Lateral diffusion of membrane-spanning and glycosylphosphatidylinositol-linked proteins: toward establishing rules governing the lateral mobility of membrane proteins.

In the plasma membrane of animal cells, many membrane-spanning proteins exhibit lower lateral mobilities than glycosylphosphatidylinositol (GPI)-linked proteins. To determine if the GPI linkage was a major determinant of the high lateral mobility of these proteins, we measured the lateral diffusion of chimeric membrane proteins composed of normally transmembrane proteins that were converted to GPI-linked proteins, or GPI-linked proteins that were converted to membrane-spanning proteins. These studies indicate that GPI linkage contributes only marginally (approximately twofold) to the higher mobility of several GPI-linked proteins. The major determinant of the high mobility of these proteins resides instead in the extracellular domain. We propose that lack of interaction of the extracellular domain of this protein class with other cell surface components allows diffusion that is constrained only by the diffusion of the membrane anchor. In contrast, cell surface interactions of the ectodomain of membrane-spanning proteins exemplified by the vesicular stomatitis virus G glycoprotein reduces their lateral diffusion coefficients by nearly 10-fold with respect to many GPI-linked proteins.     

Sorting of the human folate receptor in MDCK cells

The human folate receptor (hFR) is a glycosylphosphatidy-linositol (GPI) linked plasma membrane protein that mediates delivery of folates into cells. We studied the sorting of the hFR using transfection of the hFR cDNA into MDCK cells. MDCK cells are polarized epithelial cells that preferentially sort GPI-linked proteins to their apical membrane. Unlike other GPI-tailed proteins, we found that in MDCK cells, hFR is functional on both the apical and basolateral surfaces. We verified that the same hFR cDNA that transfected into CHO cells produces the hFR protein that is GPI-linked. We also measured the hFR expression on the plasma membrane of type III paroxysmal nocturnal hemoglobinuria (PNH) human erythrocytes. PNH is a disease that is characterized by the inability of cells to express membrane proteins requiring a GPI anchor. Despite this defect, and different from other GPI-tailed proteins, we found similar levels of hFR in normal and type III PNH human erythrocytes. The results suggest the hypothesis that there may be multiple mechanisms for targeting hFR to the plasma membrane.     

Lipid raft proteins have a random distribution during localized activation of the T-cell receptor

The extent to which lipid raft proteins are organized in functional clusters within the plasma membrane is central to the debate on structure and function of rafts. Glycosylphosphatidylinositol (GPI)-linked proteins are characteristic components of biochemically defined rafts. Several studies report a function for rafts in T-cell stimulation, but it is unclear whether molecules involved in T-cell receptor (TCR) signalling are recruited to (or excluded from) T-cell synapses through asymmetric distribution of raft microdomains or through specific protein-protein interactions. Here we used FRET analysis in live cells to determine whether GPI-linked proteins are clustered in the plasma membrane of unstimulated cells, and at regions where TCR signalling has been activated using antibody-coated beads. Multiple criteria suggested that FRET between different GPI-linked fluorescent proteins in COS-7 or unstimulated Jurkat T-cells is generated by a random, un-clustered distribution. Stimulation of TCR signalling in Jurkat cells resulted in localized increases in fluorescence of GPI-linked fluorescent proteins and cholera toxin B-subunit (CTB). However, measurements of FRET and ratio imaging showed that there was no detectable clustering and no overall enrichment of GPI-linked proteins or CTB in these regions.     

The human GPI1 gene is required for efficient glycosylphosphatidylinositol biosynthesis.

