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The clathrin-coated pit lattice is held onto the plasma membrane by an integral membrane protein that binds the clathrin AP-2 subunit with high affinity. In vitro studies have suggested that this protein controls the assembly of the pit because membrane bound AP-2 is required for lattice assembly. If so, the AP-2 binding site must be a resident protein of the coated pit and recycle with other receptors that enter cells through this pathway. Proper recycling, however, would require the switching off of AP-2 binding to allow the binding site to travel through the endocytic pathway unencumbered. Evidence for this hypothesis has been revealed by the cationic amphiphilic class of drugs (CAD), which have previously been found to inhibit receptor recycling. Incubation of human fibroblasts in the presence of these drugs caused clathrin lattices to assemble on endosomal membranes and at the same time prevented coated pit assembly at the cell surface. These effects suggest that CADs reverse an on/off switch that controls AP-2 binding to membranes. We conclude that cells have a mechanism for switching on and off AP-2 binding during the endocytic cycle.

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Glycosyl-phosphatidylinositol (GPI)-anchored proteins have been reported to reside in clusters collected over small membrane invaginations called caveolae. The detection of different GPI-anchored proteins with fluorescently labeled monoclonal antibodies showed that these proteins are not constitutively concentrated in caveolae; they enter these structures independently after cross-linking with polyclonal secondary antibodies. Analysis of the cell surface distribution of the GPI-anchored folate receptor by electron microscopy confirms these observations. Thus, multimerization of GPI-anchored proteins regulates their sequestration in caveolae, but in the absence of agents that promote clustering they are diffusely distributed over the plasma membrane.

Abstract. The folate receptor is a glycosyl-phosphatidylinositol (GPI)-anchored membrane protein that mediates the delivery of 5-methyltetrahydrofolate to the cytoplasm of MA104 cells. Ordinarily the receptor is sequestered into numerous discrete clusters that are associated with an uncoated pit membrane specialization called a caveola. By using two different methodological approaches, we found that the maintenance of both receptor clusters and caveolae depends upon the presence of cholesterol in the membrane. These results suggest that cholesterol plays a critical role in maintaining the caveola membrane domain and modulates the interaction of GPI-anchored membrane proteins via their phospholipid anchors.

Abstract. Plasmalemmal caveolae are a membrane specialization that mediates transcytosis across endothelial cells and the uptake of small molecules and ions by both epithelial and connective tissue cells. Recent findings suggest that caveolae may, in addition, be involved in signal transduction. To better understand the molecular composition of this membrane specialization, we have developed a biochemical method for purifying caveolae from chicken smooth muscle cells. Biochemical and morphological markers indicate that we can obtain ~1.5 mg of protein in the caveolae fraction from •100 g of chicken gizzard. Gel electrophoresis shows that there are more than 30 proteins enriched in caveolae relative to the plasma membrane. Among these proteins are: caveolin, a structural molecule of the caveolae coat; multiple, glycosylphosphatidylinositol-anchored membrane proteins; both G~ and Ga subunits of heterotrimeric GTP-binding protein; and the Ras-related GTP-binding protein, RaplA/B. The method we have developed will facilitate future studies on the structure and function of caveolae.T HF.RE is increasing evidence that plasmalemmal caveolae are a membrane specialization capable of sealing off from the extracellular environment to create a unique, membrane bound compartment at the cell surface. The dynamics of caveolae opening and closing is best observed in endothelial cells (46,47), where they appear to form plasmalemmal vesicles that move across the cell and fuse with the abluminal membrane. Each round of caveolaemediated transcytosis transports a portion of molecules from the blood to the tissue space without merging with other endocytic pathways. Although in other cell types the budding event has not been seen with the electron microscope, biochemical studies have shown (1%19) that caveolae can sequester membrane bound ligands away from the extracellular space and facilitate their delivery to the cytoplasm of the cell. This process is called potocytosis (3).What distinguishes potocytosis from other endocytic pathways is the use of glycosylphosphatidylinositol (GPI) 1-anchored membrane proteins to concentrate low molecular weight molecules and ions in closed caveolae (22,41). Morphological (54) and biochemical (5, 7) methods have

Abstract. The folate receptor, also known as the membrane folate-binding protein, is maximally expressed on the surfaee of folate-depleted tissue culture cells and mediates the high affinity accumulation of 5-methyltetrahydrofolic acid in the cytoplasm of these cells. Recent evidence suggests that this receptor recycles during folate internalization and that it is anchored in the membrane by a glycosyl-phosphatidylinositol linkage. Using quantitative immunocytochemistry, we now show that (a) this receptor is highly clustered on the cell surface; (b) these clusters are preferentially associated with uncoated membrane invaginations rather than clathrin-coated pits; and (c) the receptor is not present in endosomes or lysosomes. This receptor appears to physically move in and out of the cell using a novel uncoated pit pathway that does not merge with the clathrin-coated pit endocytic machinery.VER 25 different membrane receptors have been identiffed that participate in receptor-mediated endocytosis (4, 24). Characteristically, these receptors are transmembrane glycoproteins that bind protein ligands and carry them into an endocytic system. In many cases, the receptor is recycled back to the cell surface where it participates in subsequent rounds of internalization while at the same time the ligand is transported to other intracellular compartments such as the lysosome (11). The clathrin-coated pit is the staging area for this process since it controls both receptor clustering and the formation of the endosome (3, 25).The folate receptor, also known as the membrane folate binding protein, is a membrane protein that has some features of a receptor that is involved in receptor-mediated endocytosis. The molecule is maximally expressed on the surface of folate-depleted tissue culture cells and is responsible for the high affinity accumulation of 5-methyltetrahydrofolic acid (5-CH3FI-L) t in the cytoplasm of these cells (8,(26)(27)(28)35). In MA104 cells, the surface membrane has two equal sets of receptors: one set is exposed to the extracellular environment, and the other is not accessible to this space. Several lines of evidence suggest that ordinarily the two sets of receptors completely exchange with each other once every hour (27,28). Kinetic studies have shown that this exchange reaction results in the delivery of bound 5-methyltetrahydrofolic acid to the cytoplasm (28) and that delivery is blocked by 1. Abbreviations used in this paper: 5-CH3FI-I4, 5-methyltetrahydrofolic acid; DNP, dinitrophenol; GPI, glycophospholipid; LDL, low density lipoprotein; TNP, 2,4,6 trinitrophenol.ionophores that disrupt proton gradients (27). Therefore, the folate receptor internalizes 5-CH3FH4 and recycles.Other information about the folate receptor is not consistent with the receptor-mediated endocytosis paradigm. Recently, we showed (30) that this receptor is anchored in the membrane by a glycosyl-phosphatidylinositol (GPI) linkage. Although a varied of membrane proteins are membrane anchored in this manner (19,33), none of these p...