F-actin binds to the cytoplasmic surface of ponticulin, a 17-kD integral glycoprotein from Dictyostelium discoideum plasma membranes

F-actin affinity chromatography and immunological techniques are used to identify actin-binding proteins in purified Dictyostelium discoideum plasma membranes. A 17-kD integral glycoprotein (gp17) consistently elutes from F-actin columns as the major actin-binding protein under a variety of experimental conditions. The actin-binding activity of gp17 is identical to that of intact plasma membranes: it resists extraction with 0.1 N NaOH, 1 mM dithiothreitol (DTT); it is sensitive to ionic conditions; it is stable over a wide range of pH; and it is eliminated by proteolysis, denaturation with heat, or treatment with DTT and N-ethylmaleimide. gp17 may be responsible for much of the actin-binding activity of plasma membranes since monovalent antibody fragments (Fab) directed primarily against gp17 inhibit actin-membrane binding by 96% in sedimentation assays. In contrast, Fab directed against cell surface determinants inhibit binding by only 0-10%. The actin-binding site of gp17 appears to be located on the cytoplasmic surface of the membrane since Fab against this protein continue to inhibit 96% of actin-membrane binding even after extensive adsorption against cell surfaces. gp17 is abundant in the plasma membrane, constituting 0.4-1.0% of the total membrane protein. A transmembrane orientation of gp17 is suggested since, in addition to the cytoplasmic localization of the actin-binding site, extracellular determinants of gp17 are identified. gp17 is surface-labeled by sulfo-N-hydroxy-succinimido-biotin, a reagent that cannot penetrate the cell membrane. Also, gp17 is glycosylated since it is specifically bound by the lectin, concanavalin A. We propose that gp17 is a major actin-binding protein that is important for connecting the plasma membrane to the underlying microfilament network. Therefore, we have named this protein "ponticulin" from the Latin word, ponticulus, which means small bridge.     

Headgroup dynamics of an integral membrane glycoprotein.

Glycophorin, an integral membrane glycoprotein known to be a receptor for several lectins, has been spin labelled specifically on headgroup terminal sugars. The labelled derivative has been studied in solution and also in various model membranes in an attempt to determine the factors which control headgroup dynamics. Under conditions which mimic those in a living cell the oligosaccharide chains show a uniform, relatively high freedom of motion, with individual sugar correlation times on the order of 6 × 10^?10 sec to 8 × 10^?10 sec depending upon the extent of glycoprotein headgroup involvement with other glycocalyx components. They exhibit no detectable occupancy of lipid or protein hydrophobic domains. Oligosaccharide dynamics are insensitive to factors which act upon that portion of the polypeptide backbone which inserts into the membrane, however a specific recognition event markedly reduces terminal sugar mobility.     

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.     

The neutrophil glycoprotein Mo1 is an integral membrane protein of plasma membranes and specific granules

The glycoprotein Mo1 has previously been demonstrated to be on the cell surface and in the specific granule fraction of neutrophils and to be translocated to the cell surface during degranulation. It is not known, however, whether Mo1 is an integral membrane protein or a soluble, intragranular constituent loosely associated with the specific granule membrane. Purified neutrophils were disrupted by nitrogen cavitation and separated on Percoll density gradients into four fractions enriched for azurophilic granules, specific granules, plasma membrane, and cytosol, respectively. The glycoproteins in these fractions were labeled with 3H-borohydride reduction, extracted with Triton X-114, and immunoprecipitated with 60.3, an anti-Mo1 monoclonal antibody Mo1 was detected only in the specific granule and plasma membrane fractions and partitioned exclusively into the detergent-rich fraction consistent with Mo1 being an integral membrane protein. In addition, treatment of specific granule membranes with a high salt, high urea buffer to remove absorbed or peripheral proteins failed to dissociate Mo1. These data support the hypothesis that Mo1 is an integral membrane protein of plasma and specific granule membranes in human neutrophils.     

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)     

Direct interaction of the trans-Golgi network membrane protein, TGN38, with the F-actin binding protein, neurabin.