The first step in glycosylphosphatidylinositol (GPI) membrane anchor biosynthesis that is defective in paroxysmal nocturnal haemoglobinuria is mediated by an N-acetylglucosaminyl transferase expressed in the endoplasmic reticulum. Six human genes encode subunits of this enzyme, namely PIG-A, PIG-C, PIG-H, PIG-P, GPI1, and DPM2. Here, the human GPI1 gene is characterised. This gene is organised into eleven exons. The locus was mapped to chromosome 16p13.3 near the haemoglobin alpha chain locus. GPI1 is expressed ubiquitously in human cells and tissues. Expression levels are markedly elevated in haematopoietic tissues (bone marrow, foetal liver). To determine whether human GPI1 is essential for human GPI biosynthesis, antisense RNA was expressed in HEK293 cells. Transfectants exhibited a marked but incomplete decrease in the expression of a GPI-linked reporter protein, confirming that GPI1 is required for efficient GPI biosynthesis. In contrast, expression of GPI-linked proteins is normal in lymphatic cell lines from individuals with the alpha thalassaemia/mental retardation syndrome, which is characterised by large deletions from chromosome 16p removing one of the two GPI1 alleles along with the haemoglobin alpha locus. In conclusion, GPI1 plays an important role in the biosynthesis of GPI intermediates. Due to its autosomal localisation, the heterozygous deletion of GPI1 does not lead to an overt defect in the expression of GPI-linked proteins.     

Intracellular retention of glycosylphosphatidyl inositol-linked proteins in caveolin-deficient cells

The relationship between glycosylphosphatidyl inositol (GPI)-linked proteins and caveolins remains controversial. Here, we derived fibroblasts from Cav-1 null mouse embryos to study the behavior of GPI-linked proteins in the absence of caveolins. These cells lack morphological caveolae, do not express caveolin-1, and show a approximately 95% down-regulation in caveolin-2 expression; these cells also do not express caveolin-3, a muscle-specific caveolin family member. As such, these caveolin-deficient cells represent an ideal tool to study the role of caveolins in GPI-linked protein sorting. We show that in Cav-1 null cells GPI-linked proteins are preferentially retained in an intracellular compartment that we identify as the Golgi complex. This intracellular pool of GPI-linked proteins is not degraded and remains associated with intracellular lipid rafts as judged by its Triton insolubility. In contrast, GPI-linked proteins are transported to the plasma membrane in wild-type cells, as expected. Furthermore, recombinant expression of caveolin-1 or caveolin-3, but not caveolin-2, in Cav-1 null cells complements this phenotype and restores the cell surface expression of GPI-linked proteins. This is perhaps surprising, as GPI-linked proteins are confined to the exoplasmic leaflet of the membrane, while caveolins are cytoplasmically oriented membrane proteins. As caveolin-1 normally undergoes palmitoylation on three cysteine residues (133, 143, and 156), we speculated that palmitoylation might mechanistically couple caveolin-1 to GPI-linked proteins. In support of this hypothesis, we show that palmitoylation of caveolin-1 on residues 143 and 156, but not residue 133, is required to restore cell surface expression of GPI-linked proteins in this complementation assay. We also show that another lipid raft-associated protein, c-Src, is retained intracellularly in Cav-1 null cells. Thus, Golgi-associated caveolins and caveola-like vesicles could represent part of the transport machinery that is necessary for efficiently moving lipid rafts and their associated proteins from the trans-Golgi to the plasma membrane. In further support of these findings, GPI-linked proteins were also retained intracellularly in tissue samples derived from Cav-1 null mice (i.e., lung endothelial and renal epithelial cells) and Cav-3 null mice (skeletal muscle fibers).     

GPI-anchored GFP signals Ca2+ but is homogeneously distributed on the cell surface

Glycosyl-phosphatidylinositol (GPI)-anchored proteins are unique in that they penetrate only the outer leaflet of the plasma membrane but are still able to mediate intracellular signalling events following antibody-induced ligation. Detergent solubilisation studies suggest that microdomains exist at the cell surface within which are sequestered GPI-linked proteins. Here we report the construction and expression of a fluorescent GPI anchor on the surface of CHO, EL4, and U937 cells by fusing green fluorescent protein (GFP) to the GPI-attachment site of CD59. The resultant GFP-GPI has properties comparable to that of endogenously expressed GPI-anchored molecules as shown by Triton X-114 partitioning. However, sucrose gradient floatation showed that GFP-GPI was only partially resistant to detergent solubilisation. Furthermore confocal scanning laser microscopy revealed a homogeneous distribution of GFP-GPI at the cell surface, which only became clustered following cross-linking of the GPI anchor via an anti-GFP antibody. Surprisingly, GFP-GPI signalled Ca2+ change upon cross-linking demonstrating its signalling competence. Our results suggest that the GPI-anchor itself does not confer a clustered distribution to molecules but that clustering occurs following ligation with antibody, which allows the protein to become Ca2+ signalling competent.     

