Cholesterol Accessibility in Membranes

Cholesterol levels in mammalian cells are tightly regulated to lie within narrow limits. This regulation is achieved by employing multiple feedback mechanisms to regulate both synthesis and uptake of cholesterol. Most of a cell's cholesterol (~80 % of total) is in the plasma membrane (PM), but the protein machinery that senses and regulates cellular cholesterol resides in the endoplasmic reticulum (ER) membrane, which contains a very small fraction (~1% of total) of a cell's cholesterol. A carefully regulated lipid transport pathway between PM and ER allows cholesterol sensors in ER to monitor the cholesterol content of cholesterol-rich PM. This transport depends on the interactions of cholesterol with various phospholipids that control its accessibility for transport to the ER. Cholesterol in PM is organized into three different pools. One pool is accessible for transport to the ER, a second pool is sequestered by sphingomyelin (SM) and can be released by treatment with sphingomyelinase, and a third pool remains sequestered even after sphingomyelinase treatment. Here, I describe our work in developing and characterizing tools to study these different pools. The three pools were identified using bacterial toxins called cholesterol dependent cytolysins (CDCs) which selectively bind to the accessible pool of cholesterol. One example of these toxins is Anthrolysin O (ALO). To better understand the dynamics of accessible cholesterol sensing at a molecular level, we developed a stable construct of the cholesterol sensing domain of ALO (ALOD4) and performed NMR and other biophysical studies using cholesterol containing model membranes. We were able to identify residues that are significantly affected by the interaction of ALOD4 to membranes. We also developed a highly specific sensor for the SM-sequestered pool of cholesterol. This sensor is derived from a fungal toxin, Ostreolysin A (OlyA). Using X-ray crystallography, we studied the interaction of OlyA with SM and cholesterol at the atomic level. This structural analysis combined with detailed mutagenesis led us to a single point mutation in OlyA that abolishes its cholesterol specificity while retaining SM specificity. Comparing the X-ray structures of these two versions of lipid bound OlyA combined with ligand docking simulations revealed two distinct conformations of SM: one in complex with cholesterol and one free from cholesterol. Studies in live cells using OlyA and ALOD4 show that the pool of SM/cholesterol complexes in plasma membrane is maintained at a constant level across a large range of cholesterol concentrations. The development of new tools for specific forms of cholesterol (ALOD4 and OlyA) has allowed us to evaluate long-standing hypotheses regarding lipid organization in PMs and has also shed new light on lipid dynamics in the context of cellular signaling.