The GPI anchor of cell-surface proteins is synthesized on the cytoplasmic face of the endoplasmic reticulum.

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-specific 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.

Early lipid intermediates in glycosyl-phosphatidylinositol anchor assembly are synthesized in the ER and located in the cytoplasmic leaflet of the ER membrane bilayer.

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 (monosaccharides 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 subcellular 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 subcellular 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 UDP-[3H]GlcNAc, and the transmembrane distribution of the labeled lipids was probed with phosphatidylinositol-specific 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, approximately 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.

Characterization of caveolin-rich membrane domains isolated from an endothelial-rich source: implications for human disease.

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 approximately 85% of caveolin and approximately 55% of a GPI-linked marker protein, while they exclude > or = 98% of integral plasma membrane protein markers and > or = 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 1--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.

Topology of GPI biosynthesis in the endoplasmic reticulum.

Glycosylphosphatidylinositol (GPI) anchors are constructed in the endoplasmic reticulum (ER) through the action of at least seven unique enzymes. Using cell-free systems, mainly derived from African trypanosomes, it has been experimentally possible to re-create many aspects of the GPI biosynthetic pathway in vitro and to obtain a series of glycosylated phosphatidylinositol structures that correspond to biosynthetic intermediates. This approach led to the identification of the biosynthetic donors of individual components of the GPI glycan, and the discovery of unusual fatty acid re-modelling reactions in the GPI pathway in trypanosomes. Despite this progress, questions remain concerning the enzymology of the pathway, particularly the topological distribution of the different assembly steps in the ER membrane. In the work described here we have attempted to define the transbilayer orientation of different GPI biosynthetic intermediates in the ER membrane bilayer. The experiments were performed with a microsomal fraction derived from bloodstream-form Trypanosoma brucei, and standard radiolabeling procedures. The orientation of GPIs was probed with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) and the jackbean lectin Concanavalin A. Contrary to expectations based on other ER glycosylation reactions, most notably the reactions involved in the dolichol pathway of N-glycosylation, our results suggest that non-inositol-acylated (PI-PLC-sensitive) GPIs are synthesized in the cytoplasmic leaflet of the ER membrane bilayer and that the final reaction product, a phosphoethanolamine-containing GPI, flips into the luminal leaflet for transfer to protein.

Miniaturized GPCR signaling studies in 1536-well format.

G protein-coupled receptors (GPCRs) are involved in various physiological processes, such as behavior changes, mood alteration, and regulation of immune-system activity. Thus, GPCRs are popular targets in drug screening, and a well-designed assay can speed up the discovery of novel drug candidates. The Promega cAMP-Glo Assay is a homogenous bioluminescent assay to monitor changes in intracellular cyclic adenosine monophosphate (cAMP) concentrations in response to the effect of an agonist, antagonist, or test compound on GPCRs. Together with the Labcyte Echo 555 acoustic liquid handler and the Deerac Fluidics Equator HTS reagent dispenser, this setup can screen compounds in 96-, 384-, and 1536-well formats for their effects on GPCRs. Here, we describe our optimization of the cAMP-Glo assay in 1536-well format, validate the pharmacology, and assess the assay robustness for HTS. We have successfully demonstrated the use of the assay in primary screening applications of known agonist and antagonist compounds, and confirmed the primary hits via secondary screening. Implementing a high-throughput miniaturized GPCR assay as demonstrated here allows effective screening for potential drug candidates.

Soluble constituents of the ER lumen are required for GPI anchoring of a model protein.

Transfer of a glycosylphosphatidylinositol (GPI) anchor to proteins carrying a C-terminal GPI-directing signal sequence occurs after protein translocation across the endoplasmic reticulum (ER). We describe the translocation and GPI modification of a model protein, preprominiPLAP, in ER microsomes depleted of lumenal content by high pH washing. In untreated microsomes preprominiPLAP was processed to prominiPLAP and GPI-anchored miniPLAP. Both products were fully translocated, since they resisted proteinase K treatment of the microsomes, and both behaved as membrane proteins by the carbonate extraction criterion. Microsomes depleted of lumenal content were able to translocate and process preprominiPLAP to give protease-protected prominiPLAP, but were unable to convert prominiPLAP to miniPLAP. Loss of GPI anchoring capacity occurred with a wash of pH > 9.5. If the alkaline wash was performed after formation of prominiPLAP conversion to miniPLAP was relatively unimpaired. The results indicate that constituents of the ER lumen, possibly chaperones interacting with the proprotein and/or the GPI anchor precursor, are required in the initial steps of GPI anchoring.