TGN38 is a type I integral membrane protein that constitutively cycles between the trans-Golgi network (TGN) and plasma membrane. The cytosolic domain of TGN38 interacts with AP2 clathrin adaptor complexes via the tyrosine-containing motif (-SDYQRL-) to direct internalization from the plasma membrane. This motif has previously been shown to direct both internalization and subsequent TGN targeting of TGN38. We have used the cytosolic domain of TGN38 in a two-hybrid screen, and we have identified the brain-specific F-actin binding protein neurabin-I as a TGN38-binding protein. We demonstrate a direct interaction between TGN38 and the ubiquitous homologue of neurabin-I, neurabin-II (also called spinophilin). We have used a combination of yeast two-hybrid and in vitro protein interaction assays to show that this interaction is dependent on the serine (but not tyrosine) residue of the known TGN38 trafficking motif. We show that TGN38 interacts with the coiled coil region of neurabin in vitro and binds preferentially with the dimeric form of neurabin. TGN38 and neurabin also interact in vivo as demonstrated by coimmunoprecipitation from stably transfected PC12 cells. These data suggest that neurabin provides a direct physical link between TGN38-containing membranes and the actin cytoskeleton.     

Primary structure analysis of an integral membrane glycoprotein of the nuclear pore

The complete primary structure of an integral membrane glycoprotein of the nuclear pore was deduced from the cDNA sequence. The cDNA encodes a polypeptide of 204,205 D containing a 25-residue-long signal sequence, two hydrophobic segments that could function as transmembrane segments, and 13 potential N-linked oligosaccharide addition sites. Endoglycosidase H reduces the molecular mass by approximately 9 kD suggesting that not all of these 13 sites are used. We discuss possible models for the topology of this protein in the pore membrane as well as a possible role in the formation of pores and pore complexes.     

Chromogranin, an integral membrane protein

Chromogranin is the major soluble protein of the adrenal medulla chromaffin granule and is secreted upon nervous stimulation. Using antisera to pure chromogranin in immunoblotting procedures, we show that chromogranin is the major integral membrane protein as well. Extraction of chromaffin granule membranes with low salt, high salt, chelating agents, or calcium-containing solutions does not remove the chromogranin from the membranes. The membrane form of chromogranin can be purified on a C-18 semi-preparative column using high pressure liquid chromatography. Amino-terminal sequence data indicate that the membrane and soluble forms of chromogranin are identical or very similar.     

Direct modification of the glycocalyx of a cultured muscle cell line by incorporation of foreign gangliosides and an integral membrane glycoprotein

As part of a program to better understand the cause-or-effect nature of the relationship between cell surface carbohydrate and cell properties and behaviour, experiments have been carried out on direct modification of the glycocalyx of cultured cells. Modification was by incorporation of gangliosides and an integral membrane glycoprotein chosen to be dissimilar to species occurring naturally in the cell line. Two methods of incorporation were investigated: simple addition of the new components to the culture medium for various times, or assembly of the components into the walls of lipid vesicles which were subsequently fused with cells. Gangliosides from beef brain and glycophorin, the major human erythrocyte sialoglycoprotein, were successfully added to the surface of myoblasts in quantities sufficient to represent a significant perturbation. Changes in cell adhesion, morphology, and viability were observed which seem to be a direct result of glycocalyx modification.     

Structure, biosynthesis, and localization of dipeptidyl aminopeptidase B, an integral membrane glycoprotein of the yeast vacuole

