Annexins are calcium-binding proteins with a wide distribution in most polarized and nonpolarized cells that participate in a variety of membrane-membrane interactions. At the cell surface, annexin VI is thought to remodel the spectrin cytoskeleton to facilitate budding of coated pits. However, annexin VI is also found in late endocytic compartments in a number of cell types, indicating an additional important role at later stages of the endocytic pathway. Therefore overexpression of annexin VI in Chinese hamster ovary cells was used to investigate its possible role in endocytosis and intracellular trafficking of low density lipoprotein (LDL) and transferrin. While overexpression of annexin VI alone did not alter endocytosis and degradation of LDL, coexpression of annexin VI and LDL receptor resulted in an increase in LDL uptake with a concomitant increase of its degradation. Whereas annexin VI showed a wide intracellular distribution in resting Chinese hamster ovary cells, it was mainly found in the endocytic compartment and remained associated with LDL-containing vesicles even at later stages of the endocytic pathway. Thus, data presented in this study suggest that after stimulating endocytosis at the cell surface, annexin VI remains bound to endocytic vesicles to regulate entry of ligands into the prelysosomal compartment.Annexins are a family of highly conserved proteins, which are characterized by their Ca 2ϩ -dependent binding to phospholipids (1). Each annexin consists of a conserved core domain with four or eight repeats (70 amino acids) and a nonconserved, short, NH 2 -terminal domain. More than 10 different family members, several of which exist as multiple isoforms, have been described in higher vertebrates (2). Since annexins are expressed in many tissues and are located in the same cellular compartments, the understanding of the distinct physiological role of each annexin still remains elusive (1, 3). In recent years, the involvement of annexins in membrane traffic has emerged as one of their predominant functions (1, 4). Several annexins including annexin I, II, IV, VI, VII, and XIIIb have been directly implicated in different steps of the intracellular trafficking pathways (5-14) and, despite some controversy, essentially due to the variety of cells and antibodies used, they are all associated with the endocytic compartment.The enrichment of annexin VI in rat liver endosomes (12, 13), its polarized localization in the apical endosomes in rat hepatocytes (14) and WIF-B cells (15), and the colocalization with lgp120, a prelysosomal marker in normal rat kidney cells (15), indicate a potential role for annexin VI in the endocytic pathways of polarized and nonpolarized cells.In support of this hypothesis, annexin VI has been found to bind ␤-spectrin at the cell surface, which in turn recruits and activates a calpain-like protease. This cascade of events seems to open the actin-cortical cytoskeleton to facilitate the initial steps of endocytosis (16,17). The complexity of these interactions has recently...

High-density lipoprotein cholesteryl esters are taken up by many cells without simultaneous uptake of high-density lipoprotein apolipoproteins. This selective uptake was investigated in human hepatocytes in primary culture. Human high-density lipoprotein-3 (density, 1.125 to 1.21 gm/ml) was radiolabeled in both its apolipoprotein and in its cholesteryl ester moiety; uptake of these high-density lipoprotein3 tracers by hepatocytes was investigated. Apparent high-density lipoprotein3 particle uptake as measured with the cholesteryl ester tracer was in excess of that from the apolipoprotein tracer, indicating selective uptake of high-density lipoprotein3 cholesteryl esters by hepatocytes. This selective uptake is a regulated pathway in hepatocytes, as demonstrated by an inverse relationship between cell cholesterol and the rate of selective uptake. Studies on the mechanism of selective uptake have used inhibitors such as monensin, chloroquine, heparin, and a monoclonal antibody directed against low-density lipoprotein receptors. These experiments provide no evidence for a role of cell-secreted apolipoprotein E, endocytosis or retroendocytosis in selective uptake. The intracellular fate of high-density lipoprotein3-associated cholesteryl esters was investigated with [3H]cholesteryl oleate-labeled high-density lipoprotein3. Hepatocytes hydrolyzed [3H]cholesteryl oleate internalized from labeled high-density lipoprotein3; this catabolism was not inhibited by the presence of chloroquine. In parallel hepatocytes were incubated with [3H]cholesteryl oleate-labeled low-density lipoprotein. Cells hydrolyzed [3H]cholesteryl oleate taken up with low-density lipoprotein; however, this hydrolysis was inhibited by chloroquine, indicating lysosomal low-density lipoprotein cholesteryl ester catabolism. These experiments show that high-density lipoprotein3 cholesteryl esters selectively taken up by hepatocytes are hydrolyzed independently from the classical lysosomal catabolic pathway. The question was addressed if selective uptake mediates a net mass uptake of cholesterol rather than an isotope exchange phenomenon. Incubation of hepatocytes with high-density lipoprotein-3 suppressed endogenous sterol synthesis from sodium [14C]acetate. Hepatocytes were incubated in the presence of high-density lipoprotein3; medium cholesteryl esters decreased as a result of incubation with hepatocytes. These results show a net mass delivery of high-density lipoprotein cholesteryl esters to hepatocytes. In conclusion, the pathway for selective uptake of high-density lipoprotein cholesteryl esters could be demonstrated in human hepatocytes in primary culture. A role for selective uptake in high-density lipoprotein-mediated cholesterol delivery to the liver in human beings in vivo is proposed.