Segregation of glycosylphosphatidylinositol biosynthetic reactions in a subcompartment of the endoplasmic reticulum.

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.

Endoplasmic reticulum proteins involved in glycosylphosphatidylinositol-anchor attachment: photocrosslinking studies in a cell-free system.

Assembly of glycosylphosphatidylinositol (GPtdIns)-anchored proteins requires translocation of the nascent polypeptide chain across the endoplasmic reticulum (ER) membrane and replacement of the C-terminal signal sequence with a GPtdIns moiety. The anchoring reaction is carried out by an ER enzyme, GPtdIns transamidase. Genetic studies with yeast indicate that the transamidase consists of a dynamic complex of at least two subunits, Gaa1p and Gpi8p. To study the GPtdIns-anchoring reaction, we used a small reporter protein that becomes GPtdIns-anchored when the corresponding mRNA is translated in the presence of microsomes, in conjunction with site-specific photocrosslinking to identify ER membrane components that are proximal to the reporter during its conversion to a GPtdIns-anchored protein. We generated variants of the reporter protein such that upon in vitro translation in the presence of Nepsilon-(5-azido-2-nitrobenzoyl)-lysyl-tRNA, photoreactive lysine residues would be incorporated in the protein specifically near the GPtdIns-attachment site. We analyzed photoadducts resulting from UV irradiation of the samples. We show that proproteins can be crosslinked to the transamidase subunit Gpi8p, as well as to ER proteins of molecular mass approximately 60 kDa, approximately 70 kDa, and approximately 120 kDa. The identification of a photoadduct between a proprotein and Gpi8p provides the first direct evidence of an interaction between a proprotein substrate and one of the genetically identified transamidase subunits. The approximately 70-kDa protein that we identified may correspond to the other subunit Gaa1p, while the other proteins possibly represent additional, hitherto unidentified subunits of the mammalian GPtdIns transamidase complex.

A cell-free assay for glycosylphosphatidylinositol anchoring in African trypanosomes. Demonstration of a transamidation reaction mechanism.

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 GPI-anchored 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 temperature-dependent, 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.

Cell surface display and intracellular trafficking of free glycosylphosphatidylinositols in mammalian cells.

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 degrees 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.

Topology of GPI biosynthesis in the endoplasmic reticulum

Glycosylphosphatidylinositol (GPI) anchors are constructed in the endoplasmic reticulum (ER) through the action of at least seven unique enzymes. Using cell-free systems, mainly derived from African trypanosomes, it has been experimentally possible to re-create many aspects of the GPI biosynthetic pathway in vitro and to obtain a series of glycosylated phosphatidylinositol structures that correspond to biosynthetic intermediates. This approach led to the identification of the biosynthetic donors of individual components of the GPI glycan, and the discovery of unusual fatty acid re-modelling reactions in the GPI pathway in trypanosomes. Despite this progress, questions remain concerning the enzymology of the pathway, particularly the topological distribution of the different assembly steps in the ER membrane. In the work described here we have attempted to define the transbilayer orientation of different GPI biosynthetic intermediates in the ER membrane bilayer. The experiments were performed with a microsomal fraction derived from bloodstream-form Trypanosoma brucei, and standard radiolabeling procedures. The orientation of GPIs was probed with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) and the jackbean lectin Concanavalin A. Contrary to expectations based on other ER glycosylation reactions, most notably the reactions involved in the dolichol pathway of N-glycosylation, our results suggest that non-inositol-acylated (PI-PLC-sensitive) GPIs are synthesized in the cytoplasmic leaflet of the ER membrane bilayer and that the final reaction product, a phosphoethanolamine-containing GPI, flips into the luminal leaflet for transfer to protein