Deletion of the GPI pre-anchor sequence in human p97--a general approach for generating the soluble form of GPI-linked proteins

Melanotransferrin, also named p97, belongs to the transferrin-like group of iron-binding proteins. Unlike the other members of this family, p97 exists in two forms-one soluble form and one attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. The GPI-linked form plays a role in the uptake of iron, while the soluble form of p97 has the unique ability of traversing the blood-brain barrier and may be utilized to deliver drug conjugates into the brain. To investigate these possibilities, a recombinant soluble form of p97 from the GPI-linked p97 protein is required. The approach involved sequential deletions of the p97 GPI pre-anchor sequence (PAS) up to the putative site of cleavage/attachment, releasing p97 from attachment to the GPI-anchor and rendering it soluble. Transfection of the p97 deletion constructs into both the CHO and BHK TK(-) cells was performed with the aim of optimizing the production of p97 by utilizing the cell characteristics unique to each cell line. Altering the GPI PAS resulted in the generation of a recombinant soluble form that was secreted at significantly higher rates than from the full-length expressing cell lines. Increases were from 22 x 10(-9) to 241 x 10(-9)microg/cell/h for expression in the CHO cell system and from 220 x 10(-9) to 4970 x 10(-9)microg/cell/h for the BHK system. Furthermore, there appeared to be differences in the secretion rates between the various deletions suggesting the need for closer examination of the C-terminus in achieving maximum production of the altered proteins. The results of this study are likely applicable for expressing soluble forms of other GPI-linked proteins.     

Signal transducing molecules and glycosyl-phosphatidylinositol-linked proteins form a caveolin-rich insoluble complex in MDCK cells

GPI-linked protein molecules become Triton-insoluble during polarized sorting to the apical cell surface of epithelial cells. These insoluble complexes, enriched in cholesterol, glycolipids, and GPI-linked proteins, have been isolated by flotation on sucrose density gradients and are thought to contain the putative GPI-sorting machinery. As the cellular origin and molecular protein components of this complex remain unknown, we have begun to characterize these low-density insoluble complexes isolated from MDCK cells. We find that these complexes, which represent 0.4-0.8% of the plasma membrane, ultrastructurally resemble caveolae and are over 150-fold enriched in a model GPI-anchored protein and caveolin, a caveolar marker protein. However, they exclude many other plasma membrane associated molecules and organelle-specific marker enzymes, suggesting that they represent microdomains of the plasma membrane. In addition to caveolin, these insoluble complexes contain a subset of hydrophobic plasma membrane proteins and cytoplasmically-oriented signaling molecules, including: (a) GTP- binding proteins--both small and heterotrimeric; (b) annex II--an apical calcium-regulated phospholipid binding protein with a demonstrated role in exocytic fusion events; (c) c-Yes--an apically localized member of the Src family of non-receptor type protein- tyrosine kinases; and (d) an unidentified serine-kinase activity. As we demonstrate that caveolin is both a transmembrane molecule and a major phospho-acceptor component of these complexes, we propose that caveolin could function as a transmembrane adaptor molecule that couples luminal GPI-linked proteins with cytoplasmically oriented signaling molecules during GPI-membrane trafficking or GPI-mediated signal transduction events. In addition, our results have implications for understanding v- Src transformation and the actions of cholera and pertussis toxins on hetero-trimeric G proteins.     

