Molecular characterization of synaptophysin, a major calcium-binding protein of the synaptic vesicle membrane.

Synaptophysin, a mol. wt 38 000 glycopolypeptide of the synaptic vesicle membrane, was solubilized using Triton X-100 and purified by immunoaffinity or ion-exchange chromatography. From gel permeation and sucrose-density centrifugation in H2O/D2O, a Stokes radius of 7.3 nm, a partial specific volume of 0.830 and a total mol. wt of 119 000 were calculated for the native protein. Cross-linking of synaptic vesicles with glutaraldehyde, dimethylsuberimidate, or Cu2+ -o-phenantroline, resulted in the formation of a mol. wt 76 kd dimer of synaptophysin. Crosslinking of the purified protein in addition produced tri- and tetrameric adducts of the polypeptide. Native synaptophysin thus is a homooligomeric protein. Synaptophysin is N-glycosylated, since cultivation of the rat phaeochromocytoma cell line PC12 in the presence of tunicamycin reduced its mol. wt by about 6 kd. Upon transfer to nitrocellulose and incubation with 45Ca2+, synaptophysin behaved as one of the major calcium-binding proteins of the synaptic vesicle membrane. Pronase treatment of intact synaptic vesicles abolished this 45Ca2+ binding indicating that the Ca2+ binding site of synaptophysin must reside on a cytoplasmic domain of the transmembrane polypeptide. Based on these data, we propose that synaptophysin may play an important role in Ca2+-dependent neurotransmitter release.     

Studies on the biosynthesis, assembly and secretion of vitellogenin, an oestrogen-induced multicomponent protein.

1. The process by which the egg-yolk protein precursor vitellogenin is biosynthesized, assembled and secreted by Xenopus laevis (South African clawed toad) liver was studied. It was previously shown in other laboratories that vitellogenin contains the two egg-yolk proteins lipovitellin (mol.wt. 140 000) and phosvitin (mol.wt. 35 000). 2. Evidence is presented which shows that Xenopus liver microsomal fractions synthesize precursors of vitellogenin. These precursors were solubilized from the membranes with detergent and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This analysis indicated that there is only one precursor polypeptide, and this has mol.wt. approx. 200 000 +/- 20 000. This demonstrates that the egg-yolk proteins are translated as part of this larger polypeptide. 3. Experiments also demonstrate the existence of a microsomal proteinase which is able to cleave the precursor into smaller fragments. The nature of these fragments provided some indirect evidence that phosvitin and lipovitellin light chains are situated together within the precursor molecule. 4. These precursor data fit in well with structural studies on serum vitellogenin, since it has been shown that the latter protein consists of two identical subunits each with a mobility on sodium dodecyl sulphate/polyacrylamide gels identical with that shown by the microsomal precursor. This indicates that both the intracellular precursor and subunit of vitellogenin have similar (but not necessarily identical) molecular weights. 5. It was also shown that trypsin or chymotrypsin can cleave the serum vitellogenin into leucine- and serine-rich fragments which resemble lipovitellin and phosvitin respectively. Attention is, however, drawn to the fact that the serine-rich fragment is not identical with phosvitin, since it contains eight times more leucine than that expected for the authentic phosvitin molecule [Penning (1976) Ph.D. Thesis, University of Southampton].     

Ultrastructural localization of the Mr 43,000 protein and the acetylcholine receptor in Torpedo postsynaptic membranes using monoclonal antibodies

Four mouse monoclonal antibodies (mabs) were shown by immunoblotting procedures to recognize the major, basic, membrane-bound Mr 43,000 protein (43K protein) of acetylcholine receptor-rich postsynaptic membranes from Torpedo nobiliana . These mabs and a mab against an extracellular determinant on the acetylcholine receptor were used to localize the two proteins in electroplax (Torpedo californica) and on unsealed postsynaptic membrane fragments at the ultrastructural level. Bound mabs were revealed with a rabbit anti-mouse Ig serum and protein A-colloidal gold. The anti-43K mabs bound only to the cytoplasmic surface of the postsynaptic membrane. The distributions of the receptor and the 43K protein along the membrane were found to be coextensive. Distances between the membrane center and gold particles were very similar for anti-receptor and anti-43K mabs (29 +/- 7 nm and 26 to 29 +/- 7 to 10 nm, respectively). These results show that the 43K protein is a receptor-specific protein having a restricted spatial relationship to the membrane. They thus support models in which the 43K protein is associated with the cytoplasmic domains of the receptor molecule.     

