann-Pick type C disease (NPC) is a rare, autosomal recessive lipid storage disorder. NPC1 is the gene mainly responsible for the disease. The principle cellular lesion of NPC cells are defects in intracellular trafficking of cholesterol and delayed sterol homeostatic responses. Free cholesterol is accumulated in late endosome/lysosome system, which is suggestive of the cholesterol traffic defect by membrane traffic pathway. NCR1 (Niemann-Pick C related 1) is the yeast homologue of NPC1. To explore the role of Ncr1p in membrane trafficking we tested some of the vesicular traffic pathways including endocytosis, vacuole protein biosynthesis and MVB sorting, in ncr1D as well as in two class E vps mutants, vta1D and vps20D. VTA1 and VPS20 are two novel VPS4 interactors involving transport through both early endosome and late endosome and they are also important for efficient sorting of newly-synthesized vacuole proteins. ncr1D shows no obvious defects in these pathways which were tested by receptor-mediated endocytosis (alpha factor uptake and degradation assay), bulk membrane endocytosis (FM4-64 staining), fluid phase endocytosis (Lucifer yellow), vacuole protein sorting (CPY maturation), multivesicular body sorting (localization of CPS1p and Sna3p), and recycling of Golgi markers between endosome and Golgi (Kex2p, Vps10p localization). vta1D and vps20D shows defects in these pathways. The results support the existence of NPC1 compartment in cells. In this model the NPC1 vesicles recognize and package free cholesterol in the late endosome/lysosome and transport them to other organelles in the cell by transient contact between NPC vesicles and the acceptant compartment. The cargo recycling from intracellular compartments to the plasma membrane need to be checked in ncr1D in the future to further explore its function in membrane trafficking. We also showed the fluid phase endocytosis defect in arv1D, a strain having strong defects in sterol homeostasis. This supports our hypnosis that membrane trafficking plays an important role in sterol trafficking and homeostasis.