Clustering and immobilization of acetylcholine receptors by the 43-kD protein: a possible role for dystrophin-related protein

Recombinant acetylcholine receptors (AChRs) expressed on the surface of cultured fibroblasts become organized into discrete membrane domains when the 43-kD postsynaptic protein (43k) is co-expressed in the same cells (Froehner, S.C., C. W. Luetje, P. B. Scotland, and J. Patrick, 1990. Neuron. 5:403-410; Phillips, W. D., M. C. Kopta, P. Blount, P. D. Gardner, J. H. Steinbach, and J. P. Merlie. 1991. Science (Wash. DC). 251:568-570). Here we show that AChRs present on the fibroblast cell surface prior to transfection of 43k are recruited into 43k-rich membrane domains. Aggregated AChRs show increased resistance to extraction with Triton X-100, suggesting a 43k-dependent linkage to the cytoskeleton. Myotubes of the mouse cell line C2 spontaneously display occasional AChR/43k-rich membrane domains that ranged in diameter up to 15 microns, but expressed many more when 43k was overexpressed following transfection of 43k cDNA. However, the membrane domains induced by recombinant 43k were predominantly small (< or = 2 microns). We were then interested in whether the cytoskeletal component, dystrophin related protein (DRP; Tinsley, J. M., D. J. Blake, A. Roche, U. Fairbrother, J. Riss, B. C. Byth, A. E. Knight, J. Kendrick-Jones, G. K. Suthers, D. R. Love, Y. H. Edwards, and K. E. Davis, 1992. Nature (Lond.). 360:591-593) contributed to the development of AChR clusters. Immunofluorescent anti-DRP staining was present at the earliest stages of AChR clustering at the neuromuscular synapse in mouse embryos and was also concentrated at the large AChR-rich domains on nontransfected C2 myotubes. Surprisingly, anti-DRP staining was concentrated mainly at the large, but not the small AChR clusters on C2 myotubes suggesting that DRP may be principally involved in permitting the growth of AChR clusters.     

A novel 87,000-Mr protein associated with acetylcholine receptors in Torpedo electric organ and vertebrate skeletal muscle

To identify proteins associated with nicotinic postsynaptic membranes, mAbs have been prepared to proteins extracted by alkaline pH or lithium diiodosalicylate from acetylcholine receptor-rich (AChR) membranes of Torpedo electric organ. Antibodies were obtained that recognized two novel proteins of 87,000 Mr and a 210,000:220,000 doublet as well as previously described proteins of 43,000 Mr, 58,000 (51,000 in our gel system), 270,000, and 37,000 (calelectrin). The 87-kD protein copurified with acetylcholine receptors and with 43- and 51-kD proteins during equilibrium centrifugation on continuous sucrose gradients, whereas a large fraction of the 210/220-kD protein was separated from AChRs. The 87-kD protein remained associated with receptors and 43-kD protein during velocity sedimentation through shallow sucrose gradients, a procedure that separated a significant amount of 51-kD protein from AChRs. The 87- and 270-kD proteins were cleaved by Ca++- activated proteases present in crude preparations and also in highly purified postsynaptic membranes. With the exception of anti-37-kD antibodies, some of the monoclonals raised against Torpedo proteins also recognized determinants in frozen sections of chick and/or rat skeletal muscle fibers and in permeabilized chick myotubes grown in vitro. Anti-87-kD sites were concentrated at chick and rat endplates, but the antibodies also recognized determinants present at lower site density in the extrasynaptic membrane. Anti-210:220-kD labeled chick endplates, but studies of neuron-myotube cocultures showed that this antigen was located on neurites rather than the postsynaptic membrane. As reported in other species, 43-kD determinants were restricted to chick endplates and anti-51-kD and anti-270-kD labeled extrasynaptic as well as synaptic membranes. None of the cross reacting antibodies recognized determinants on intact (unpermeabilized) myotubes, so the antigens must be located on the cytoplasmic aspect of the surface membrane. The role that each intracellular determinant plays in AChR immobilization at developing and mature endplates remains to be investigated.     

