Sterol-modulated Glycolipid Sorting Occurs in Niemann-Pick C1 Late Endosomes

Abstract

The Niemann-Pick C1 (NPC1) protein and endocytosed low density lipoprotein (LDL)-derived cholesterol were shown to enrich separate subsets of vesicles containing lysosomal associated membrane protein 2. Localization of Rab7 in the NPC1-containing vesicles and enrichment of lysosomal hydrolases in the cholesterolcontaining vesicles confirmed that these organelles were late endosomes and lysosomes, respectively. Lysobisphosphatidic acid, a lipid marker of the late endosomal pathway, was found in the cholesterol-enriched lysosomes. Recruitment of NPC1 to Rab7 compartments was stimulated by cellular uptake of cholesterol. The NPC1 compartment was shown to be enriched in glycolipids, and internalization of GalNAc␤1-4[NeuAc␣2-3]Gal␤1-4Glc␤1-1-ceramide (G M2 ) into endocytic vesicles depends on the presence of NPC1 protein. The glycolipid profiles of the NPC1 compartment could be modulated by LDL uptake and accumulation of lysosomal cholesterol. Expression in cells of biologically active NPC1 protein fused to green fluorescent protein revealed rapidly moving and flexible tubular extensions emanating from the NPC1-containing vesicles. We conclude that the NPC1 compartment is a dynamic, sterolmodulated sorting organelle involved in the trafficking of plasma membrane-derived glycolipids as well as plasma membrane and endocytosed LDL cholesterol.

Cessation of rapid late endosomal tubulovesicular trafficking in Niemann–Pick type C1 disease

“…Endocytosed cell surface receptors and their ligands enter an early tubular endosomal pathway that sorts ligand-receptor complexes recycling to the plasma membrane from others destined to travel through the later arm of the endosomal pathway (21). Endocytic uptake of LDL recruits NPC1 protein into a Rab 7 positive late endosomal compartment (7). NPC1-GFP is visualized to be trafficking in living cells between late endosomal compartments and lysosomes, via an extensive system of branching tubules with rapid rates of movement in the range of 1-5 m͞s.…”

“…Cells were fixed with 3% paraformaldehyde in PBS for 30 min to 1 h, washed in PBS, and immunostained with rabbit anti-NPC1 antiserum (7,9), mouse IgG against CHO LAMP1 and CHO LAMP2 (UH1 and UH3) (monoclonal antibodies were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by the Department of Biological Sciences, University of Iowa, Iowa City), and monoclonal IgG against ␣-tubulin (clone DM1A; Sigma).…”

“…Retroendocytic clearance of fluid phase markers, such as sucrose, from late endosomes is retarded by LDL-derived cellular enrichment of cholesterol (9). We have recently shown that cholesterol modulates glycolipid sorting from NPC1-containing late endosomes into lysosomes (7). In this report, we have visualized functional green fluorescent protein-labeled NPC1 protein (NPC1-GFP) trafficking in living cells.…”

“…Cellular cholesterol enrichment, through endocytic uptake of low-density lipoprotein (LDL), alters the intracellular distribution of NPC1 protein from a finely dispersed granular pattern to sequestration in prominent cytoplasmic vesicles (7,8). These NPC1-containing vesicles are positive for lysosomal-associated membrane protein 2 (LAMP2) (7,9), are not enriched in lysosomal hydrolases (7), and are positive for the late endosomal marker proteins Rab7 (7) and Rab 9 (10). NPC1-containing late endosomes are critical for the retroendocytic trafficking of multiple lysosomal cargo (4,9).…”

“…After biosynthesis of NPC1 in the endoplasmic reticulum, the protein is processed further in the Golgi apparatus (6); at steady state, it is associated with late endosomes. Cellular cholesterol enrichment, through endocytic uptake of low-density lipoprotein (LDL), alters the intracellular distribution of NPC1 protein from a finely dispersed granular pattern to sequestration in prominent cytoplasmic vesicles (7,8). These NPC1-containing vesicles are positive for lysosomal-associated membrane protein 2 (LAMP2) (7, 9), are not enriched in lysosomal hydrolases (7), and are positive for the late endosomal marker proteins Rab7 (7) and Rab 9 (10).…”