Identification of a second member of the ponticulin gene family and its differential expression pattern

We have identified a homologue (ponB) of the ponticulin gene (ponA), an F-actin binding protein, in the expressed sequence tag library generated to mRNA isolated from fusion-competent cells of Dictyostelium discoideum. PonB is predicted to have many of the same characteristics as ponticulin. Both proteins are predicted to possess a cleaved signal peptide, a glycosyl anchor, an amphipathic beta-strand structure and six conserved cysteines. Because of the sequence similarity and predicted conserved structures, this gene constitutes the second member of a ponticulin gene family. Unlike ponticulin, ponB is not expressed in axenically grown cells or during the asexual reproductive phase of D. discoideum. PonB is expressed by cells grown on bacterial lawns and by cells induced to be fusion-competent, i.e., gametes. The expression of ponB correlates with the appearance of a new F-actin binding activity in cell lysates of bacterially grown ponA(-) cells. By immunofluorescence microscopy, ponB appears to be localized to vesicles and to the plasma membrane of bacterially grown cells. Because ponticulin is the major high-affinity link between the plasma membrane and the cytoskeleton, the ponticulin gene family is likely to be part of the redundant system of proteins involved in connecting the cytoskeleton to the plasma membrane.     

Ponticulin, a developmentally-regulated plasma membrane glycoprotein, mediates actin binding and nucleation

Ponticulin is a 17,000-dalton transmembrane glycoprotein that is involved in the binding and nucleation of actin filaments by Dictyostelium discoideum plasma membranes. The major actin-binding protein isolated from these membranes by F-actin affinity chromatography, ponticulin also binds F-actin on blot overlays. The actin-binding activity of ponticulin in vitro is identical to that observed for purified plasma membranes: it resists extraction with 0.1 N NaOH, is sensitive to high salt concentrations, and is destroyed by heat, proteolysis, and thiol reduction and alkylation. A cytoplasmic domain of ponticulin mediates binding to actin because univalent antibody fragments directed against the cytoplasmic surface of this protein inhibit 96% of the actin-membrane binding in sedimentation assays. Antibody specific for ponticulin removes both ponticulin and the ability to reconstitute actin nucleation activity from detergent extracts of solubilized plasma membranes. Levels of plasma membrane ponticulin increase 2- to 3-fold during aggregation streaming, when cells adhere to each other and are highly motile. Although present throughout the plasma membrane, ponticulin is preferentially localized to some actin-rich membrane structures, including sites of cell-cell adhesion and arched regions of the plasma membrane reminiscent of the early stages of pseudopod formation. Ponticulin also is present but not obviously enriched at phagocytic cups of log-phase amebae. These results indicate that ponticulin may function in vivo to attach and nucleate actin filaments at the cytoplasmic surface of the plasma membrane. A 17,000-dalton analogue of ponticulin has been identified in human polymorphonuclear leukocyte plasma membranes by immunoblotting and immunofluorescence microscopy.(ABSTRACT TRUNCATED AT 250 WORDS)     

The integral membrane protein, ponticulin, acts as a monomer in nucleating actin assembly

Ponticulin, an F-actin binding transmembrane glycoprotein in Dictyostelium plasma membranes, was isolated by detergent extraction from cytoskeletons and purified to homogeneity. Ponticulin is an abundant membrane protein, averaging approximately 10(6) copies/cell, with an estimated surface density of approximately 300 per microns2. Ponticulin solubilized in octylglucoside exhibited hydrodynamic properties consistent with a ponticulin monomer in a spherical or slightly ellipsoidal detergent micelle with a total molecular mass of 56 +/- 6 kD. Purified ponticulin nucleated actin polymerization when reconstituted into Dictyostelium lipid vesicles, but not when a number of commercially available lipids and lipid mixtures were substituted for the endogenous lipid. The specific activity was consistent with that expected for a protein comprising 0.7 +/- 0.4%, by mass, of the plasma membrane protein. Ponticulin in octylglucoside micelles bound F- actin but did not nucleate actin assembly. Thus, ponticulin-mediated nucleation activity was sensitive to the lipid environment, a result frequently observed with transmembrane proteins. At most concentrations of Dictyostelium lipid, nucleation activity increased linearly with increasing amounts of ponticulin, suggesting that the nucleating species is a ponticulin monomer. Consistent with previous observations of lateral interactions between actin filaments and Dictyostelium plasma membranes, both ends of ponticulin-nucleated actin filaments appeared to be free for monomer assembly and disassembly. Our results indicate that ponticulin is a major membrane protein in Dictyostelium and that, in the proper lipid matrix, it is sufficient for lateral nucleation of actin assembly. To date, ponticulin is the only integral membrane protein known to directly nucleate actin polymerization.     