Cytoplasmic components of acetylcholine receptor clusters of cultured rat myotubes: the 58-kD protein

A 58-kD protein, identified in extracts of postsynaptic membrane from Torpedo electric organ, is enriched at sites where acetylcholine receptors (AChR) are concentrated in vertebrate muscle (Froehner, S. C., A. A. Murnane, M. Tobler, H. B. Peng, and R. Sealock. 1987. J. Cell Biol. 104:1633-1646). We have studied the 58-kD protein in AChR clusters isolated from cultured rat myotubes. Using immunofluorescence microscopy we show that the 58-kD protein is highly enriched at AChR clusters, but is also present in regions of the myotube membrane lacking AChR. Within clusters, the 58-kD protein codistributes with AChR, and is absent from adjacent membrane domains involved in myotube-substrate contact. Semiquantitative fluorescence measurements suggest that molecules of the 58-kD protein and AChR are present in approximately equal numbers. Differential extraction of peripheral membrane proteins from isolated AChR clusters suggests that the 58-kD protein is more tightly bound to cluster membrane than is actin or spectrin, but less tightly bound than the receptor-associated 43-kD protein. When AChR clusters are disrupted either in intact cells or after isolation, the 58-kD protein still codistributes with AChR. Clusters visualized by electron microscopy after immunogold labeling and quick-freeze, deep-etch replication show that, within AChR clusters, the 58-kD protein is sharply confined to AChR-rich domains, where it is present in a network of filaments lying on the cytoplasmic surface of the membrane. Additional actin filaments overlie, and are attached to, this network. Our results suggest that within AChR domains of clusters, the 58-kD protein lies between AChR and the receptor-associated 43-kD protein, and the membrane-skeletal proteins, beta-spectrin, and actin.     

Clustering of the acetylcholine receptor by the 43-kD protein: involvement of the zinc finger domain

A postsynaptic membrane-associated protein of M(r) 43,000 (43-kD protein) is involved in clustering of the nicotinic acetylcholine receptor (AChR) at the neuromuscular junction. Previous studies have shown that recombinant mouse 43-kD protein forms membrane-associated clusters when expressed in Xenopus oocytes. Coexpression with the AChR results in colocalization of the receptor with the 43-kD protein clusters (Froehner, S. C., C. W. Luetje, P. B. Scotland, and J. Patrick, 1990. Neuron. 5:403-410). To understand the mechanism of this clustering, we have studied the role of the carboxy-terminal region of the 43-kD protein. The amino acid sequence of this region predicts two tandem zinc finger structures followed by a serine phosphorylation site. Both Torpedo 43-kD protein and the carboxy-terminal region of the mouse 43-kD protein bind radioisotopic zinc. Mutation of two histidine residues in this predicted domain greatly attenuates zinc binding, lending support to the proposal that this region forms zinc fingers. When expressed in oocytes, the ability of this mutant 43-kD protein to form clusters is greatly reduced. Its ability to interact with AChR, however, is retained. In contrast, a mutation that eliminates the potential serine phosphorylation site has no effect on clustering of the 43-kD protein or on interaction with the AChR. These findings suggest that protein interactions via the zinc finger domain of the 43- kD protein may be important for AChR clustering at the synapse.     

Increased expression of the 43-kD protein disrupts acetylcholine receptor clustering in myotubes

The 43-kD protein is a peripheral membrane protein that is in approximately 1:1 stoichiometry with the acetylcholine receptor (AChR) in vertebrate muscle cells and colocalizes with it in the postsynaptic membrane. To investigate the role of the 43-kD protein in AChR clustering, we have isolated C2 muscle cell lines in which some cells overexpress the 43-kD protein. We find that myotubes with increased levels of the 43-kD protein have small AChR clusters and that those with the highest levels of expression have a drastically reduced number of clusters. Our results suggest that the 1:1 stoichiometry of AChR and 43-kD protein found in muscle cells is important for AChR cluster formation.     

Association of cytoskeletal proteins with newly formed acetylcholine receptor aggregates induced by embryonic brain extract