We have characterized the structure, biogenesis, and localization of dipeptidyl aminopeptidase B (DPAP B), a membrane protein of the yeast vacuole. An antibody specific for DPAP B recognizes a 120-kD glycoprotein in yeast that behaves like an integral membrane protein in that it is not removed from membranes by high pH Na2CO3 treatment. Inspection of the deduced amino acid sequence of DPAP B reveals a hydrophobic domain near the NH2 terminus that could potentially span a lipid bilayer. The in vitro enzymatic activity and apparent molecular weight of DPAP B are unaffected by the allelic state of PEP4, a gene essential for the proteolytic activation of a number of soluble vacuolar hydrolases. DPAP B is synthesized as a glycosylated precursor that is converted to the mature 120-kD species by carbohydrate addition. The precursor form of DPAP B accumulates in sec mutants (Novick, P., C. Field, and R. Schekman. 1980. Cell. 21:205-215) that are blocked at the ER (sec18) or Golgi apparatus (sec7), but not at secretory vesicles (sec1). Immunolocalization of DPAP B in wild-type or sec1 mutant cells shows that the protein resides in the vacuolar membrane. However, it is present in non-vacuolar compartments in sec18 and sec7 cells, confirming that the delivery of DPAP B is blocked in these mutants. Interestingly, DPAP B appears to stain the nuclear envelope in a sec18 mutant, which is consistent with the accumulation of DPAP B in the ER membrane at the restrictive temperature. These results suggest that soluble and membrane-bound vacuolar proteins use the same stages of the secretory pathway for their transport.     

Ligand binding rapidly induces disulfide-dependent dimerization of glycoprotein VI on the platelet plasma membrane

Thrombus formation in hemostasis or thrombotic disease is initiated by adhesion of circulating platelets to damaged blood vessel walls. Exposed subendothelial collagen interacting with platelet glycoprotein (GP) VI leads to platelet activation and integrin alpha(IIb)beta(3)-mediated aggregation. We previously showed that ligand binding to GPVI also induces metalloproteinase-dependent shedding, generating an approximately 55-kDa soluble ectodomain fragment and an approximately 10-kDa membrane-associated remnant. Here, treatment of platelets with collagen or the GPVI-targeting rattlesnake toxin convulxin also induces rapid (10-30 s) formation of a high molecular weight GPVI complex (GPVIc) under nonreducing conditions, as detected by immunoblotting with anti-GPVI antibodies. The appearance of an approximately 20-kDa remnant detectable using a polyclonal antibody against the GPVI cytoplasmic tail under nonreducing, but not reducing, conditions after ectodomain shedding and nonreduced/reduced two-dimensional SDS-polyacrylamide gel analysis of biotinylated platelets confirmed that that GPVIc was a homodimer. Formation of disulfide-linked GPVIc was prolonged in the presence of metalloproteinase inhibitor GM6001 and was independent of GPVI signaling because it was unaffected by inhibitors of Src kinases, Syk, or phosphoinositide 3-kinase. To identify the thiol involved in disulfide bond formation, wild-type or mutant GPVI, where two available sulfhydryls (Cys-274 and Cys-338) were individually mutated to serine, was expressed in rat basophilic leukemia cells. Dimerization of wild-type and C274S GPVI, but not the C338S mutant, was observed after treating cells with convulxin. We conclude that (i) a subpopulation of GPVI forms a constitutive dimer on the platelet surface, facilitating rapid disulfide cross-linking, (ii) convulxin or other GPVI agonists induce disulfide-linked GPVI dimerization independent of GPVI signaling, and (iii) the penultimate residue of the GPVI cytoplasmic tail, Cys-338, mediates disulfide-dependent dimer formation.     

Identification and partial purification of ABGP205, an integral membrane glycoprotein from brain that binds ankyrin.

1. A procedure was devised that allows the membrane-skeletal proteins brain spectrin and ankyrin to be extracted selectively from a membrane-skeletal preparation, together with some actin, an Mr-103,000 protein and a population of glycoproteins. 2. Ankyrin-binding activities of the glycoproteins were investigated by affinity chromatography. We detected only one, Mr 205,000, that binds ankyrin and is prevented from binding by the cytoplasmic domain of Band 3, the established erythrocyte-membrane-binding site for ankyrin. The Mr-205,000 glycoprotein, designated ABGP205, may be a candidate for a membrane-binding site for ankyrin.     