Novel applications for glycosylphosphatidylinositol-anchored proteins in pharmaceutical and industrial biotechnology

Glycosylphosphatidylinositol (GPI)-anchored proteins have been regarded as typical cell surface proteins found in most eukaryotic cells from yeast to man. They are embedded in the outer plasma membrane leaflet via a carboxy-terminally linked complex glycolipid GPI structure. The amphiphilic nature of the GPI anchor, its compatibility with the function of the attached protein moiety and the capability of GPI-anchored proteins for spontaneous insertion into and transfer between artificial and cellular membranes initially suggested their potential for biotechnological applications. However, these expectations have been hardly fulfilled so far. Recent developments fuel novel hopes with regard to: (i) Automated online expression, extraction and purification of therapeutic proteins as GPI-anchored proteins based on their preferred accumulation in plasma membrane lipid rafts, (ii) multiplex custom-made protein chips based on GPI-anchored cell wall proteins in yeast, (iii) biomaterials and biosensors with films consisting of sets of distinct GPI-anchored binding-proteins or enzymes for sequential or combinatorial catalysis, and (iv) transport of therapeutic proteins across or into relevant tissue cells, e.g., enterocytes or adipocytes. Latter expectations are based on the demonstrated translocation of GPI-anchored proteins from plasma membrane lipid rafts to cytoplasmic lipid droplets and eventually further into microvesicles which upon release from donor cells transfer their GPI-anchored proteins to acceptor cells. The value of these technologies, which are all based on the interaction of GPI-anchored proteins with membranes and surfaces, for the engineering, production and targeted delivery of biomolecules for a huge variety of therapeutic and biotechnological purposes should become apparent in the near future.     

Flotillin-1 defines a clathrin-independent endocytic pathway in mammalian cells

Previous studies provide evidence for an endocytic mechanism in mammalian cells that is distinct from both clathrin-coated pits and caveolae, and is not inhibited by overexpression of GTPase-null dynamin mutants. This mechanism, however, has been defined largely in these negative terms. We applied a ferro-fluid-based purification of endosomes to identify endosomal proteins. One of the proteins identified in this way was flotillin-1 (also called reggie-2). Here, we show that flotillin-1 resides in punctate structures within the plasma membrane and in a specific population of endocytic intermediates. These intermediates accumulate both glycosylphosphatidylinositol (GPI)-linked proteins and cholera toxin B subunit. Endocytosis in flotillin-1-containing intermediates is clathrin-independent. Total internal reflection microscopy and immuno-electron microscopy revealed that flotillin-1-containing regions of the plasma membrane seem to bud into the cell, and are distinct from clathrin-coated pits and caveolin-1-positive caveolae. Flotillin-1 small interfering RNA (siRNA) inhibited both clathrin-independent uptake of cholera toxin and endocytosis of a GPI-linked protein. We propose that flotillin-1 is one determinant of a clathrin-independent endocytic pathway in mammalian cells.     

Caveolin forms a hetero-oligomeric protein complex that interacts with an apical GPI-linked protein: implications for the biogenesis of caveolae

Glycosyl-phosphatidylinositol (GPI)-linked proteins are transported to the apical surface of epithelial cells where they undergo cholesterol- dependent clustering in membrane micro-invaginations, termed caveolae or plasmalemmal vesicles. However, the sorting machinery responsible for this caveolar-clustering mechanism remains unknown. Using transfected MDCK cells as a model system, we have identified a complex of cell surface molecules (80, 50, 40, 22-24, and 14 kD) that interact in a pH- and cholesterol-dependent fashion with an apical recombinant GPI-linked protein. A major component of this hetero-oligomeric protein complex is caveolin, a type II transmembrane protein. As this hetero- oligomeric caveolin complex is detectable almost immediately after caveolin synthesis, our results suggest that caveolae may assemble intracellularly during transport to the cell surface. As such, our studies have implications for understanding both the intracellular biogenesis of caveolae and their subsequent interactions with GPI- linked proteins in epithelia and other cell types.     