Indirect immunofluorescence localization of ponticulin in motile cells

Ponticulin is the major actin-binding integral glycoprotein in plasma membranes isolated from log-phase Dictyostelium discoideum amebae. As such, this protein appears to be an important link between the plasma membrane and actin filaments (Wuestehube and Luna: Journal of Cell Biology 105:1741-1751, 1987). In this study, indirect immunofluorescence microscopy was used to examine the distribution of ponticulin in randomly moving D. discoideum amebae and in amebae engaged in cell migration and phagocytosis. Ponticulin is distributed throughout the plasma membrane and also is present in intracellular vesicles associated with the microtubule-organizing center-Golgi complex adjacent to the nucleus. In aggregating amebae, ponticulin is concentrated in regions of lateral cell-cell contact and in arched regions of the plasma membrane. Ponticulin also is present, but not obviously enriched, in filopodia, in the actin-rich anterior end of polarized cells, and in detergent-insoluble cytoskeletons. In amebae engaged in phagocytosis of yeast, ponticulin is present but not enriched in phagocytic cups and is associated with intracellular vesicles around engulfed yeast. These results suggest that ponticulin is stably associated with actin filaments in certain regions of the plasma membrane and that the actin-binding activity of ponticulin may be tightly controlled. Indirect immunofluorescence microscopy and immunoblot analysis demonstrate that human polymorphonuclear leukocytes also contain a 17 kD protein that specifically cross-reacts with antibodies affinity-purified against D. discoideum ponticulin. As in D. discoideum, the mammalian 17 kD ponticulin-analog appears to be localized in plasma membrane and is evident in actin-rich cell extensions. These results indicate that ponticulin-mediated linkages between the plasma membrane and actin may be present in higher eukaryotic cells.     

A Golgi retention signal in a membrane-spanning domain of coronavirus E1 protein

The E1 glycoprotein from an avian coronavirus is a model protein for studying retention in the Golgi complex. In animal cells expressing the protein from cDNA, the E1 protein is targeted to cis Golgi cisternae (Machamer, C. E., S. A. Mentone, J. K. Rose, and M. G. Farquhar. 1990. Proc. Natl. Acad. Sci. USA. 87:6944-6948). We show that the first of the three membrane-spanning domains of the E1 protein can retain two different plasma membrane proteins in the Golgi region of transfected cells. Both the vesicular stomatitis virus G protein and the alpha-subunit of human chorionic gonadotropin (anchored to the membrane by fusion with the G protein membrane-spanning domain and cytoplasmic tail) were retained in the Golgi region of transfected cells when their single membrane-spanning domains were replaced with the first membrane-spanning domain from E1. Single amino acid substitutions in this sequence released retention of the chimeric G protein, as well as a mutant E1 protein which lacks the second and third membrane-spanning domains. The important feature of the retention sequence appears to be the uncharged polar residues which line one face of a predicted alpha helix. This is the first retention signal to be defined for a resident Golgi protein. The fact that it is present in a membrane-spanning domain suggests a novel mechanism of retention in which the membrane composition of the Golgi complex plays an instrumental role in retaining its resident proteins.     

Signal-anchor domains of proteins of the outer membrane of mitochondria: structural and functional characteristics

We have studied the topogenesis of a class of mitochondrial outer membrane proteins that expose a hydrophilic domain to the cytosol and are anchored to the membrane by a single transmembrane domain in the N-terminal region. To determine the role of these latter sequences in the targeting and insertion of such proteins we took two approaches. First, a functional complementation assay was used to define the structural elements that together with the anchor domain make up the topogenic signal. Moderate hydrophobicity of the transmembrane domain was found to be the most important requirement. Variants with a scrambled sequence of the membrane-spanning segment were only partially functional suggesting that specificity in the amino acid sequence is also of considerable importance. A net positive charge at both flanking regions of the transmembrane domain contributes to the efficiency of targeting and membrane integration but is not an essential structural feature of this signal. Second, chimeras of Tom20, Tom70, and OM45 were generated that contained the cytosolic domain of Tom20 or Tom70 and the anchor domain of one of the other members of the class. These hybrid proteins were able to rescue the growth of cells lacking Tom20 or Tom70. Thus, anchor domains of outer membrane proteins are functionally interchangeable. They play only a minor role in the specific function of these proteins, but have a decisive role in topogenic signaling.     

