title: Cholesterol accumulation sequesters Rab9 and disrupts late endosome function in NPC1-deficient cells.
abstract: Niemann-Pick type C disease is an autosomal recessive disorder that leads to massive accumulation of cholesterol and glycosphingolipids in late endosomes and lysosomes. To understand how cholesterol accumulation influences late endosome function, we investigated the effect of elevated cholesterol on Rab9-dependent export of mannose 6-phosphate receptors from this compartment. Endogenous Rab9 levels were elevated 1.8-fold in Niemann-Pick type C cells relative to wild type cells, and its half-life increased 1.6-fold, suggesting that Rab9 accumulation is caused by impaired protein turnover. Reduced Rab9 degradation was accompanied by stabilization on endosome membranes, as shown by a reduction in the capacity of Rab9 for guanine nucleotide dissociation inhibitor-mediated extraction from Niemann-Pick type C membranes. Cholesterol appeared to stabilize Rab9 directly, as liposomes loaded with prenylated Rab9 showed decreased extractability with increasing cholesterol content. Rab9 is likely sequestered in an inactive form on Niemann-Pick type C membranes, as cation-dependent mannose 6-phosphate receptors were missorted to the lysosome for degradation, a process that was reversed by overexpression of GFP-tagged Rab9. In addition to using primary fibroblasts isolated from Niemann-Pick type C patients, RNA interference was utilized to recapitulate the disease phenotype in cultured cells, greatly facilitating the analysis of cholesterol accumulation and late endosome function. We conclude that cholesterol contributes directly to the sequestration of Rab9 on Niemann-Pick type C cell membranes, which in turn, disrupts mannose 6-phosphate receptor trafficking.
authors.affiliation: Department of Biochemistry, Stanford University School of Medicine, 279 Campus Drive, Stanford, CA 94305-5307, USA.
authors.forename: Ian G
authors.initials: IG
authors.lastname: Ganley
authors.forename: Suzanne R
authors.initials: SR
authors.lastname: Pfeffer
mesh_terms: D000818:Animals; D001692:Biological Transport / Q000502:physiology; D002352:Carrier Proteins / Q000235:genetics*; D002784:Cholesterol / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology; D006028:Glycosphingolipids / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007313:Insecta / Q000166:cytology; D007425:Intracellular Membranes / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins / Q000172:deficiency / Q000235:genetics*; D000077296:Niemann-Pick C1 Protein; D009542:Niemann-Pick Diseases / Q000473:pathology / Q000503:physiopathology*; D010641:Phenotype; D017368:Protein Prenylation; D021381:Protein Transport / Q000502:physiology; D017527:Receptor, IGF Type 2 / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D006028:Glycosphingolipids; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; C106881:NPC1 protein, human; D000077296:Niemann-Pick C1 Protein; D017527:Receptor, IGF Type 2; D002784:Cholesterol; C079856:RAB9A protein, human; D020691:rab GTP-Binding Proteins