Transport of newly synthesized sterol to the sterol-enriched plasma membrane occurs via nonvesicular equilibration.

The mechanism by which newly synthesized sterols are transported from their site of synthesis, the endoplasmic reticulum (ER), to the sterol-enriched plasma membrane (PM) is not fully understood. Studies in mammalian cells suggest that newly synthesized cholesterol is transported to the PM in Golgi-bypassing vesicles and/or via a nonvesicular process. Using the yeast Saccharomyces cerevisiae as a model system, we now rule out an essential role for known vesicular transport pathways in transporting the major yeast sterol, ergosterol, from its site of synthesis to the PM. We use a cyclodextrin-based sterol capture assay to show that transport of newly synthesized ergosterol to the PM is unaltered in cells defective in Sec18p, a protein required for almost all intracellular vesicular trafficking events; we also show that transport is not blocked in cells that are defective in formation of transport vesicles at the ER or in vesicle fusion with the PM. Our data suggest instead that transport occurs by equilibration (t(1/2) approximately 10-15 min) of ER and PM ergosterol pools via a bidirectional, nonvesicular process that is saturated in wild-type exponentially growing yeast. To reconcile an equilibration process with the high ergosterol concentration of the PM relative to ER, we note that a large fraction of PM ergosterol is found condensed with sphingolipids in membrane rafts that coexist with free sterol. We propose that the concentration of free sterol is similar in the PM and ER and that only free (nonraft) sterol molecules have access to a nonvesicular transport pathway that connects the two organelles. This is the first description of biosynthetic sterol transport in yeast.

Niemann-Pick type C disease: importance of N-glycosylation sites for function and cellular location of the NPC2 protein.

Niemann-Pick disease type C (NPC), a neurovisceral disorder characterized by accumulation of cholesterol and glycolipids in the lysosomal/late endosomal system, is due to mutations on either the NPC1 or the NPC2 genes. Although NPC1 and NPC2 proteins appear essential for proper cellular cholesterol trafficking, their precise functions and relationship have remained elusive. Mutation identification in NPC2 patients did not provide insights into structure-function relationships, but recent studies brought important information on the cholesterol-binding site of the NPC2 protein. The present work was focused on localization and N-glycosylation of NPC2, considering that glycosylation is often essential for targeting, stability and biological function of proteins. Using immunocytofluorescence in cultured human fibroblasts, we found that the native NPC2 protein is essentially lysosomal, at variance with the late endosomal location of NPC1. Expression of cDNA mutants affecting each of the three potential NPC2 N-glycosylation sites in NPC2-/- fibroblasts showed that only two sites are used. The intracellular human NPC2 protein occurred as two N-glycosylated forms, with either one single oligosaccharide chain attached to Asn 58 or two oligosaccharides attached to Asn 58 and 135. The oligosaccharidic chains were of the hybrid and/or high mannose type, with no complex chains. Further studies on the cellular location of Asn 58 and Asn 135 mutant proteins and their respective effect on restoration of normal cholesterol traficking in NPC2-/- cells led to the conclusion that only the oligosaccharide chain carried by Asn 58 is responsible for proper targeting of NPC2 to lysosomes, and is crucial for NPC2 function.