Abstract. Caveolae are 50-100-nm membrane microdomains that represent a subcompartment of the plasma membrane. Previous morphological studies have implicated caveolae in (a) the transcytosis of macromolecules (including LDL and modified LDLs) across capillary endothelial cells, (b) the uptake of small molecules via a process termed potocytosis involving GPI-linked receptor molecules and an unknown anion transport protein, (c) interactions with the actin-based cytoskeleton, and (d) the compartmentalization of certain signaling molecules, including G-protein coupled receptors. Caveolin, a 22-kD integral membrane protein, is an important structural component of caveolae that was first identified as a major v-Src substrate in Rous sarcoma virus transformed cells. This finding initially suggested a relationship between caveolin, transmembrane signaling, and cellular transformation.We have recently developed a procedure for isolating caveolin-rich membrane domains from cultured cells. To facilitate biochemical manipulations, we have applied this procedure to lung tissue-an endothelial and caveolin-rich source-allowing large scale preparation of these complexes. These membrane domains retain *85 % of caveolin and ,',,55 % of a GPI-linked marker protein, while they exclude I>98% of integral plasma membrane protein markers and t>99.6% of other organelle-specific membrane markers tested. Characterization of these complexes by micro-sequencing and immuno-blotting reveals known receptors for modified forms of LDL (scavenger receptors: CD 36 and RAGE), multiple GPI-linked proteins, an anion transporter (plasma membrane porin), cytoskeletal elements, and cytoplasmic signaling molecules-including Src-like kinases, hetero-trimeric G-proteins, and three members of the Rap family of small GTPases (Rap I-the Ras tumor suppressor protein, Rap 2, and TC21). At least a fraction of the actin in these complexes appeared monomeric (G-actin), suggesting that these domains could represent membrane bound sites for microfilament nucleation/assembly during signaling. Given that the majority of these proteins are known molecules, our current studies provide a systematic basis for evaluating these interactions in vivo.

Abstract. Glycosylated phosphoinositides serve as membrane anchors for numerous eukaryotic cell surface glycoproteins. Recent biochemical and genetic studies indicate that the glycolipids are assembled by sequential addition of components (monosacchafides and phosphoethanolamine) to phosphatidylinositol. The biosynthetic steps are presumed to occur in the ER, but formal proof of this is lacking. We describe experiments designed to establish the subeellular location of the initial steps in glycosyl-phosphatidylinositol (GPI) anchor biosynthesis and to define the transmembrane distribution of early biosynthetic lipid intermediates. The experiments were performed with the thymoma cell line BW5147.3. A subeellular fractionation protocol was used to show that early biosynthetic steps in GPI assembly, i.e., synthesis and deacetylation of N-acetylglucosaminyl phosphatidylinositol, occur in the ER. GPI biosynthetic intermediates were synthesized by incubating the microsomes with UDPpH]GlcNAc, and the transmembrane distribution of the labeled lipids was probed with phosphatidylinositolspecific phospholipase C (PI-PLC). Treatment of the radiolabeled microsomes with PI-PLC showed that >70% of the N-acetylglucosaminyl phosphatidylinositol and glucosaminyl phosphatidylinositol could be hydrolyzed, indicating that the two lipids were primarily distributed in the cytoplasmic (outer) leaflet of the microsomes. Similar cleavage results were obtained using Streptolysin O-permeabilized thymoma cells. When permeabilized cells were incubated with UDP-[3H]GlcNAc and treated with PI-PLC, ,x,85 % of the radiolabeled N-acetylglucosaminyl phosphatidylinositol and glucosaminyl phosphatidylinositol could be cleaved, indicating that they were accessible to the enzyme. The cumulative data indicate that early GPI intermediates are primarily located in the cytoplasmic leaflet of the ER, and are probably synthesized from PI located in the cytoplasmic leaflet and UDP-GlcNAc synthesized in the cytosol.LYCOLIPIDS containing the structural motif Mant~l-4GlcNotl-6myo Inositol-l-P-lipid are ubiquitous in the eukaryotes (Ferguson et al., 1992). These lipids, termed glycosyl-phosphatidylinositols (GPIs) I, were originally discovered covalenfly linked to eukaryotic cell-surface glycoproteins and recognized to be an important alterhate mechanism for anchoring proteins to cell membranes (Low et al., 1986). All protein-linked GPIs contain the extended structure Ethanolamine-P-6Manotl-2Mant~l-6Manod-4GlcNod-6myo Inositol-l-P-lipid. The EtN residue is amide linked to the carboxyl-terminal amino acid of the protein, and the trimannose core glycan can be modified by a variety of 1. Abbreviations used in this paper: 5BrdUMP, 5-bmmo-2'-deoxyuridine