O-glycosylation of mucin-like domain retains the neutral ceramidase on the plasma membranes as a type II integral membrane protein

Ceramidase is a key enzyme involved in regulating cellular levels of ceramide, sphingosine, and possibly sphigosine 1-phosphate and thus could modulate sphingolipid signaling. Here we report that O-glycosylation of the mucin-like domain of neutral ceramidases was required for localization to the surface of plasma membranes. The deduced amino acid sequences of the mammalian enzymes contain a serine-threonine-rich domain (mucin box), which follows the signal/anchor sequence, whereas those of bacterial and invertebrate enzymes completely lack a mucin box, suggesting that the specific domain has been acquired during evolution. In HEK293 cells overexpressing ceramidase, the enzyme was not only secreted into the medium after cleavage of the NH(2)-terminal signal/anchor sequence but also localized at the plasma membrane as a type II integral membrane protein. Lectin blot analysis using peanut agglutinin revealed that the mucin box of the enzyme is highly glycosylated with O-glycans. Interestingly, a mutant lacking the mucin box or possible O-glycosylation sites in the mucin box was secreted into the medium but not localized at the surface of the cells. Furthermore, a mucin box-fused chimera green fluorescent protein (GFP), but not GFP itself, with the signal/anchor sequence was distributed on the surface of the cells. These results suggest that O-glycosylation of the mucin box retains proteins on the plasma membranes. We also found that the 112-kDa membrane-bound enzyme from mouse kidney is O-glycosylated, whereas the 94-kDa soluble enzyme from liver is not. These results clearly indicate that post-translational modification of the enzyme with O-glycans is tissue-specific and helps the enzyme to localize at the surface of plasma membranes as a type II membrane protein.     

Specialized membrane-localized chaperones prevent aggregation of polytopic proteins in the ER

The integral endoplasmic reticulum (ER) membrane protein Shr3p is required for proper plasma membrane localization of amino acid permeases (AAPs) in yeast. In the absence of Shr3p AAPs are uniquely retained in the ER with each of their twelve membrane-spanning segments correctly inserted in the membrane. Here, we show that the membrane domain of Shr3p specifically prevents AAPs from aggregating, and thus, plays a critical role in assisting AAPs to fold and correctly attain tertiary structures required for ER exit. Also, we show that the integral ER proteins, Gsf2p, Pho86p, and Chs7p, function similarly to Shr3p. In cells individually lacking one of these components only their cognate substrates, hexose transporters, phosphate transporters, and chitin synthase-III, respectively, aggregate and consequently fail to exit the ER membrane. These findings indicate that polytopic membrane proteins depend on specialized membrane-localized chaperones to prevent inappropriate interactions between membrane-spanning segments as they insert and fold in the lipid bilayer of the ER membrane.     

The transmembrane domain and cytoplasmic tail of herpes simplex virus type 1 glycoprotein H play a role in membrane fusion

Herpes simplex virus glycoprotein H (gH) is one of the four virion envelope proteins which are required for virus entry and for cell-cell fusion in a transient system. In this report, the role of the transmembrane and cytoplasmic tail domains of gH in membrane fusion was investigated by generating chimeric constructs in which these regions were replaced with analogous domains from other molecules and by introducing amino acid substitutions within the membrane-spanning sequence. gH molecules which lack the authentic transmembrane domain or cytoplasmic tail were unable to mediate cell-cell fusion when coexpressed with gB, gD, and gL and were unable to rescue the infectivity of a gH-null virus as efficiently as a wild-type gH molecule. Many amino acid substitutions of specific amino acid residues within the transmembrane domain also affected cell-cell fusion, in particular, those introduced at a conserved glycine residue. Some gH mutants that were impaired in cell-cell fusion were nevertheless able to rescue the infectivity of a gH-negative virus, but these pseudotyped virions entered cells more slowly than wild-type virions. These results indicate that the fusion event mediated by the coexpression of gHL, gB, and gD in cells shares common features with the fusion of the virus envelope with the plasma membrane, they point to a likely role for the membrane-spanning and cytoplasmic tail domains of gH in both processes, and they suggest that a conserved glycine residue in the membrane-spanning sequence is crucial for efficient fusion.     

