Niemann-Pick type C (NPC) proteins are essential for sterol homeostasis, believed to drive sterol integration into the vacuolar/lysosomal membrane before redistribution to other cellular membranes. Here, using a combination of crystallography, cryo-electron microscopy, biochemical and in vivo studies on the Saccharomyces cerevisiae NPC system, NCR1/NPC2, we present a framework for sterol membrane integration. Sterols are transferred between hydrophobic pockets of vacuolar NPC2 and membrane-protein NCR1. NCR1 has its N terminal domain (NTD) positioned to deliver a sterol to a tunnel connecting NTD to the luminal membrane leaflet 50 Å away. A sterol is caught inside this tunnel during transport, and a proton-relay network of charged residues in the transmembrane region is linked to this tunnel supporting a proton-driven transport mechanism. We propose a model for sterol integration which clarifies the role of NPC proteins in this essential eukaryotic pathway and which rationalizes mutations in patients with Niemann-Pick disease Type C.

Cholesterol plays an important role in determining the biophysical properties of biological membranes, and its concentration is tightly controlled by homeostatic processes. The intracellular transport of cholesterol among organelles is a key part of the homeostatic mechanism, but sterol transport processes are not well understood. Fluorescence microscopy is a valuable tool for studying intracellular transport processes, but this method can be challenging for lipid molecules because addition of a fluorophore may alter the properties of the molecule greatly. We discuss the use of fluorescent molecules that can bind to cholesterol to reveal its distribution in cells. We also discuss the use of intrinsically fluorescent sterols that closely mimic cholesterol, as well as some minimally modified fluorophore-labeled sterols. Methods for imaging these sterols by conventional fluorescence microscopy and by multiphoton microscopy are described. Some label-free methods for imaging cholesterol itself are also discussed briefly.

The correct intracellular distribution of cholesterol among cellular membranes is essential for many biological functions of mammalian cells, including signal transduction and membrane traffic. Intracellular trafficking plays a major role in the proper disposition of internalized cholesterol and in the regulation of cholesterol efflux. Despite the importance of the transport and distribution of cholesterol within cells for normal physiology and in pathological conditions, many fundamental aspects of intracellular cholesterol movement are not well understood. For instance, the relative roles of vesicular and nonvesicular transport have not been fully determined, and the asymmetric distribution of cholesterol between the two leaflets of biological membranes is poorly characterized in many cases. Also, while it is clear that small, cholesterol-enriched microdomains (often referred to as rafts; see Simons and Ehehalt, this Perspective series, ref. 1) occur in many biological membranes, the composition, size, and dynamics of these microdomains remain uncertain. Here, we will discuss the current understanding of intracellular cholesterol transport.

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