Abstract. Glycosylphosphatidylinositol (GPI) membrane protein anchors are synthesized from sugar nucleotides and phospholipids in the ER and transferred to newly synthesized proteins destined for the cell surface. The topology of GPI synthesis in the ER was investigated using sealed trypanosome microsomes and the membrane-impermeant probes phosphatidylinositol-speeific phospholipase C, Con A, and proteinase K. All the GPI biosynthetic intermediates examined were found to be located on the external face of the microsomal vesicles suggesting that the principal steps of GPI assembly occur in the cytoplasmic leaflet of the ER. Protease protection experiments showed that newly GPI-modified trypanosome variant surface glycoprotein was primarily oriented towards the ER lumen, consistent with eventual expression at the cell surface. The unusual topographical arrangement of the GPI assembly pathway suggests that a biosynthetic intermediate, possibly the phosphoethanolamine-containing anchor precursor, must be translocated across the ER membrane bilayer in the process of constructing a GPI anchor.

We established an in vitro assay for the addition of glycosyl-phosphatidylinositol (GPI) anchors to proteins using procyclic trypanosomes engineered to express GPI-anchored variant surface glycoprotein (VSG). The assay is based on the premise that small nucleophiles, such as hydrazine, can substitute for the GPI moiety and effect displacement of the membrane anchor of a GPIanchored protein or pro-protein causing release of the protein into the aqueous medium. Cell membranes containing pulse-radiolabeled VSG were incubated with hydrazine, and the VSG released from the membranes was measured by carbonate extraction, immunoprecipitation, and SDS-polyacrylamide gel electrophoresis/fluorography. Release of VSG was time-and temperaturedependent, was stimulated by hydrazine, and occurred only for VSG molecules situated in early compartments of the secretory pathway. No nucleophile-induced VSG release was seen in membranes prepared from cells expressing a VSG variant with a conventional transmembrane anchor (i.e. a nonfunctional GPI signal sequence). Pro-VSG was shown to be a substrate in the reaction by assaying membranes prepared from cells treated with mannosamine, a GPI biosynthesis inhibitor. When a biotinylated derivative of hydrazine was used instead of hydrazine, the released VSG could be precipitated with streptavidin-agarose, indicating that the biotin moiety was covalently incorporated into the protein. Hydrazine was shown to block the C terminus of the released VSG hydrazide because the released material, unlike a truncated form of VSG lacking a GPI signal sequence, was not susceptible to proteolysis by carboxypeptidases. These results firmly establish that the released material in our assay is VSG hydrazide and strengthen the proof that GPI anchoring proceeds via a transamidation reaction mechanism. The reaction could be inhibited with sulfhydryl alkylating reagents, suggesting that the transamidase enzyme contains a functionally important sulfhydryl residue. Genes encoding glycosylphosphatidylinositol (GPI)1 -anchored proteins specify two signal sequences in the primary translation product: an N-terminal signal sequence for targeting the protein to the endoplasmic reticulum (ER) and a Cterminal GPI-directing signal sequence directing the attachment of a GPI anchor (1). Both sequences are removed during processing of the preproprotein to the mature GPI-anchored form, but cleavage of the N-terminal signal sequence is not strictly necessary (1, 2). The assembly of GPI-anchored proteins requires translocation of the nascent polypeptide chain across the ER membrane and replacement of the C-terminal signal sequence with a preassembled, ethanolamine-containing GPI moiety attached to the newly exposed carboxyl group. The reaction is presumed to be catalyzed by an ER-localized transamidase enzyme (3, 4).GPI anchoring can be reproduced in cell-free systems that rely on endogenous, membrane protein acceptors for GPI anchors (3) or, alternatively, that involve an in vitro translation system to load microsomal membrane...