Identification of Reggie-1 and Reggie-2 as plasmamembrane-associated proteins which cocluster with activated GPI-anchored cell adhesion molecules in non-caveolar micropatches in neurons

Neurons are believed to possess plasmalemmal microdomains and proteins analogous to the caveolae and caveolin of nonneuronal cells. Caveolae are plasmalemmal invaginations where activated glycosyl-phosphatidylinositol (GPI)-anchored proteins preferentially assemble and where transmembrane signaling may occur. Molecular cloning of rat reggie-1 and -2 (80% identical to goldfish reggie proteins) shows that reggie-2 is practically identical to mouse flotillin-1. Flotillin-1 and epidermal surface antigen (ESA) (flotillin-2) are suggested to represent possible membrane proteins in caveolae. Rat reggie-1 is 99% homologous to ESA in overlapping sequences but has a 49-amino-acid N-terminus not present in ESA. Antibodies (ABs) which recognize reggie-1 or -2 reveal that both proteins cluster at the plasmamembrane and occur in micropatches in neurons [dorsal root ganglia (DRGs), retinal ganglion, and PC-12 cells] and in nonneuronal cells. In neurons, reggie micropatches occur along the axon and in lamellipodia and filopodia of growth cones, but they do not occur in caveolae. By quantitative electronmicroscopic analysis we demonstrate the absence of caveolae in (anti-caveolin negative) neurons and show anti-reggie-1 immunogold-labeled clusters at the plasmamembrane of DRGs. When ABs against the GPI-anchored cell adhesion molecules (CAMs) F3 and Thy-1 are applied to live DRGs, the GPI-linked CAMs sequester into micropatches. Double immunofluorescence shows a colocalization of the CAMs with micropatches of anti-reggie antibodies. Thus, reggie-1 and reggie-2 identify sites where activated GPI-linked CAMs preferentially accumulate and which may represent noncaveolar micropatches (domains). © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 502–523, 1998     

Investigating the role of murine epididymosomes and uterosomes in GPI-linked protein transfer to sperm using SPAM1 as a model

Sperm uptake of glycosyl phosphatidylinositol (GPI)-linked proteins from luminal fluids has been shown to occur in male and estrous female reproductive tracts. In males, this is attributed to membranous vesicles secreted into the epididymis and prostate. While epididymosomes have been characterized, there have been no reports of the presence of vesicles in female luminal fluids. Here we report the presence of vesicles, characterized as "uterosomes," in the murine estrous female reproductive fluid; and use Sperm Adhesion Molecule 1 (SPAM1/PH-20), a well-known hyaluronidase found in male and female fluids, as a model to investigate vesicle-mediated GPI-linked protein transfer to sperm. Epididymosomes and uterosomes isolated after ultracentrifugation of epididymal (ELF) and uterine luminal fluid (ULF) were analyzed by electron microscopy and shown to be approximately 10-70 and approximately 15-50 nm in diameter. The structural integrity of uterosomes was confirmed by their resistance to hypo-osmotic and freeze/thaw stresses; and immunogold labeling localized SPAM1 to their outer membrane surface, as was the case for epididymosomes. SPAM1 was acquired by caudal sperm during incubation in epididymosomes and uterosomes; uptake was abolished when the GPI anchor was enzymatically cleaved. Sperm analyzed by confocal and transmission electron microscopy (TEM) after incubation in fluorescently labeled vesicles revealed the label on the membrane over the acrosome and midpiece of the flagella, where SPAM1 normally resides. High magnification TEM images demonstrated vesicles juxtaposed to the sperm plasma membrane potentially transferring SPAM1. Taken together, these results implicate vesicular docking as the mechanism of vesicle-mediated GPI-linked protein transfer to sperm from murine reproductive fluids.     

Uromodulin (Tamm-Horsfall glycoprotein/uromucoid) is a phosphatidylinositol-linked membrane protein