Association of the postsynaptic 43K protein with newly formed acetylcholine receptor clusters in cultured muscle cells

The postsynaptic membrane from Torpedo electric organ contains, in addition to the acetylcholine receptor (AChR), a major peripheral membrane protein of approximately 43,000 mol wt (43K protein). Previous studies have shown that this protein is closely associated with AChR and may be involved in anchoring receptors to the postsynaptic membrane. In this study, binding sites for monoclonal antibodies (mabs) to the 43K protein have been compared to the distribution of AChR in Xenopus laevis muscle cells in culture. In double label immunofluorescence experiments, clusters of AChR that occur spontaneously on these cells were stained with anti-43K mabs. Newly formed receptor clusters induced with positive polypeptide-coated latex beads were also stained with anti-43K mabs as early as 12 h after the application of the beads. Exact correspondence in the distribution of the anti-43K protein binding sites and the AChR was found in both types of clusters. These results suggest that the 43K protein becomes associated with AChR clusters during a period of active postsynaptic membrane differentiation. Thus, this protein may participate in the clustering process.     

Water-soluble acetylcholine receptor from Torpedo californica. Solubilization, purification and characterization

The nicotinic acetylcholine receptor from electrogenic tissue of Torpedo californica was solubilized by tryptic digestion of membrane fragments obtained from autolysed tissue, without use of detergent. The water-soluble acetylcholine receptor was purified by affinity chromatography on a cobra-toxin-Sepharose resin. The purified receptor bound 4000-6000 pmol per mg protein of alpha-[125I]bungarotoxin, and toxin-binding was specifically inhibited by cholinergic ligands. Gel filtration revealed a single molecular species of Stokes radius 125 +/- 10 A and on sucrose gradient centrifugation one major peak was observed of 20-22 S. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and beta-mercaptoethanol revealed two major polypeptides of mol. wt. 30 000 and 48 000.     

Interaction of the 43K protein with components of Torpedo postsynaptic membranes

Interactions of the major Mr 43 000 peripheral membrane protein (43K protein) with components of Torpedo postsynaptic membranes have been examined. Treatment of membranes with copper o-phenanthroline promotes the polymerization of 43K protein to dimers and higher oligomers. These high molecular weight forms of 43K protein can be converted to monomers by reduction with dithiothreitol and do not contain any of the other major proteins found in these membranes, including the subunits of the acetylcholine receptor, as shown by immunoblotting with monoclonal antibodies. To study directly its interactions with the membrane, the 43K protein was radioiodinated and purified by immunoaffinity chromatography. Purified 43K protein binds tightly to pure liposomes of various compositions in a manner that is not inhibited by KCl concentrations up to 0.75 M. The binding can be reversed by adjusting the pH of the reaction to 11, the same treatment that removes 43K protein from postsynaptic membranes. Unlabeled 43K protein solubilized from Torpedo membranes with cholate can be reconstituted with exogenously added lipids in the absence of the receptor. The results suggest that 43K protein molecules are amphipathic and that they may interact with each other and with the lipid bilayer. These interactions cannot explain the coextensive distribution of 43K proteins with acetylcholine receptors in situ. However, they could account for the association of the 43K protein with the postsynaptic membrane and may contribute to the maintenance of the structure of the cytoplasmic specialization of which this protein is a major component.     

Water-soluble acetylcholine receptor from Torpedo californica. Solubilization,purification and characterization

The nicotinic acetylcholine receptor from electrogenic tissue of Torpedo californica was solubilized by tryptic digestion of membrane fragments obtained from autolysed tissue, without use of detergent. The water-soluble acetylcholine receptor was purified by affinity chromatography on a cobra-toxin-Sepharose resin. The purified receptor bound 4000–6000 pmol per mg protein of α-[¹²⁵]bungarotoxin, and toxin-binding was specifically inhibited by cholinergic ligands. Gel filtration revealed a single molecular species of Stokes radius $\text{125 ± 10}$ Å and on sucrose gradient centrifugation one major peak was observed of 20–22 S. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and β-mercaptoethanol revealed two major polypeptides of mol. wt. 30 000 and 48 000.     

Heterogeneity of the filamentous haemagglutinin of Bordetella pertussis studied with monoclonal antibodies

The filamentous haemagglutinin of Bordetella pertussis has been purified from static, liquid culture supernatants and from extracts of cells grown on a solid medium. SDS-PAGE of the purified protein has shown multiple polypeptides with molecular weights ranging from 220 000 to about 58 000. By transferring the SDS-dissociated polypeptides to nitrocellulose paper and reacting with several monoclonal antibodies, it has been shown that many of the polypeptides are probably fragments of the polypeptide of highest molecular weight.     