Aggregates of acetylcholine receptors (AChR) in muscle cell membranes are associated with accumulations of certain cytoskeletal and peripheral membrane proteins. We treated cultured rat myotubes briefly with embryonic brain extract (EBX) to promote AChR aggregation and determined the distribution of several of these proteins at early stages of aggregation. EBX-treated and control cultures were stained with tetramethylrhodamine-alpha-bungarotoxin to identify AChR aggregates and were then frozen and sectioned on a cryostat. These sections were stained with primary antibodies and fluoresceinated secondary antibodies to localize cytoskeletal proteins. The distributions of AChRs and cytoskeletal proteins was examined qualitatively and analyzed by a semiquantitative assay. Qualitatively, the 43K protein had a distribution that was virtually identical to that of AChR in both control and EBX-treated cultures, and it always colocalized with early AChR aggregates. The 58K protein similarly colocalized with early AChR aggregates, but it was also in aggregate-free areas of muscle membrane. The association of vinculin with the aggregates was quantitatively similar to that of the 43K and 58K proteins, but, qualitatively, its distribution did not follow that of the AChR as closely. Like the 58K protein and vinculin, alpha-actinin, filamin, and actin were concentrated in AChR aggregates and were also enriched elsewhere. However, they were less closely associated with the aggregates, both quantitatively and qualitatively. These results show that AChR aggregates induced by EBX tend to be enriched in the same cytoskeletal proteins that are present at the neuromuscular junction in vivo and at AChR clusters formed at sites of cell-substrate adhesion in vitro. Semiquantitative analysis also revealed that the fractional area of the cell surface associated with vinculin, alpha-actinin, and the 58K protein was the same in controls and EBX-treated myotubes, although the area enriched in AChR and the 43K protein increased about three-fold upon EBX treatment. These results suggest that AChR aggregates may form preferentially in membrane regions that are already enriched in these proteins.     

Identification of a MAP 2-like ATP-binding protein associated with axoplasmic vesicles that translocate on isolated microtubules

Axoplasmic vesicles were purified and observed to translocate on isolated microtubules in an ATP-dependent, trypsin-sensitive manner, implying that ATP-binding polypeptides essential for force generation were present on the vesicle surface. To identify these proteins [alpha 32P]8-azidoadenosine 5'-triphosphate ([alpha 32P]8-N3ATP), a photoaffinity analogue of ATP, was used. The results presented here identify and characterize a vesicle-associated polypeptide having a relative molecular mass of 292 kD that bound [alpha 32P]8-N3ATP. The incorporation of label is ultraviolet light-dependent and ATP-sensitive. Moreover, the 292-kD polypeptide could be isolated in association with vesicles or microtubules, depending on the conditions used, and the data indicate that the 292-kD polypeptide is similar to mammalian brain microtubule-associated protein 2 (MAP 2) for the following reasons: The 292-kD polypeptide isolated from either squid axoplasm or optic lobe cross-reacts with antiserum to porcine brain MAP 2. Furthermore, it purifies with taxol-stabilized microtubules and is released with salt. Based on these characteristics, the 292-kD polypeptide is distinct from the known force-generating molecules myosin and flagellar dynein, as well as the 110-130-kD kinesin-like polypeptides that have recently been described (Brady, S. T., 1985, Nature (Lond.), 317:73-75; Vale, R. D., T. S. Reese, and M. P. Sheetz, 1985b, Cell, 42:39-50; Scholey, J. M., M. E. Porter, P. M. Grissom, and J. R. McIntosh, 1985, Nature (Lond.), 318:483-486). Because the 292-kD polypeptide binds ATP and is associated with vesicles that translocate on purified MAP-free microtubules in an ATP-dependent fashion, it is therefore believed to be involved in vesicle-microtubule interactions that promote organelle motility.     

Mutagenesis of the 43-kD postsynaptic protein defines domains involved in plasma membrane targeting and AChR clustering

The postsynaptic membrane of the neuromuscular junction contains a myristoylated 43-kD protein (43k) that is closely associated with the cytoplasmic face of the nicotinic acetylcholine receptor (AChR)-rich plasma membrane. Previously, we described fibroblast cell lines expressing recombinant AChRs. Transfection of these cell lines with 43k was necessary and sufficient for reorganization of AChR into discrete 43k-rich plasma membrane domains (Phillips, W. D., C. Kopta, P. Blount, P. D. Gardner, J. H. Steinbach, and J. P. Merlie. 1991. Science (Wash. DC). 251:568-570). Here we demonstrate the utility of this expression system for the study of 43k function by site-directed mutagenesis. Substitution of a termination codon for Asp254 produced a truncated (28-kD) protein that associated poorly with the cell membrane. The conversion of Gly2 to Ala2, to preclude NH2-terminal myristoylation, reduced the frequency with which 43k formed plasma membrane domains by threefold, but did not eliminate the aggregation of AChRs at these domains. Since both NH2 and COOH-termini seemed important for association of 43k with the plasma membrane, a deletion mutant was constructed in which the codon Gln15 was fused in-frame to Ile255 to create a 19-kD protein. This mutated protein formed 43k-rich plasma membrane domains at wild-type frequency, but the domains failed to aggregate AChRs, suggesting that the central part of the 43k polypeptide may be involved in AChR aggregation. Our results suggest that membrane association and AChR interactions are separable functions of the 43k molecule.     