Membrane association of the RNA-dependent RNA polymerase is essential for hepatitis C virus RNA replication

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), represented by nonstructural protein 5B (NS5B), belongs to a class of integral membrane proteins termed tail-anchored proteins. Its membrane association is mediated by the C-terminal 21 amino acid residues, which are dispensable for RdRp activity in vitro. For this study, we investigated the role of this domain, termed the insertion sequence, in HCV RNA replication in cells. Based on a structural model and the amino acid conservation among different HCV isolates, we designed a panel of insertion sequence mutants and analyzed their membrane association and RNA replication. Subgenomic replicons with a duplication of an essential cis-acting replication element overlapping the sequence that encodes the C-terminal domain of NS5B were used to unequivocally distinguish RNA versus protein effects of these mutations. Our results demonstrate that the membrane association of the RdRp is essential for HCV RNA replication. Interestingly, certain amino acid substitutions within the insertion sequence abolished RNA replication without affecting membrane association, indicating that the C-terminal domain of NS5B has functions beyond serving as a membrane anchor and that it may be involved in critical intramembrane protein-protein interactions. These results have implications for the functional architecture of the HCV replication complex and provide new insights into the expanding spectrum of tail-anchored proteins.     

Direct binding of F-actin to ponticulin, an integral plasma membrane glycoprotein

We have developed an 125I-labeled F-actin blot overlay assay for the identification of F-actin-binding proteins after transfer to nitrocellulose from SDS-polyacrylamide gels. Two major F-actin-binding proteins from Dictyostelium discoideum, a cytoplasmic 30 kDa protein and a 17 kDa integral membrane protein, and two minor membrane polypeptides of 19 kDa and 15 kDa were detected by this method. Using F-actin affinity and immunoaffinity chromatography, the 17 kDa polypeptide was identified as ponticulin, a previously described actin-binding glycoprotein from D. discoideum plasma membranes (Wuestehube, L.J., and Luna, E.J., [1987]: J. Cell Biol. 105:1741-1751). The binding of F-actin to ponticulin on blots is specific because unlabeled F-actin competes with 125I-labeled F-actin and because G-actin does not bind. Nitrocellulose-bound ponticulin displays binding characteristics similar to those of purified plasma membranes in solution, e.g., F-actin binding is sensitive to high salt and to elevated temperatures. Under optimal conditions, 125-I-labeled F-actin blot overlays are at least as sensitive as are immunoblots with an antibody specific for ponticulin. When blotted onto nitrocellulose after 2-D gel electrophoresis, all isoforms of ponticulin and of the 19 kDa and 15 kDa polypeptides appear to bind F-actin in proportion to their abundance. Thus the actin-binding activies of these proteins do not appear to be regulated by modifications that affect isoelectric point. However, the actin-binding activity of nitrocellulose-bound ponticulin is diminished when the protein is exposed to reducing agents, suggesting an involvement of disulfide bond(s) in ponticulin function. The 125I-labeled F-actin blot overlay assay also may enable us to identify F-actin-binding proteins in other cell types and should provide a convenient method for monitoring the purification of these proteins.     

Subcellular localization of human neutral ceramidase expressed in HEK293 cells

We previously reported that rat and mouse neutral ceramidases were mainly localized to plasma membranes as a type II integral membrane protein and partly detached from the cells via processing of the N-terminal/anchor sequence when expressed in HEK293 cells [M. Tani, H. Iida, M. Ito, O-glycosylation of mucin-like domain retains the neutral ceramidase on the plasma membranes as a type II integral membrane protein, J. Biol. Chem. 278 (2003) 10523-10530]. In contrast, the human homologue was exclusively detected in mitochondria when expressed in HEK293 and MCF7 cells as a fusion protein with green fluorescent protein at the N-terminal of the enzyme [S.E. Bawab, P. Roddy, T. Quian, A. Bielawska, J.J. Lemasters, Y.A. Hannun, Molecular cloning and characterization of a human mitochondrial ceramidase, J. Biol. Chem. 275 (2000) 21508-21513]. Given this discrepancy, we decided to clone the neutral ceramidase from human kidney cDNA and re-examine the intracellular localization of the enzyme when expressed in HEK293 cells. The putative amino acid sequence of the newly cloned enzyme was identical to that reported for human neutral ceramidase except at the N-terminal; the new protein was 19 amino acids longer at the N-terminal. We found that the putative full-length human neutral ceramidase was transported to plasma membranes, but not to mitochondria, possibly via a classical ER/Golgi pathway and localized mainly in plasma membranes when expressed in HEK293 cells. The N-terminal-truncated mutant, previously reported as a human mitochondrial ceramidase, was also weakly expressed in HEK293 cells but mainly released into the medium possibly due to the insufficient signal/anchor sequence.     