In vitro synthesis and membrane insertion of bovine MP26, an integral protein from lens fiber plasma membrane

Synthesis of MP26, the principal protein of lens fiber plasma membranes, was directed in the reticulocyte lysate system by poly A mRNA enriched from whole bovine lens RNA using oligo (dt)-cellulose chromatography. Synthesized MP26 was enriched by immune precipitation. The in vitro-synthesized MP26 had an electrophoretic mobility indistinguishable from that of the native molecule. MP26 showed a cotranslational requirement for dog pancreas microsomes in order for membrane association to occur. Microsome-associated in vitro- synthesized MP26 showed a sensitivity to digestion with chymotrypsin which was similar to the sensitivity of native MP26 in isolated lens fiber plasma membranes, indicating correct insertion of the MP26 into the microsome. Synthesis and membrane insertion of MP26 using N-formyl- [35S]methionyl tRNA as label demonstrated that no proteolytic processing or significant glycosylation accompanied membrane insertion. Chymotryptic cleavage of membrane-inserted, N-formyl-[35S]methionine- labeled MP26 resulted in loss of label, suggesting that the N-terminal of the in vitro-synthesized MP26 faces the cytoplasm.     

Modulation of an RGDS binding site on the platelet membrane glycoprotein IIb-IIIa complex

Fibronectin, von Willebrand factor, and fibrinogen each bind to the glycoprotein IIb-IIIa complex on activated platelets via an arg-gly-asp-ser (RGDS) sequence present within the adhesive proteins. Both the IIb and IIIa polypeptides of the IIb-IIIa complex on thrombin activated platelets are specifically and extensively labeled by a radiolabeled, photoactivatable arylazide derivative of the RGDS sequence when the labeling is performed in the presence of concentrations of Ca++ or Mg++ approaching 0.5 mM. In contrast, labeling of unactivated platelets, ADP activated platelets, or thrombin activated platelets in the presence of low concentrations of divalent cations resulted in restriction of labeling to the IIb polypeptide of the complex.     

Molecular cloning of syndecan, an integral membrane proteoglycan

We describe cDNA clones for a cell surface proteoglycan that bears both heparan sulfate and chondroitin sulfate and that links the cytoskeleton to the interstitial matrix. The cDNA encodes a unique core protein of 32,868 D that contains several structural features consistent with its role as a glycosamino-glycan-containing matrix anchor. The sequence shows discrete cytoplasmic, transmembrane, and NH2-terminal extracellular domains, indicating that the molecule is a type I integral membrane protein. The cytoplasmic domain is small and similar in size but not in sequence to that of the beta-chain of various integrins. The extracellular domain contains a single dibasic sequence adjacent to the extracellular face of the transmembrane domain, potentially serving as the protease-susceptible site involved in release of this domain from the cell surface. The extracellular domain contains two distinct types of putative glycosaminoglycan attachment sites; one type shows sequence characteristics of the sites previously described for chondroitin sulfate attachment (Bourdon, M. A., T. Krusius, S. Campbell, N. B. Schwartz, and E. Ruoslahti. 1987. Proc. Natl. Acad. Sci. USA. 84:3194-3198), but the other type has newly identified sequence characteristics that potentially correspond to heparan sulfate attachment sites. The single N-linked sugar recognition sequence is within the putative chondroitin sulfate attachment sequence, suggesting asparagine glycosylation as a mechanism for regulating chondroitin sulfate chain addition. Both 5' and 3' regions of this cDNA have sequences substantially identical to analogous regions of the human insulin receptor cDNA: a 99-bp region spanning the 5' untranslated and initial coding sequences is 67% identical and a 35-bp region in the 3' untranslated region is 81% identical in sequence. mRNA expression is tissue specific; various epithelial tissues show the same two sizes of mRNA (2.6 and 3.4 kb); in the same relative abundance (3:1), the cerebrum shows a single 4.5-kb mRNA. This core protein cDNA describes a new class of molecule, an integral membrane proteoglycan, that we propose to name syndecan (from the Greek syndein, to bind together).     