Protein composition and structure of human placental microvillous membrane. External surface, sialic acid containing, extrinsic and intrinsic components.

The microvillous membrane of human placenta is in direct contact with maternal blood and thus plays a vital role in many essential functions of the placenta. As an initial step in understanding the membrane proteins, and their relationship to these functions and to the structure of the membrane, we have investigated an isolated membrane preparation. Ten major peptide bands and an approximately equal number of minor bands were seen with sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Sialoglycoproteins were labeled with periodate (PA-³H) borohydride and external surface components with lactoperoxidase-[¹²⁵I] (LP-¹²⁵I). One principal (69 000 mol. wt) and several minor (100 000, 45 000, and 38–40 000 mol. wt) bands were labeled as Sialoglycoproteins and found to be exposed on the surface of the membrane. Approx. 50% of the membrane protein and all of the sialic acid was tightly bound to membrane lipid and resistant to extraction with dimethyl maleic anhydride (DMMA). Electron microscopy demonstrated extraction by DMMA of microfilaments presumptively identified as actin and other electron dense components from the villous core. The extracted supernate and the residual pellet differed markedly in protein composition. The supernatant contained bands of 180 000, 115 000, 85 000, 70–72 000, 45 000, and 38–40 000 mol. wt whereas the lipid pellet contained components of 200 000, 150 000, 100 000, 69 000, and 64 000 mol. wt. The lipid matrix with which these proteins were associated contained phosphatidyl choline and sphingomyelin and was similar in composition to other plasma membranes. Thus by using a variety of experimental approaches the proteins of the human placental microvillous membrane can be divided into groups based on their sialic acid content, exposure on the external surface, tightness of binding to the membrane lipid, and relation to membrane structure.     

Extraction of peripheral proteins from nicotinic acetylcholine receptor-enriched membranes

The solubilisation of membrane proteins from nicotinic acetylcholine receptor-enriched membranes from the electric organ of Torpedo marmorata was studied. Chaotropic ions were shown to be ineffective in extracting peripheral proteins from these membranes. Two different anhydrides, 2,3-dimethylmaleic and 3,4,5,6-tetrahydrophthalic anhydride, released certain peripheral membrane proteins but not the integral receptor protein. Treatment of membranes containing $\text{> 3}\text{nmol}$ α-bungarotoxin binding sites per mg protein with anhydride resulted in a 43 kDa polypeptide as the major constituent of the solubilised material. The nature of the 43 kDa polypeptide is discussed. Gentle anhydride treatment did not change the α-bungarotoxin and carbamoylcholine binding properties of the receptor.     

Characterization of molecules involved in protein translocation using a specific antibody

The vectorial translocation of nascent proteins through the membrane of the rough endoplasmic reticulum has been shown to require a specific membrane-bound protein whose cytoplasmic domain can be proteolytically cleaved and isolated as an active peptide of mol wt 60,000 (Meyer and Dobberstein, 1980, J. Cell Biol. 87:503-508). Rabbit antibodies raised against this peptide were used to further characterize the membrane- bound molecule. Immunoprecipitation of solubilized, radiolabeled rough microsomal proteins yielded a single polypeptide of mol wt 72,000, representing the membrane-bound protein from which the 60,000-mol wt peptide was proteolytically derived. The antibody could also be used to remove exclusively the 60,000-mol wt peptide, and thus the translocation activity, from elastase digests tested in a reconstituted system. Moreover, immunoprecipitation of elastase extracts alkylated with [14C] N-ethylmaleimide selected a single species of mol wt 60,000. Immunoprecipitation of in vivo radiolabeled proteins from the appropriate cell type yielded the 72,000-mol wt membrane protein irrespective of the duration of labeling, or if followed by a chase. Subsequent treatment with protease generated the 60,000-mol wt fragment. In addition, the antibody could be used to visualize reticular structures in intact cells which correspond to endoplasmic reticulum at the ultrastructural level. It is thus clear that one membrane component required in the vectorial translocation of nascent secretory (and membrane) proteins is a peptide of mol wt 72,000.     

Isolation and renaturation of alpha 2-macroglobulin receptor from diploid human fibroblasts.

alpha 2-Macroglobulin receptor was extracted from human diploid fibroblasts and purified by affinity chromatography in a single step. The receptor had mol.wt. 125 000 after sodium dodecyl sulphate (SDS)/polyacrylamide-gel electrophoresis. The isolated receptor was separated by SDS/polyacrylamide-gel electrophoresis, transferred on to nitrocellulose sheets and subsequently renatured, as shown by a specific binding test, by incubation with Nonidet P40.     

