, we showed that SR-BI-mediated cholesterol efflux was highly correlated (r 2 ‫؍‬ 0.985) with HDL phosphatidylcholine content. The effects of varying HDL phospholipid composition on SR-BI-mediated free cholesterol flux were not correlated with changes in either the K d or B max values for high affinity binding to SR-BI. We conclude that SR-BI-mediated free cholesterol flux is highly sensitive to HDL phospholipid composition. Thus, factors that regulate cellular SR-BI expression and the local modification of HDL phospholipid composition will have a large impact on reverse cholesterol transport.The deposition of cholesterol in peripheral cells is opposed by the process of reverse cholesterol transport (RCT) 1 where high density lipoproteins (HDL) remove free cholesterol (FC) from cells and deliver it back to the liver for excretion (1-3). The flux of FC between cells and HDL is bi-directional. Depending on the direction of the FC concentration gradient between cells and lipoproteins, either net efflux or net influx of cholesterol can occur (4, 5). The creation of a cholesterol gradient depends upon many properties of the acceptors and the cell plasma membrane. Such factors include the cholesterol and phospholipid content of the acceptors and plasma membrane (4, 5), the existence of cholesterol domains within the plasma membrane (6 -10), and the size, number, and composition of acceptor particles (11-13).Recent studies have shown that, when cells express scavenger receptor BI (SR-BI), the bi-directional flux of FC between cells and HDL is accelerated (8,14,15). The mechanism by which SR-BI mediates FC flux is uncertain. However, recent studies from our laboratory demonstrated that binding of the acceptor particles to SR-BI is not a requirement for SR-BImediated cholesterol efflux (7,8). Rather SR-BI induces a reorganization of the plasma membrane cholesterol, and this reorganization is linked to enhanced FC flux (7,8,16). Regardless of the mechanism, evidence is accumulating to support the importance of SR-BI-mediated FC flux in RCT. Recent studies of Ji and colleagues (17) showed that either attenuation or overexpression of hepatic SR-BI in mice led to significantly decreased or increased delivery of HDL FC into bile. In addition, the expression of SR-BI in peripheral cells and in foam cells of the arterial wall suggests a role for SR-BI in the removal of FC from the periphery (15, 18, 19).SR-BI-mediated FC flux requires phospholipid in the acceptor (15), and studies have shown that cholesterol efflux from cells is highly correlated with the concentration of HDL phospholipid in serum (20,21). Also, the stimulation of cholesterol efflux upon phospholipid supplementation of serum is closely linked to the levels of SR-BI among cell types (14). These observations are consistent with epidemiological data demonstrating that humans with low HDL phospholipid levels have a high incidence of coronary artery disease (22). These findings suggest that changes in HDL phospholipid content may alter SR-BI-mediated FC flux. The cu...

Scavenger receptor BI (SR-BI) mediates the selective uptake of high density lipoprotein (HDL) cholesteryl ester (CE), a process by which HDL CE is taken into the cell without internalization and degradation of the HDL particle. The biochemical mechanism by which SR-BI mediates the selective uptake of HDL CE is poorly understood. Given that CE transfer will occur to some extent from HDL to protein-free synthetic membranes, one hypothesis is that the role of SR-BI is primarily to tether HDL close to the cell surface to facilitate CE transfer from the particle to the plasma membrane. In the present study, this hypothesis was tested by comparing the selective uptake of HDL CE mediated by mouse SR-BI (mSR-BI) with that mediated by rat CD36 (rCD36), a closely related class B scavenger receptor. Both mSR-BI and rCD36 bind HDL with high affinity, and both receptors mediate HDL CE selective uptake. However, SR-BI mediates selective uptake of HDL CE with a 7-fold greater efficiency than rCD36. HDL CE selective uptake mediated by rCD36 is dependent on HDL binding to the receptor, since a mutation that blocks HDL binding also blocks HDL CE selective uptake. These data lead us to hypothesize that one component of HDL CE selective uptake is the tethering of HDL particles to the cell surface. To explore the molecular domains responsible for the greater efficiency of selective uptake by mSR-BI, we compared binding and selective uptake among mSR-BI, scavenger receptor BII, and various chimeric receptors formed from mSR-BI and rCD36. The results show that the extracellular domain of mSR-BI is essential for efficient HDL CE uptake, but the C-terminal cytoplasmic tail also has a major influence on the selective uptake process.The risk for developing atherosclerosis and cardiovascular disease is inversely related to plasma concentrations of high density lipoprotein (HDL) 1 cholesterol (1). Although the mechanism of this protective effect remains uncertain, it has been known for some time that HDL plays a pivotal role in the transport of free cholesterol and cholesteryl esters (CE) through the plasma. HDL participates in reverse cholesterol transport (2), a process involving the uptake of free cholesterol from peripheral tissue and its subsequent delivery (as free cholesterol or CE) to steroidogenic (for hormone synthesis) and hepatic (for bile acid synthesis) tissues. HDL provides CE to cells via the selective uptake pathway in which HDL CE is taken into the cell without the internalization and lysosomal degradation of the HDL particle (3-8). Recent studies identified a cell surface receptor, scavenger receptor BI (SR-BI), which binds HDL particles and mediates the selective uptake of HDL CE in transfected cells (9). Immunochemical analysis of SR-BI in rodents indicates that it is expressed most abundantly in the liver and in steroidogenic cells of the adrenal gland and ovary (9 -11), where the selective uptake of HDL CE is greatest (4, 7). SR-BI expression is regulated by gonadotropins and adrenocorticotropic hormone coordinately w...

No abstract

Full-length cDNAs for DNA ligase IV and the alpha and beta isoforms of DNA ligase III were cloned from Xenopus laevis to permit study of the genes encoding mitochondrial DNA ligase. DNA ligase III alpha and III beta share a common NH(2) terminus that encodes a mitochondrial localization signal capable of targeting green fluorescent protein to mitochondria while the NH(2) terminus of DNA ligase IV does not. Reverse transcriptase-polymerase chain reaction analyses with adult frog tissues demonstrate that while DNA ligase III alpha and DNA ligase IV are ubiquitously expressed, DNA ligase III beta expression is restricted to testis and ovary. Mitochondrial lysates from X. laevis oocytes contain both DNA ligase III alpha and III beta but no detectable DNA ligase IV. Gel filtration, sedimentation, native gel electrophoresis, and in vitro cross-linking experiments demonstrate that mtDNA ligase III alpha exists as a high molecular weight complex. We discuss the possibility that DNA ligase III alpha exists in mitochondria in association with novel mitochondrial protein partners or as a homodimer.