P E Fielding

Reverse cholesterol transport (RCT) is the pathway by which peripheral cell cholesterol can be returned to the liver for catabolism. Evidence of specific functions for molecular structures within individual plasma lipoprotein species has rapidly accumulated from recent studies using molecular and cellular physiology techniques. The removal of cholesterol from cells, like its delivery, appears to be specific and well regulated. Although further research will be needed, RCT can now be understood in molecular terms.

Recent data on the roles of vesicle- and 'raft'-mediated pathways in intracellular free cholesterol (FC) transport are reviewed. Cholesterol internalized from plasma lipoproteins is transferred via endocytic vesicles to the trans-Golgi network (TGN), consistent with prior data indicating a key role for this organelle in protein and lipid sorting and transport. Newly synthesized and lipoprotein-derived FC are returned to the cell surface by a common raft-dependent pathway. Intracellular FC transport promotes the delivery of GPI-anchored proteins to the cell surface; it is also an additional mechanism to regulate cell FC content. Many peripheral cells express caveolin, an FC-binding protein localized to plasma membrane caveolae. FC delivery to cell surface caveolae is accelerated by caveolin. Caveolar FC becomes targeted to small, lipid-poor (prebeta-) high density lipoprotein particles. Caveolin may protect quiescent cells, regulating FC efflux more efficiently in response to changing medium lipoprotein concentrations. Overall, these recent findings suggest that cell FC content can be regulated at the levels of both influx and efflux, and indicate key roles for the TGN and in cells expressing caveolin, cell-surface caveolae.

Immunoadsorption affinity chromatography has been used to define the structure of lipoproteins in human plasma containing lecithin:cholesterol acyltransferase (EC 2.3.1.43) (LCAT) and transfer protein (apo D). The whole of LCAT was absorbed by antibodies specific for apo D and for apo A-1, indicating that the enzyme is present in plasma exclusively as a complex with its cofactor (apo A-1) and product transfer protein (apo D). About 80% of apo D (but no LCAT) was removed by antibody to apo A-2, indicating the presence of most of apo D in the form of an enzyme-free complex will apo A-1 and apo A-2. After removal of LCAT with antibody to apo D, plasma was unreactive as a substrate with isolated LCAT, but substrate activity was generated by ultracentrifugal flotation with either intact or adsorbed plasma. The apparent stoichiometry of the complex with LCAT (LCAT:apo A-1:apo D) was 1.0:0.9:1.8; that of the complex containing apo A-1, apo A-2, and apo D was 3.9:2.2:1.0.

Immunoaffinity chromatography has been used to study the determinants of sterol efflux and net transport from cultured fibroblasts to human plasma medium. Sterol efflux was highly (approximately 80%) dependent upon a minor lipoprotein fraction containing apolipoprotein A-I unassociated with other apolipoproteins. The remaining activity was associated with the lipoprotein-free fraction of plasma and could be replaced by apoprotein-free albumin. Efflux was independent of lecithin:cholesterol acyltransferase (EC 2.3.1.43) activity. Net transport (i.e., the excess of efflux over influx) was completely inhibited by inhibition of lecithin:cholesterol acyltransferase or its removal by affinity chromatography on immobilized antibodies to apolipoprotein A-I or D (components of the transfer complex in human plasma). In uninhibited plasma, efflux and net transport rates had similar kinetics, suggesting that these were linked functions and that net transport was initiated by a carrier-dependent efflux step that, in the absence of lecithin:cholesterol acyltransferase activity, was associated with an equivalent influx of free sterol to the cells and that, in the presence of lecithin:cholesterol acyltransferase, was associated with esterification and transfer protein activity. The cholesterol carrier lipoprotein function (approximately 5% of plasma apolipoprotein A-I) appears to be the first step of lecithin:cholesterol acyltransferase-linked sterol transport from cells.