Abstract
High density lipoprotein-associated cholesteryl esters (HDL CE) are taken up by many cells without parallel uptake of HDL apoproteins, a pathway we have termed "selective uptake." The first step in this pathway, the reversible incorporation of HDL CE into the plasma membrane, is the subject of the present study. To examine the role of membrane proteins, the rate of HDL CE incorporation into isolated rat liver plasma membrane was compared with the rate of incorporation into synthetic membranes devoid of protein. Both membrane systems exhibited saturable uptake of CE, and at rates that were similar (t1/2 approximately 2 h, measured with 50 micrograms of HDL protein). Addition of unlabeled HDL to "chase" CE tracer from the biological and synthetic membranes revealed two kinetically distinct CE pools (t1/2 approximately 0.5 h and t1/2 approximately 30 h). Both biological and synthetic membranes accepted similar amounts of CE into both pools, with a maximum incorporation of 2-4 mol % relative to membrane phospholipids. CE transfer between HDL and membranes was kinetically second-order, in contrast to the first-order transfer of unesterified cholesterol. There was no evidence for direct participation of any apolipoprotein in CE uptake; CE in HDL or in protein-free microemulsions of similar particle size transferred to membranes at similar rates. To examine the possibility that CE transfer requires transient fusion of the HDL amphipathic coat with the membrane outer leaflet, radiolabeled cardiolipin was incorporated into either HDL particles or into synthetic membranes as an amphipathic coat marker that does not diffuse through the aqueous phase; transfer between HDL and membranes was not observed. Thus, CE are transferred between HDL and cell membranes in a collision-mediated process that does not involve amphipathic coat fusion and is not dependent on either membrane protein or apolipoproteins.
Highlights
(HDL cholesteryl esters (CE)) are taken up by many cells without parallel thereby allowing continued eMuofxfree cholesterol from memuptake of HDL apoproteins, a pathway we have termed branes
Amphipathic coat with the membrane outer leaflet, radiolabeled cardiolipin was incorporated into either HDL particles or into synthetic membranes as an amphipathic coat marker that does not diffusethrough the aqueous phase; transfer between HDL and membranes are transferred to membranes duricnogllision events in a process not dependent on membrane proteionr apolipoprotein and that t h e process does not require the fusion of the membrane leaflet with the HDL phospholipid coat
Pansfer of €€DL CE to Biological and Synthetic Membranes-The transfer of HDL CE into membranes was measuredinterms of themembrane association of CE tracer ([3H]cholesteryl-oleylether) in excess of that accounted for by membrane-associated HDL particles; association of HDL particles with membranes was measured in terms of1251-apoA-I
Summary
(HDL CE) are taken up by many cells without parallel thereby allowing continued eMuofxfree cholesterol from memuptake of HDL apoproteins, a pathway we have termed branes. To examine the role of membrane proteins, the rateof HDL CE incorporation into isolated rat liver plasma membrane was compared withthe rate of incorporation into synthetic membranes devoid of protein. CE transfer between HDL and membranes was kinetically second-ordeinr, contrast to directly take up CE from HDL(2) i n a process independent of CETP (3),termed “selective uptake.A” wide range of cell types from several species exhibit this pathwinayvitro.The liver and steroidogenic tissue exhibithe pathway in vivoin normalrats (4).Selective uptakeis a saturable process (5)with no specific apoprotein requirement ( 6 ) .The processis regulated according to cholesterol statuins both cultured cell(s7) a n d in the tissues of intact rats (7). To 2 months. [3HlCholesterol, [‘4Clcholesteroal,nd NalZ5Iwere obtained from Amersham Corp. [‘4C101eic acid was obtained from ICN (Irvine, CA), bovine serum albumin and casein from Sigma, and HPLC grade
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