The identification of proteins in the proximity of signal-anchor sequences during their targeting to and insertion into the membrane of the ER

Using a photocross-linking approach we have investigated the cytosolic and membrane components involved in the targeting and insertion of signal-anchor proteins into the membrane of the ER. The nascent chains of both type I and type II signal-anchor proteins can be cross-linked to the 54-kD subunit of the signal recognition particle. Upon addition of rough microsomes the type I and type II signal-anchor proteins interact with a number of components. Both types of protein interact with an integral membrane protein, the signal sequence receptor, previously identified by its proximity to preprolactin during its translocation (Wiedmann, M., T.V. Kurzchalia, E. Hartmann, and T.A. Rapoport. 1987. Nature [Lond.] 328:830-833). Three proteins, previously unidentified, were found to be cross-linked to the nascent chains of the signal-anchor proteins. Among them was a 37-kD protein that was found to be the main component interacting with the type I SA protein used. These proteins were not seen in the absence of membranes suggesting they are components of the ER. The ability of the nascent chains to be cross-linked to these identified proteins was shown to be abolished by prior treatment with agents known to disrupt translocation intermediates or ribosomes. We propose that the newly identified proteins function either in the membrane insertion of only a subset of proteins or only at a specific stage of insertion.     

Three-dimensional structure of the acetylcholine receptor by cryoelectron microscopy and helical image reconstruction

Long tubular vesicles have been grown from isolated Torpedo postsynaptic membranes, in which the receptors are arranged helically on the vesicle surface. The structures of these tubes have been analyzed by cryoelectron microscopy of specimens embedded in thin films of ice, combined with helical image reconstruction. Complete data sets from tubes belonging to several helical families have been obtained to a resolution of 17 A in all directions. Confirming a preliminary study (Toyoshima, C., and N. Unwin. 1988. Nature (Lond.). 336:247-250), the central ion channel has an almost constant diameter throughout the molecule except for the portion extending through the hydrophobic part of the lipid bilayer, where the pore is too small to be resolved. However, the density on the pseudo fivefold axis running through the pore is consistently highest in the cytoplasmic half of the bilayer, suggesting the gate is located in that region. The path followed by each subunit has been identified throughout the length of the receptor. The two alpha subunits follow equivalent paths. All subunits have similar features which change in character at the same level relative to the membrane.     

The signal sequence receptor has a second subunit and is part of a translocation complex in the endoplasmic reticulum as probed by bifunctional reagents

Bifunctional cross-linking reagents were used to probe the protein environment in the ER membrane of the signal sequence receptor (SSR), a 24-kD integral membrane glycoprotein (Wiedmann, M., T. V. Kurzchalia, E. Hartmann, and T. A. Rapoport. 1987. Nature [Lond.]. 328:830-833). The proximity of several polypeptides was demonstrated. A 22-kD glycoprotein was identified tightly bound to the 34-kD SSR even after membrane solubilization. The 34-kD polypeptide, now termed alpha SSR, and the 22-kD polypeptide, the beta SSR, represent a heterodimer. We report on the sequence of the beta SSR, its membrane topology, and on the mechanism of its integration into the membrane. Cross-linking also produced dimers of the alpha-subunit of the SSR indicating that oligomers of the SSR exist in the ER membrane. Various bifunctional cross-linking reagents were used to study the relation to ER membrane proteins of nascent chains of preprolactin and beta-lactamase at different stages of their translocation through the membrane. The predominant cross-linked products obtained in high yields contained the alpha SSR, indicating in conjunction with previous results that it is a major membrane protein in the neighborhood of translocating nascent chains of secretory proteins. The results support the existence of a translocon, a translocation complex involving the SSR, which constitutes the specific site of protein translocation across the ER membrane.     