The Role of the Membrane-spanning Domain Sequence in Glycoprotein-mediated Membrane Fusion

The role of glycoprotein membrane-spanning domains in the process of membrane fusion is poorly understood. It has been demonstrated that replacing all or part of the membrane-spanning domain of a viral fusion protein with sequences that encode signals for glycosylphosphatidylinositol linkage attachment abrogates membrane fusion activity. It has been suggested, however, that the actual amino acid sequence of the membrane-spanning domain is not critical for the activity of viral fusion proteins. We have examined the function of Moloney murine leukemia virus envelope proteins with substitutions in the membrane-spanning domain. Envelope proteins bearing substitutions for proline 617 are processed and incorporated into virus particles normally and bind to the viral receptor. However, they possess greatly reduced or undetectable capacities for the promotion of membrane fusion and infectious virus particle formation. Our results imply a direct role for the residues in the membrane-spanning domain of the murine leukemia virus envelope protein in membrane fusion and its regulation. They also support the thesis that membrane-spanning domains possess a sequence-dependent function in other protein-mediated membrane fusion events.     

Evidence for similar function of transmembrane segments in receptor and membrane-anchored proteins

Transmembrane (TM) regions of receptor proteins should have unique structural and/or chemical characteristics if these regions contain residues functional in TM signal transduction. However, in a survey of the membrane-occurring residues in 37 integral membrane proteins, we found that amino acid compositions of TM regions of receptor proteins (n = 11) could not be distinguished statistically from corresponding regions of membrane-anchored proteins (e.g., recognition molecules) with a functional external domain attached to a single hydrophobic membrane-spanning anchor segment (n = 16). TM regions in both categories of proteins differed from the compositions of TM regions in membrane-transport proteins (n = 10). The analysis implies that TM regions in receptor proteins may function mainly to anchor (and position) receptors in their cellular membranes, and therefore residues in receptors that participate in signal transduction need not be restricted to these regions. In addition to mechanisms involving receptor aggregation, ligand-activated conformational perturbation of a receptor external aqueous domain, resulting in membrane penetration of hydrophobic segment(s) of this domain to produce intramembranous contact with its cytoplasmic domain, is hypothesized as a further possible mode of signal transduction.     

Ponticulin is the major high affinity link between the plasma membrane and the cortical actin network in Dictyostelium

Interactions between the plasma membrane and underlying actin-based cortex have been implicated in membrane organization and stability, the control of cell shape, and various motile processes. To ascertain the function of high affinity actin-membrane associations, we have disrupted by homologous recombination the gene encoding ponticulin, the major high affinity actin-membrane link in Dictyostelium discoideum amoebae. Cells lacking detectable amounts of ponticulin message and protein also are deficient in high affinity actin-membrane binding by several criteria. First, only 10-13% as much endogenous actin cosediments through sucrose and crude plasma membranes from ponticulin- minus cells, as compared with membranes from the parental strain. Second, purified plasma membranes exhibit little or no binding or nucleation of exogenous actin in vitro. Finally, only 10-30% as much endogenous actin partitions with plasma membranes from ponticulin-minus cells after these cells are mechanically unroofed with polylysine- coated coverslips. The loss of the cell's major actin-binding membrane protein appears to be surprisingly benign under laboratory conditions. Ponticulin-minus cells grow normally in axenic culture and pinocytose FITC-dextran at the same rate as do parental cells. The rate of phagocytosis of particles by ponticulin-minus cells in growth media also is unaffected. By contrast, after initiation of development, cells lacking ponticulin aggregate faster than the parental cells. Subsequent morphogenesis proceeds asynchronously, but viable spores can form. These results indicate that ponticulin is not required for cellular translocation, but apparently plays a role in cell patterning during development.     

