| Description |
Cholesterol is an essential component of animal cell plasma membrane. In the plasma membrane, cholesterol governs membrane fluidity and effects the function of membrane proteins. Cells have two ways to obtain cholesterol: They can synthesize cholesterol from acetate, or they can take it up through receptor-mediated endocytosis of low-density lipoproteins (LDL). Cholesterol esters present in the endocystosed LDL are hydrolyzed in lysosomes and the free cholesterol is rapidly transported to the plasma membrane where >90% of the cellular cholesterol resides. I have developed an in vitro assay to monitor the movement of cholesterol to the plasma membrane. Using centrifugation, cells are divided into two fractions, the pellet (PlO) and supernatant (S 10) of a 1O,000xg spin. The PlO contains plasma membrane, while the S 10 contains cytosol, ER, endosomes, and other organelles. S 1 0 fractions containing radiolabeled cholesterol are combined with PlO fractions without label and incubated to recreate cellular conditions. The fractions are then re-separated and the relative amounts of labeled cholesterol in each fraction, determined. Using this assay, I have determined several key characteristics of cholesterol trafficking to the plasma membrane. Cholesterol movement is ATP-dependent, and in the presence of ATP, more than 90% of the labeled cholesterol moves to the plasma membrane. This transport is rapid, reaching completion in approximately 15 minutes. This transport is correlated with an increase in the density of the fraction that contains the radiolabeled cholesterol. The transport of cholesterol to the plasma membrane is dependent on at least one cytosolic protein, as treatment of the S 10 with proteinase K destroys transport and the transport can be restored by the addition of untreated cytosolic fractions. |