Saccharomyces cerevisiae and Schizosaccharomyces pombe contain a homologue to the 54-kD subunit of the signal recognition particle that in S. cerevisiae is essential for growth

We have isolated and sequenced genes from Saccharomyces cerevisiae (SRP54SC) and Schizosaccharomyces pombe (SRP54sp) encoding proteins homologous to both the 54-kD protein subunit (SRP54mam) of the mammalian signal recognition particle (SRP) and the product of a gene of unknown function in Escherichia coli, ffh (Romisch, K., J. Webb, J. Herz, S. Prehn, R. Frank, M. Vingron, and B. Dobberstein. 1989. Nature (Lond.). 340:478-482; Bernstein H. D., M. A. Poritz, K. Strub, P. J. Hoben, S. Brenner, P. Walter. 1989. Nature (Lond.). 340:482-486). To accomplish this we took advantage of short stretches of conserved sequence between ffh and SRP54mam and used the polymerase chain reaction (PCR) to amplify fragments of the homologous yeast genes. The DNA sequences predict proteins for SRP54sc and SRP54sp that are 47% and 52% identical to SRP54mam, respectively. Like SRP54mam and ffh, both predicted yeast proteins contain a GTP binding consensus sequence in their NH2-terminal half (G-domain), and methionine-rich sequences in their COOH-terminal half (M-domain). In contrast to SRP54mam and ffh the yeast proteins contain additional Met-rich sequences inserted at the COOH-terminal portion of the M-domain. SRP54sp contains a 480- nucleotide intron located 78 nucleotides from the 5' end of the open reading frame. Although the function of the yeast homologues is unknown, gene disruption experiments in S. cerevisiae show that the gene is essential for growth. The identification of SRP54sc and SRP54sp provides the first evidence for SRP related proteins in yeast.     

Ribosome binding to the endoplasmic reticulum: a 180-kD protein identified by crosslinking to membrane-bound ribosomes is not required for ribosome binding activity

We have used the membrane-impermeable, thiol-cleavable, crosslinker 3,3'-dithio bis (sulfosuccinimidylpropionate) to identify proteins that are in the vicinity of membrane-bound ribosomes of the RER. A specific subset of RER proteins was reproducibly crosslinked to the ribosome. Immunoblot analysis of the crosslinked products with antibodies raised against signal recognition particle receptor, ribophorin I, and the 35-kD subunit of the signal sequence receptor demonstrated that these translocation components had been crosslinked to the ribosome, but each to a different extent. The most prominent polypeptide among the crosslinked products was a 180-kD protein that has recently been proposed to be a ribosome receptor (Savitz, A.J., and D.I. Meyer, 1990. Nature (Lond.). 346: 540-544). RER membrane proteins were reconstituted into liposomes and assayed with radiolabeled ribosomes to determine whether ribosome binding activity could be ascribed to the 180-kD protein. Differential detergent extraction was used to prepare soluble extracts of microsomal membrane vesicles that either contained or lacked the 180-kD protein. Liposomes reconstituted from both extracts bound ribosomes with essentially identical affinity. Additional fractionation experiments demonstrated that the bulk of the ribosome binding activity present in detergent extracts of microsomal membranes could be readily resolved from the 180-kD protein by size exclusion chromatography. Taken together, we conclude that the 180-kD protein is in the vicinity of membrane bound ribosomes, yet does not correspond to the ribosome receptor.     

Recombinant neuromuscular synapses

The developing neuromuscular junction has provided an important paradigm for studying synapse formation. An outstanding feature of neuromuscular differentiation is the aggregation of acetylcholine receptors (AChRs) at high density in the postsynaptic membrane. While AChR aggregation is generally believed to be induced by the nerve, the mechanisms underlying aggregation remain to be clarified. A 43-kD protein (43k) normally associated with the cytoplasmic aspect of AChR clusters has long been suspected of immobilizing AChRs by linking them to the cytoskeleton. In recent studies, the AChR clustering activity of 43k has, at last, been demonstrated by expressing recombinant AChR and 43k in non-muscle cells. Mutagenesis of 43k has revealed distinct domains within the primary structure which may be responsible for plasma membrane targeting and AChR binding. Other lines of study have provided clues as to how nerve-derived (extracellular) AChR-cluster inducing factors such as agrin might activate 43k-driven postsynaptic membrane specialization.     