Uromodulin, originally identified as an immunosuppressive glycoprotein in the urine of pregnant women, has been previously shown to be identical to human Tamm-Horsfall glycoprotein (THP). THP is synthesized by the kidney and localizes to the renal thick ascending limb and early distal tubule. It is released into the urine in large quantities and thus represents a potential candidate for a protein secreted in a polarized fashion from the apical plasma membrane of epithelial cells in vivo. After introduction of the full-length cDNA encoding uromodulin/THP into HeLa, Caco-2, and Madin-Darby canine kidney cells by transfection, however, the expressed glycoprotein was almost exclusively cell-associated, as determined by immunoprecipitation after radioactive labeling of the cells. By immunofluorescence, THP was localized to the plasma membranes of transfected cells. In transfected cell extracts, THP also remained primarily in the detergent phase in a Triton X-114 partitioning assay, indicating that it has a hydrophobic character, in contrast to its behavior after isolation from human urine. Triton X-114 detergent-associated THP was redistributed to the aqueous phase after treatment of cell extracts with phosphatidylinositol-specific phospholipase C. Treatment of intact transfected HeLa cells with phosphatidylinositol-specific phospholipase C also resulted in the release of THP into the medium, suggesting that it is a glycosylphosphatidylinositol (GPI)-linked membrane protein. Similar to other known GPI-linked proteins, uromodulin/THP contains a stretch of 16 hydrophobic amino acids at its extreme carboxyl terminus which could function as a GPI addition signal and was shown to label with [3H]ethanolamine. The results indicate that THP is a member of this class of lipid-linked membrane proteins and is released into the urine after the loss of its hydrophobic anchor, probably by the action of a phospholipase or protease.     

The glycosylphosphatidyl inositol-anchored adhesion molecule F3/contactin is required for surface transport of paranodin/contactin-associated protein (caspr)

Paranodin/contactin-associated protein (caspr) is a transmembrane glycoprotein of the neurexin superfamily that is highly enriched in the paranodal regions of myelinated axons. We have investigated the role of its association with F3/contactin, a glycosylphosphatidyl inositol (GPI)-anchored neuronal adhesion molecule of the Ig superfamily. Paranodin was not expressed at the cell surface when transfected alone in CHO or neuroblastoma cells. Cotransfection with F3 resulted in plasma membrane delivery of paranodin, as analyzed by confocal microscopy and cell surface biotinylation. The region that mediates association with paranodin was mapped to the Ig domains of F3 by coimmunoprecipitation experiments. The association of paranodin with F3 allowed its recruitment to Triton X-100-insoluble microdomains. The GPI anchor of F3 was necessary, but not sufficient for surface expression of paranodin. F3-Ig, a form of F3 deleted of the fibronectin type III (FNIII) repeats, although GPI-linked and expressed at the cell surface, was not recovered in the microdomain fraction and was unable to promote cell surface targeting of paranodin. Thus, a cooperative effect between the GPI anchor, the FNIII repeats, and the Ig regions of F3 is required for recruitment of paranodin into lipid rafts and its sorting to the plasma membrane.     

Requirements for glycosylphosphatidylinositol attachment are similar but not identical in mammalian cells and parasitic protozoa

The general features of the glycosylphosphatidylinositol (GPI) signal have been conserved in evolution. To test whether the requirements for GPI attachment are indeed the same in mammalian cells and parasitic protozoa, we expressed the prototype GPI-linked protein of Trypanosoma brucei, the variant surface glycoprotein (VSG), in COS cells. Although large amounts of VSG were produced, only a small fraction became GPI linked. This impaired processing is not caused by the VSG ectodomain, since replacement of the VSG GPI signal with that of decay accelerating factor (DAF) produced GPI-linked VSG. Furthermore, whereas fusion of the DAF GPI signal to the COOH terminus of human growth hormone (hGH) produces GPI-linked hGH, an analogous hGH fusion using the VSG GPI signal does not, indicating that the VSG GPI signal functions poorly in mammalian cells. By constructing chimeric VSG-DAF GPI signals and fusing them to the COOH terminus of hGH, we show that of the two critical elements that comprise the GPI-signal--the cleavage/attachment site and the COOH terminal hydrophobic domain--the former is responsible for the impaired activity of the VSG GPI signal in COS cells. To confirm this, we show that the VSG GPI signal can be converted to a viable signal for mammalian cells by altering the amino acid configuration at the cleavage/attachment site. We also show that when fused to the COOH terminus of hGH, the putative GPI signal from the malaria circumsporozoite (CS) protein produces low levels of GPI- anchored hGH, suggesting that the CS protein is indeed GPI linked, but that the CS protein GPI signal, like the VSG-signal, functions poorly in COS cells. The finding that the requirements for GPI attachment are similar but not identical in parasitic protozoa and mammalian cells may allow for the development of selective inhibitors of GPI-anchoring that might prove useful as antiparasite therapeutics.     