Cytoplasmic domains of cellular and viral integral membrane proteins substitute for the cytoplasmic domain of the vesicular stomatitis virus glycoprotein in transport to the plasma membrane

Oligonucleotide-directed mutagenesis was used to construct chimeric cDNAs that encode the extracellular and transmembrane domains of the vesicular stomatitis virus glycoprotein (G) linked to the cytoplasmic domain of either the immunoglobulin mu membrane heavy chain, the hemagglutinin glycoprotein of influenza virus, or the small glycoprotein (p23) of infectious bronchitis virus. Biochemical analyses and immunofluorescence microscopy demonstrated that these hybrid genes were correctly expressed in eukaryotic cells and that the hybrid proteins were transported to the plasma membrane. The rate of transport to the Golgi complex of G protein with an immunoglobulin mu membrane cytoplasmic domain was approximately sixfold slower than G protein with its normal cytoplasmic domain. However, this rate was virtually identical to the rate of transport of micron heavy chain molecules measured in the B cell line WEHI 231. The rate of transport of G protein with a hemagglutinin cytoplasmic domain was threefold slower than wild type G protein and G protein with a p23 cytoplasmic domain, which were transported at similar rates. The combined results underscore the importance of the amino acid sequence in the cytoplasmic domain for efficient transport of G protein to the cell surface. Also, normal cytoplasmic domains from other transmembrane glycoproteins can substitute for the G protein cytoplasmic domain in transport of G protein to the plasma membrane. The method of constructing precise hybrid proteins described here will be useful in defining functions of specific domains of viral and cellular integral membrane proteins.     

Isolation of plasma membrane glycoprotein from bovine thymocytes

Glycoprotein was isolated from a purified thymocyte membrane preparation by two methods: lithium diiodosalicylate-phenol extraction and hot 75% ethanol extraction. A higher yield of membrane sialic acid was obtained by the latter method. The preparations had similar apparent molecular weights on sodium dodecyl sulfate gel electrophoresis. Both had similar receptor activities against a panel of hemagglutinins, although the 75% ethanol extract was more active on a weight basis. However, there were significant differences in carbohydrate and amino acid compositions of the two thymocyte extracts. The lithium diiodosalicylate-extracted material had much more glucose, ribose, and glycine than the ethanol extract. The glycoprotein preparations from thymocytes were quite distinct from the glycoprotein of bovine erythrocytes in both composition and receptor properties.     

Post-translational processing of an O-glycosylated protein, the human CD8 glycoprotein, during the intracellular transport to the plasma membrane.

The biosynthesis of human CD8 glycoprotein in transfected rat epithelial cells produces an unglycosylated precursor, an intermediate species only initially O-glycosylated, and a doublet mature form carrying neutral and sialylated O-linked oligosaccharides with the core-2 structure (Pascale, M. C., Malagolini, N., Serafini-Cessi, F., Migliaccio, G., Leone, A., and Bonatti, S. (1992) J. Biol. Chem. 267, 9940-9947). In this study the most relevant post-translational events: dimerization, addition of the first O-linked GalNAc, fulfillment of O-linked chains, as well as expression of involved glycosyltransferases, have been examined and correlated with the localization and transit rate of CD8 through the exocytic pathway. The glycosyltransferase activities measured in rat epithelial cells transfected with human CD8 DNA are entirely consistent with the primary structure assigned to CD8 oligosaccharides. The half-time of appearance of the initially O-glycosylated precursor and mature form was estimated to be 4 and 14 min, respectively, and the half-time for delivery of mature CD8 to the cell surface was found to be about 30 min, indicating a very fast routing. Pulse experiments with [35S]cysteine at 37 degrees C followed by chase-periods at low temperatures showed that folding/dimerization occurs before routing to the Golgi apparatus, whereas the addiction of O-linked GalNAc appears to take place later, very likely in cis-Golgi cisternae. Treatment of cells with monensin accumulated the intermediate CD8 form carrying non-elongated O-linked GalNAc, whereas brefeldin A treatment produced a sialylated glycoprotein species with a mobility faster than the mature CD8. These results indicate that the two drugs affect assembly of O-linked chains at different time of their processing.