Merlin,the neurofibromatosis type 2 gene product,and β1 integrin associate in isolated and differentiating Schwann cells

Neurofibromatosis type 2, a disease characterized by the formation of multiple nervous system tumors, especially schwannomas, is caused by mutation in the gene-encoding merlin/schwannomin. The molecular mechanism by which merlin functions as a tumor suppressor is unknown, but is hypothesized to involve plasma membrane and cytoskeleton interaction. Several merlin antibodies were used to study merlin expression, localization, and protein association in primary cultures of rat sensory neurons, Schwann cells (SCs), and SCs grown with neurons (SC/N cultures) before and during differentiation into myelinating cells. Western blot analysis revealed that neurons predominantly expressed a 68-kD protein, but SCs expressed two additional 88- and 120-kD related proteins. Extensive immunological characterization demonstrated that the 88-kD protein shared three domains with the 68-kD merlin protein. Western blot analysis of soluble and insoluble culture fractions demonstrated that the majority of merlin and related proteins were soluble in isolated SCs and undifferentiated SC/N cultures, but became insoluble in myelinating SC/N cultures. Double immunofluorescence staining suggested that merlin translocated from the perinuclear cytoplasm in undifferentiated SCs to the subplasmalemma in differentiating SCs and partially colocalized with β1 integrin. Finally, β1 integrin antibody coimmunoprecipitated 68-kD merlin from isolated SC and undifferentiated SC/N cultures, but predominantly the 88-kD protein from differentiating SC/N cultures. Together, these results provide evidence that merlin interacts with β1 integrin and that merlin localization changes from a cytosolic to cytoskeletal compartment during SC differentiation. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 487–501, 1998     

Treatments that extract the 43K protein from acetylcholine receptor clusters modify the conformation of cytoplasmic domains of all subunits of the receptor.

The nicotinic acetylcholine receptor (AChR) of Torpedo electric organ and vertebrate skeletal muscle is closely associated with a Mr 43,000 protein (43K). In this study, we have examined the effects on the AChR of treatments which remove the 43K protein. We used semiquantitative fluorescence techniques to measure the binding of antibodies to clustered AChR in cultured rat myotubes. We found that labeling by antibodies to the cytoplasmic portions of each of the four receptor polypeptides increased significantly upon extraction of the 43K protein. Labeling by an antibody to an extracellular epitope of the alpha subunits was not affected by removal of the 43K protein, suggesting that changes were restricted to the cytoplasmic domains of the AChR. Increases in labeling by antibodies were more limited following protease treatment, which removes most cytoskeletal structures but leaves the 43K protein bound to the membrane. Competition between an antibody to the beta subunit and an antibody to the gamma and delta subunits suggests that the cytoplasmic portion of the AChR still retains a degree of native structure in the absence of the 43K protein. Our results suggest that, although some of these changes may be due to simply exposing additional epitopes on the AChR, the cytoplasmic portions of all the subunits of the AChR undergo significant conformational changes upon extraction of the 43K protein.     

Monoclonal antibody 7H6 reacts with a novel tight junction-associated protein distinct from ZO-1, cingulin and ZO-2

The tight junction is an essential element of the intercellular junctional complex; yet its protein composition is not fully understood. At present, only three proteins, ZO-1 (Stevenson, B. R., J. D. Siliciano, M. S. Mooseker, and D. A. Goodenough. 1986. J. Cell Biol. 103:755-766), cingulin (Citi, S., H. Sabanay, R. Jakes, B. Geiger, and J. Kendrick-Jones. 1988. Nature (Lond.). 333:272-275) and ZO-2 (Gumbiner, B., T. Lowenkopf, and D. Apatira. 1991. Proc. Natl. Acad. Sci. USA. 88:3460-3464) are known to be associated with the tight junction. We have generated a monoclonal antibody (7H6) against a bile canaliculus-rich membrane fraction prepared from rat liver. This 7H6 antigen was preferentially localized by immunofluorescence at the junctional complex regions of hepatocytes and other epithelia, and 7H6- affiliated gold particles were shown electron microscopically to localize at the periphery of tight junctions. Immunoblot analysis of a bile canaliculus-rich fraction of rat liver using 7H6, anti-ZO-1 antibody (R26.4C), and anti-cingulin antibody revealed that 7H6 reacted selectively with a 155-kD protein, whereas R26.4C reacted only with a 225-kD protein. Anti-cingulin antibody reacted solely with 140 and 108- kD proteins, indicating that the protein recognized by 7H6 is immunologically different from ZO-1 and cingulin. Immunoprecipitation of detergent extracts obtained from metabolically labeled MDCK cells with R26.4C coprecipitated a 160-kD protein, which corresponds to ZO-2, with ZO-1. However, 7H6 did not react with the 160-kD protein. These results strongly suggest that the 7H6 antibody recognizes a novel tight junction-associated protein different from ZO-1, cingulin and ZO-2.     

Assignment of segments of the bacteriorhodopsin sequence to positions in the structural map.