In addition to serving as membrane anchors for cell surface proteins, glycosylphosphatidylinositols (GPIs) can be found abundantly as free glycolipids in mammalian cells. In this study we analyze the subcellular distribution and intracellular transport of metabolically radiolabeled GPIs in three different cell lines. We use a variety of membrane isolation techniques (subcellular fractionation, plasma membrane vesiculation to isolate pure plasma membrane fractions, and enveloped viruses to sample cellular membranes) to provide direct evidence that free GPIs are not confined to their site of synthesis, the endoplasmic reticulum, but can redistribute to populate other subcellular organelles. Over short labeling periods (2.5 h), radiolabeled GPIs were found at similar concentration in all subcellular fractions with the exception of a mitochondria-enriched fraction where GPI concentration was low. Pulse-chase experiments over extended chase periods showed that although the total amount of cellular radiolabeled GPIs decreased, the plasma membrane complement of labeled GPIs increased. GPIs at the plasma membrane were found to populate primarily the exoplasmic leaflet as detected using periodate oxidation of the cell surface. Transport of GPIs to the cell surface was inhibited by Brefeldin A and blocked at 15°C, suggesting that GPIs are transported to the plasma membrane via a vesicular mechanism. The rate of transport of radiolabeled GPIs to the cell surface was found to be comparable with the rate of secretion of newly synthesized soluble proteins destined for the extracellular space.

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Neuronal calcium sensor-1 (NCS-1), the mammalian orthologue of frequenin, belongs to a family of EF-handcontaining Ca 2؉ sensors. NCS-1/frequenin has been shown to enhance synaptic transmission in PC12 cells and Drosophila and Xenopus, respectively. However, the precise molecular mechanism for the enhancement of exocytosis is largely unknown. In PC12 cells, NCS-1 potentiated exocytosis evoked by ATP, an agonist to phospholipase C-linked receptors, but had no effect on depolarization-evoked release. NCS-1 also enhanced exocytosis triggered by ionomycin,

Glycosylphosphatidylinositols (GPIs) are synthesized in the endoplasmic reticulum (ER) via the sequential addition of monosaccharides, fatty acid, and phosphoethanolamine(s) to phosphatidylinositol (PI). While attempting to establish a mammalian cell-free system for GPI biosynthesis, we found that the assembly of mannosylated GPI species was impaired when purified ER preparations were substituted for unfractionated cell lysates as the enzyme source. To explore this problem we analyzed the distribution of the various GPI biosynthetic reactions in subcellular fractions prepared from homogenates of mammalian cells. The results indicate the following: (i) the initial reaction of GPI assembly, i.e. the transfer of GlcNAc to PI to form GlcNAc-PI, is uniformly distributed in the ER; (ii) the second step of the pathway, i.e. de-N-acetylation of GlcNAc-PI to yield GlcN-PI, is largely confined to a subcompartment of the ER that appears to be associated with mitochondria; (iii) the mitochondria-associated ER subcompartment is enriched in enzymatic activities involved in the conversion of GlcN-PI to H5 (a singly mannosylated GPI structure containing one phosphoethanolamine side chain; and (iv) the mitochondria-associated ER subcompartment, unlike bulk ER, is capable of the de novo synthesis of H5 from UDP-GlcNAc and PI. The confinement of these GPI biosynthetic reactions to a domain of the ER provides another example of the compositional and functional heterogeneity of the ER. The implications of these findings for GPI assembly are discussed. Glycosylphosphatidylinositols (GPIs)1 were originally discovered covalently linked to eukaryotic cell-surface glycoproteins and were recognized to be an important alternate mechanism for anchoring proteins to cell membranes (1, 2). GPI-anchored proteins have the general structure protein-(ethanolamine-P-6Man␣1-2Man␣1-6Man␣1-4GlcN␣1-6myo-inositol-1-P-lipid), where the structure within parentheses represents a minimal GPI moiety, and the carboxyl terminus of the protein is amide-linked to the GPI ethanolamine residue. GPIs can also be found in cells as free glycolipids representing protein anchor precursors or biosynthetic intermediates. Cellular pools of non-protein-linked GPIs vary depending on their structure as well as cell type, and their physiological significance remains to be determined.The structure and biosynthesis of GPIs has been reviewed recently (3-9). GPI biosynthesis involves the sequential addition of monosaccharides and phosphoethanolamine (EtN-P) to phosphatidylinositol (PI) yielding the minimal structure EtN-P-Man 3 GlcN-PI (Fig. 1). The assembly of GPI anchors in mammalian cells is initiated on the cytoplasmic leaflet of the endoplasmic reticulum (ER) (10) by the formation of GlcNAc-PI from UDP-GlcNAc and PI (11). GlcNAc-PI is then de-N-acetylated (to yield GlcN-PI) (11), acylated on the inositol residue (12-15), and elaborated by the addition of three mannose residues and one or more EtN-P groups linked to the mannose residues (16 -20). Once the terminal EtN-P resid...