We have recently described a novel recycling pathway of triglyceride-rich lipoprotein (TRL)-associated apolipoprotein (apo) E in human hepatoma cells. We now demonstrate that not only TRL-derived apoE but also lipoprotein lipase (LPL) is efficiently recycled in vitro and in vivo. Similar recycling kinetics of apoE and LPL in normal and low density lipoprotein receptor-negative human fibroblasts also indicate that the low density lipoprotein receptor-related protein seems to be involved. Intracellular sorting mechanisms are responsible for reduced lysosomal degradation of both ligands after receptor-mediated internalization. Immediately after internalization in rat liver, TRLs are disintegrated, and apoE and LPL are found in endosomal compartments, whereas TRL-derived phospholipids accumulate in the perinuclear region of hepatocytes. Subsequently, substantial amounts of both proteins can be found in purified recycling endosomes, indicating a potential resecretion of these TRL components. Pulse-chase experiments of perfused rat livers with radiolabeled TRLs demonstrated a serum-induced release of internalized apoE and LPL into the perfusate. Analysis of the secreted proteins identified ϳ80% of the recycled TRLderived proteins in the high density lipoprotein fractions. These results provide the first evidence that recycling of TRL-derived apoE and LPL could play an important role in the modulation of lipoproteins in vivo.Triglycerides are transported mainly by two distinct classes of triglyceride-rich lipoproteins (TRLs), 1 the chylomicrons and the very low density lipoproteins (VLDLs). After assembly in the intestine, chylomicrons are transported via lymph into the bloodstream, where they are converted at the endothelial surface to remnant lipoproteins through the catalytic action of lipoprotein lipase (LPL) (for review, see Refs. 1 and 2). After lipolysis, LPL remains associated with the chylomicron remnants and, in concert with apolipoprotein (apo) E (3-5), facilitates their clearance into hepatocytes (6) via LDL receptor (LDLR) and the LDLR-related protein (LRP) (7-10). The essential role for both receptors in TRL removal in vivo has been demonstrated in gene knockout and gene transfer experiments (Refs. 11 and 12; for a recent review, see Ref. 13).Several studies have used different "model particles" to investigate the intracellular processing of TRL constituents. In contrast to the lysosomal degradation of LDL-derived apoB (14), ␤-VLDL-derived apoE was identified in widely distributed vesicles and showed a slow protein degradation in mouse macrophages (15, 16). However, in the same cells, ␤-VLDLderived lipids were delivered to perinuclear, lysosomal compartments (17). Delayed transport and degradation of TRL proteins were also observed in hepatoma cells (18,19). In recent studies, we have been able to demonstrate that the altered transport and retarded degradation of internalized TRLs is due to intracellular disintegration and sorting of TRL components in a peripheral cellular compartment. Whereas lipids are ...

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