Specific amino acid sequence segments have been assigned to locations in the structural map of bacteriorhodopsin using two-dimensional neutron diffraction data and a model building analysis. Models are constructed computationally by building specific regions of the amino acid sequence as alpha helices and then positioning the helices on axes indicated by the density map of Henderson and Unwin (Nature [Lond.]. 1975, 257:28-32). Neutron diffraction data were collected from samples of stacked, oriented "native" purple membranes as well as purple membranes containing different kinds of deuterated amino acids. Models differing in the assignments of helices to specific axes and in rotations of the helices about those axes were tested against the neutron data using a weighted residual factor to rank the models. This residual factor was calculated between observed and predicted intensity differences for pairs of data sets. Using this approach, a small set of related models has been found that predicts the observed intensity changes between five independent data sets. These models are inconsistent with the proposed locations of the retinal chromophore and the carboxyl terminus and with any of the previously proposed models for bacteriorhodopsin.     

Common structural domains in the sarcoplasmic reticulum Ca-ATPase and the transverse tubule Mg-ATPase

Transverse tubule (TT) membranes isolated from chicken skeletal muscle possess a very active magnesium-stimulated ATPase (Mg-ATPase) activity. The Mg-ATPase has been tentatively identified as a 102-kD concanavalin A (Con A)-binding glycoprotein comprising 80% of the integral membrane protein (Okamoto, V.R., 1985, Arch. Biochem. Biophys., 237:43-54). To firmly identify the Mg-ATPase as the 102-kD TT component and to characterize the structural relationship between this protein and the closely related sarcoplasmic reticulum (SR) Ca-ATPase, polyclonal antibodies were raised against the purified SR Ca-ATPase and the TT 102-kD glycoprotein, and the immunological relationship between the two ATPases was studied by means of Western immunoblots and enzyme-linked immunosorbent assays (ELISA). Anti-chicken and anti-rabbit SR Ca-ATPase antibodies were not able to distinguish between the TT 102-kD glycoprotein and the SR Ca-ATPase. The SR Ca-ATPase and the putative 102-kD TT Mg-ATPase also possess common structural elements, as indicated by amino acid compositional and peptide mapping analyses. The two 102-kD proteins exhibit similar amino acid compositions, especially with regard to the population of charged amino acid residues. Furthermore, one-dimensional peptide maps of the two proteins, and immunoblots thereof, show striking similarities indicating that the two proteins share many common epitopes and peptide domains. Polyclonal antibodies raised against the purified TT 102-kD glycoprotein were localized by indirect immunofluorescence exclusively in the TT-rich I bands of the muscle cell. The antibodies substantially inhibit the Mg-ATPase activity of isolated TT vesicles, and Con A pretreatment could prevent antibody inhibition of TT Mg-ATPase activity. Further, the binding of antibodies to intact TT vesicles could be reduced by prior treatment with Con A. We conclude that the TT 102-kD glycoprotein is the TT Mg-ATPase and that a high degree of structural homology exists between this protein and the SR Ca-ATPase.     

Microfilaments and actin-associated proteins at sites of membrane-substrate attachment within acetylcholine receptor clusters

Rat myotubes in tissue culture form broad areas of close contact with the substrate. These areas often display two distinct, interdigitating sets of membrane domains. One, the "contact domain", is close to the substrate; the other, termed the "AChR domain", is further from the substrate and is rich in acetylcholine receptors (AChR). We have used fluorescence techniques to study the organization of the cytoskeleton in these areas. Substrate-apposed membrane of the myotubes was exposed either by shearing or by permeabilizing the cells with a neutral detergent. Phalloidin derivatives and affinity-purified polyclonal or monoclonal antibodies specific for cytoskeletal proteins were then applied to the samples. Sheared samples were observed by epifluorescence microscopy; detergent-permeabilized samples were observed by total internal reflection fluorescence microscopy. We found that, like antivinculin, fluorescent phalloidin derivatives and antibodies to alpha-actinin, filamin, and talin preferentially labeled the contact domains. This suggests that bundles of microfilaments associate with the membrane at sites of myotube-substrate attachment. In contrast, a 43K protein, closely associated with AChR, was present only at AChR domains. A monoclonal antibody to actin labeled both AChR and contact domains, suggesting that actin is enriched over both regions. Our results suggest that, like the plasma membrane of AChR clusters, the underlying membrane skeleton is organized into at least two distinct domains.