Cytoskeletal proteins at the cholinergic synapse: distribution of desmin, actin, fodrin, neurofilaments, and tubulin in Torpedo electric organ

Treatment of the electric organ of Torpedo marmorata with Triton X-100 in the presence of 2 mM MgCl2 generated a cytoskeletal fraction in which a 54 kDa polypeptide is a major constituent. This 54 kDa polypeptide accounted for about 8% of the cellular protein when total electric organ tissue was analyzed by two-dimensional gel electrophoresis. Immunoblotting experiments showed that this protein reacts with monoclonal antibodies to desmin, the major intermediate filament protein of avian and mammalian muscle tissue. Negative stain analysis revealed that filaments of about 10 nm diameter are the major structural elements of the electric organ cytoskeleton. In the presence of Ca2+ there was a rapid degradation of the desmin-like protein and intermediate filaments due to a Ca2+-activated protease. Some of the resulting fragments retained antigenic activity against the desmin antibodies. Immunoblotting of membrane fractions enriched in acetylcholine receptor revealed desmin in addition to some actin. A further cytoskeletal component was identified from biochemical and immunological properties as a homologue of the mammalian neurofilament L-polypeptide. Thus Torpedo expresses proteins homologous to the mammalian desmin and neurofilament L-protein which can be detected using immunological approaches. Immunofluorescence microscopy was used to map the location of various cytoskeletal proteins of the cholinergic synapse on paraffin sections and on en face preparations of membranes. Desmin staining was restricted to electrocytes and in en face preparations was seen associated with both the ventral receptor-containing membrane and with the non-innervated dorsal membrane. Antibodies to neurofilament L-protein stained only the axons and not the electrocytes. Staining for fodrin, a non-erythrocyte spectrin, resulted in submembraneous decoration of both the axons and the electrocytes. Axonal staining for neurofilaments and microtubules did not extend into the ends of the nerve terminal arborizations.     

Identification by cross-linking of a beta-bungarotoxin binding polypeptide in chick brain membranes.

beta-Bungarotoxin (beta-BTX) is a snake venom neurotoxin which inhibits neurotransmitter release from different types of nerve terminals. To identify presynaptic membrane components potentially important in neurosecretion, 125I-labeled beta-BTX (mol. wt. 21 000) was cross-linked to a high-affinity binding site in synaptic membrane fractions of chick brain using the photoactivable cross-linker N-succinimidyl-6(4'-azido-2'-nitrophenylamino)-hexanoate. Electrophoretic analysis of the cross-linked membrane proteins under both reducing and non-reducing conditions revealed a single [125I]beta-BTX-polypeptide adduct of apparent mol. wt. 116 000 (+/- 2000). The labeling of this band was prevented under conditions previously shown to inhibit the binding of [125I]beta-BTX to its high-affinity binding site. It is concluded that the cross-linking procedure identified a polypeptide of the presynaptic binding site for beta-BTX, and that this polypeptide has a mol. wt. of 95 000.     

Immunological localization of a major karyoskeletal protein in nucleoli of oocytes and somatic cells of Xenopus laevis

Oocyte nuclei of Xenopus laevis contain two major karyoskeletal proteins characterized by their resistance to extractions in high salt buffers and the detergent Triton X-100, i.e. a polypeptide of 68,000 mol wt which is located in the core complex-lamina structure and a polypeptide of 145,000 mol wt enriched in nucleolar fractions. Both proteins are also different by tryptic peptide maps and immunological determinants. Mouse antibodies were raised against insoluble karyoskeletal proteins from Xenopus oocytes and analyzed by immunoblotting procedures. Affinity purified antibodies were prepared using antigens bound to nitrocellulose paper. In immunofluorescence microscopy of Xenopus oocytes purified antibodies against the polypeptide of 145,000 mol wt showed strong staining of nucleoli, with higher concentration in the nucleolar cortex, and of smaller nucleoplasmic bodies. In various other cells including hepatocytes, Sertoli cells, spermatogonia, and cultured kidney epithelial cells antibody staining was localized in small subnucleolar granules. The results support the conclusion that this "insoluble" protein is a major nucleus-specific protein which is specifically associated with--and characteristic of--nucleoli and certain nucleolus-related nuclear bodies. It represents the first case of a positive localization of a karyoskeletal protein in the nuclear interior, i.e. away from the pore complex-lamina structure of the nuclear cortex.     