Elongation of axolotl tailbud embryos requires GPI-linked proteins and organizer-induced, active, ventral trunk endoderm cell rearrangements

Application of phosphatidylinositol-specific phospholipase C to early tailbud stage axolotl embryos reveals that a specific subset of morphogenetic movements requires glycosylphosphatidylinositol (GPI)-linked cell-surface proteins. These include pronephric duct extension, "gill bulge" formation, and embryonic elongation along the anteroposterior axis. The work of Kitchin (1949, J. Exp. Zool. 112, 393-416) led to the conclusion that extension of the notochord provided the motive force driving anteroposterior stretching in axolotl embryos, elongation of other tissues being a passive response. We therefore conjectured that axial mesoderm cells might display the GPI-linked proteins required for elongation of the embryo. However, we show here that removal of most of the neural plate and axial and paraxial mesoderm prior to neural tube closure does not prevent elongation of ventrolateral tissues. Tissue-extirpation and tissue-marking experiments indicate that elongation of the ventral trunk occurs via active, directed tissue rearrangements within the endoderm, directed by signals emanating from the blastopore region. Extension of both dorsal and ventral tissues requires GPI-linked proteins. We conclude that elongation of axolotl embryos requires active cell rearrangements within ventral as well as axial tissues. The fact that both types of elongation are prevented by removal of GPI-linked proteins implies that they share a common molecular mechanism.     

Cholesterol depletion suppresses the translational diffusion of class II major histocompatibility complex proteins in the plasma membrane

Glycosylphosphatidylinositol (GPI)-linked and native major histocompatibility complex class II I-E(k) were used as probes to determine the effect of varying cholesterol concentration on the mobility of proteins in the plasma membrane. These proteins were imaged in Chinese hamster ovary cells using single-molecule fluorescence microscopy. Observed diffusion coefficients of both native and GPI-linked I-E(k) proteins were found to depend on cholesterol concentration. As the cholesterol concentration decreases the diffusion coefficients decrease by up to a factor of 7 for native and 5 for GPI-linked I-E(k). At low cholesterol concentrations, after sphingomyelinase treatment, the diffusion coefficients are reduced by up to a factor of 60 for native and 190 for GPI-linked I-E(k). The effect is reversible on cholesterol reintroduction. Diffusion at all studied cholesterol concentrations, for both proteins, appears to be predominantly Brownian for time lags up to 2.5 s when imaged at 10 Hz. A decrease in diffusion coefficients is observed for other membrane proteins and lipid probes, DiIC12 and DiIC18. Fluorescence recovery after photobleaching measurements shows that the fraction of immobile lipid probe increases from 8 to approximately 40% after cholesterol extraction. These results are consistent with the previous work on cholesterol-phospholipid interactions. That is, cholesterol extraction destroys liquid cholesterol-phospholipid complexes, leaving solid-like high melting phospholipid domains that inhibit the lateral diffusion of membrane components.     

PIG-S and PIG-T, essential for GPI anchor attachment to proteins, form a complex with GAA1 and GPI8

Many eukaryotic cell surface proteins are anchored to the plasma membrane via glycosylphosphatidylinositol (GPI). The GPI transamidase mediates GPI anchoring in the endoplasmic reticulum, by replacing a protein's C-terminal GPI attachment signal peptide with a pre-assembled GPI. During this transamidation reaction, the GPI transamidase forms a carbonyl intermediate with a substrate protein. It was known that the GPI transamidase is a complex containing GAA1 and GPI8. Here, we report two new components of this enzyme: PIG-S and PIG-T. To determine roles for PIG-S and PIG-T, we disrupted these genes in mouse F9 cells by homologous recombination. PIG-S and PIG-T knockout cells were defective in transfer of GPI to proteins, particularly in formation of the carbonyl intermediates. We also demonstrate that PIG-S and PIG-T form a protein complex with GAA1 and GPI8, and that PIG-T maintains the complex by stabilizing the expression of GAA1 and GPI8. Saccharomyces cerevisiae Gpi16p (YHR188C) and Gpi17p (YDR434W) are orthologues of PIG-T and PIG-S, respectively.