Cell surface anchorage and ligand-binding domains of the Saccharomyces cerevisiae cell adhesion protein alpha-agglutinin, a member of the immunoglobulin superfamily.

alpha-Agglutinin is a cell adhesion glycoprotein expressed on the cell wall of Saccharomyces cerevisiae alpha cells. Binding of alpha-agglutinin to its ligand a-agglutinin, expressed by a cells, mediates cell-cell contact during mating. Analysis of truncations of the 650-amino-acid alpha-agglutinin structural gene AG alpha 1 delineated functional domains of alpha-agglutinin. Removal of the C-terminal hydrophobic sequence allowed efficient secretion of the protein and loss of cell surface attachment. This cell surface anchorage domain was necessary for linkage to a glycosyl phosphatidylinositol anchor. A construct expressing the N-terminal 350 amino acid residues retained full a-agglutinin-binding activity, localizing the binding domain to the N-terminal portion of alpha-agglutinin. A 278-residue N-terminal peptide was inactive; therefore, the binding domain includes residues between 278 and 350. The segment of alpha-agglutinin between amino acid residues 217 and 308 showed significant structural and sequence similarity to a consensus sequence for immunoglobulin superfamily variable-type domains. The similarity of the alpha-agglutinin-binding domain to mammalian cell adhesion proteins suggests that this structure is a highly conserved feature of adhesion proteins in diverse eukaryotes.     

Transposition of domains between the M2 and HN viral membrane proteins results in polypeptides which can adopt more than one membrane orientation

The influenza A virus M2 polypeptide is a small integral membrane protein that does not contain a cleaved signal sequence, but is unusual in that it assumes the membrane orientation of a class I integral membrane protein with an NH2-terminal ectodomain and a COOH-terminal cytoplasmic tail. To determine the domains of M2 involved in specifying membrane orientation, hybrid genes were constructed and expressed in which regions of the M2 protein were linked to portions of the paramyxovirus HN and SH proteins, two class II integral membrane proteins that adopt the opposite orientation in membranes from M2. A hybrid protein (MgMH) consisting of the M2 NH2-terminal and membrane-spanning domains linked precisely to the HN COOH-terminal ectodomain was found in cells in two forms: integrated into membranes in the M2 topology or completely translocated across the endoplasmic reticulum membrane and ultimately secreted from the cell. The finding of a soluble form suggested that in this hybrid protein the anchor function of the M2 signal/anchor domain can be overridden. A second hybrid which contained the M2 NH2 terminus linked to the HN signal anchor and ectodomain (MgHH) was found in both the M2 and the HN orientation, suggesting that the M2 NH2 terminus was capable of reversing the topology of a class II membrane protein. The exchange of the M2 signal/anchor domain with that of SH resulted in a hybrid protein which assumed only the M2 topology. Thus, all these data suggest that the NH2-terminal 24 residues to M2 are important for directing the unusual membrane topology of the M2 protein. These data are discussed in relationship to the loop model for insertion of proteins into membranes and the role of charged residues as a factor in determining orientation.     

Cellular receptor for urokinase plasminogen activator. Carboxyl-terminal processing and membrane anchoring by glycosyl-phosphatidylinositol.

The cellular receptor for human urokinase-type plasminogen activator (u-PAR) is shown by several independent criteria to be a true member of a family of integral membrane proteins, anchored to the plasma membrane exclusively by a COOH-terminal glycosyl-phosphatidylinositol moiety. 1) Amino acid analysis of u-PAR after micropurification by affinity chromatography and N-[2-hydroxy-1,1-bis(hydroxymethyl)-ethyl]glycine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of 2-3 mol of ethanolamine/mol protein. 2) Membrane-bound u-PAR is efficiently released from the surface of human U937 cells by trace amounts of purified bacterial phosphatidylinositol-specific phospholipase C. This soluble form of u-PAR retains the binding specificity toward both u-PA and its amino-terminal fragment holding the receptor-binding domain. 3) Treatment of purified u-PAR with phosphatidylinositol-specific phospholipase C or mild alkali completely alters the hydrophobic properties of the receptor as judged by temperature-induced detergent-phase separation and charge-shift electrophoresis. 4) Biosynthetic labeling of u-PAR was obtained with [3H]ethanolamine and myo-[3H]inositol. 5) Finally, comparison of amino acid compositions derived from cDNA sequence and amino acid analysis shows that a polypeptide of medium hydrophobicity is excised from the COOH terminus of the nascent u-PAR. A similar proteolytic processing has been reported for other proteins that are linked to the plasma membrane by a glycosyl-phosphatidylinositol membrane anchor.     

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.