Distribution of actin, myosin, actin-binding protein and gelsolin in cultured lymphoid cells

Myosin, actin-binding protein (ABP) and gelsolin are proteins interacting with actin in vitro and may control the movement of amoeboid cells. The distribution of these proteins was examined by indirect immunofluorescence (IFL) of smeared, acetone-fixed lymphoid cells. Actin, ABP and gelsolin were found in filopodia and in the cytoplasm, whereas myosin was mainly in the cortical cytoplasm. In the presence of Ca²⁺ the staining of the filopodia by anti-actin, anti-ABP and anti-gelsolin antibodies were abolished. A 91 000 daltons (D) polypeptide reacting with anti-gelsolin antibodies and a 43 000 D polypeptide reacting with anti-actin antibodies were eluted from acetone-treated cells in presence of Ca²⁺. This effect of Ca²⁺ on the staining could be due to the action of gelsolin, which severs actin filaments. The shortened filaments would then elute from the cells during the staining procedures.     

Selective photoaffinity labeling of acetylcholine receptor using a cholinergic analogue

A bisazido derivative was synthesized from bis(3-aminopyridinium)-1,10-decane diiodide and it was shown that it was bound (KD congruent to 2.2 muM) specifically to purified acetylcholine receptor and fulfilled the requirements for a photoaffinity label. Like the parent compound the derivative could transform membrane-bound receptor from a low ligand affinity conformation(s) to a high ligand affinity form (s), a transition which is thought to resemble desensitization processes observed in vivo. Photolysis of 3H-labeled bisazido reagent was carried out in the presence of the receptor. After dodecyl sulfate-polyacrylamide gel electrophoresis of labeled purified receptor two of the four subunits (mol wt 40 000 and 60 000) contained 90% of the bound radioactivity while for membrane-bound receptor the subunits of mol wt 40 000 and 50 000 were labeled. The results favor the assumption that the specific ligand binding sites are located on mol wt 40 000 subunits and labeling of the other subunits reflects (a) their proximity to the ligand-binding site and (b) alterations in subunit topography between membrane-bound and solubilized states.     

Identification of a cDNA clone coding for the acetylcholine binding subunit of Torpedo marmorata acetylcholine receptor.

A recombinant DNA plasmid has been constructed that contains sequences of the gene coding for the acetylcholine binding subunit (alpha-subunit, 40 000 daltons) of Torpedo marmorata acetylcholine receptor protein (AChR). Polyadenylated RNA purified from Torpedo electric organ was used to construct a cDNA library. The AChR alpha-subunit cDNA clone was then identified by a two-step screening of 700 recombinant clones. As AChR is present in Torpedo electric organ but not in Torpedo liver or spleen, differential screening led to the selection of 12 clones specific for the electric organ. We then tested the ability of cDNA inserts to hybridize alpha-subunit mRNA specifically, as judged by cell-free translation and immunoprecipitation. The insert from one clone, p alpha-1, selectively hybridized with a mRNA species which elicited the synthesis of a 38 000 mol. wt. polypeptide. This polypeptide was precipitated by: (1) a rabbit serum raised against purified denatured alpha-subunit (the pure alpha-subunit displaced the complex); and (2) a rat monoclonal antibody specific for the denatured alpha-subunit. It was thus identified as a precursor of the alpha chain. Blot hybridization analysis of polyadenylated RNA from Torpedo electric organ with the p alpha-1 probe revealed a major species of 2.0 kb, which thus contains approximately 800 non-coding nucleotides.     

Phosphoproteins: structural components of oncornaviruses.

Oncornaviruses, which contain a virion-associated protein kinase, were found to possess phosphoproteins as virion structural components. One major phosphoprotein common to strains of laboratory and wild mouse oncornaviruses and a strain of feline leukemia virus was shown to be a polypeptide of about 12, 000 mol wt. In addition to this, the Kirsten strain of murine sarcoma virus contained a second major phosphoprotein of about 10, 000 mol wt, and mouse erythroblastosis virus contained a second major phosphoprotein that was either identical to or comigrated with the virion glycoprotein of about 74, 000 mol wt. The major phosphoprotein of RD-114 virus was found to be of about 16, 000 mol wt. The major phosphoamino acid of the 12, 000-mol wt polypeptide of the mouse erythroblastosis virus was identified as phosphoserine, and that of the 16, 000-mol wt polypeptide of the RD-114 virus was identified as phosphothreonine.