Targeting of transmembrane and GPI-anchored forms of N-CAM to opposite domains of a polarized epithelial cell.

The calcium-independent neural cell adhesion molecule N-CAM is expressed transiently during development in many tissues, including epithelia. The three naturally occurring principal isoforms of N-CAM differ in the way in which they associate with the membrane and in their cytoplasmic domains. These isoforms are generated by developmentally regulated alternative splicing of a single gene: the large cytoplasmic domain (ld) form (relative molecular mass 180,000 (Mr 180K] is specific for post-mitotic neurons; the 120K small cytoplasmic domain (ssd) and 140K small surface domain (sd) forms also occur on other cell types. One function of the different isoforms could be to specify cellular localization; for example, glycosyl phosphatidyl inositol (GPI)-membrane anchoring acts as a targeting signal for expression on the apical surface of polarized epithelial cells. Neurons and epithelial cells may use similar mechanisms for polarizing their plasma membrane proteins. We have therefore investigated the targeting of GPI-anchored (ssd N-CAM, 120K) and transmembrane forms of N-CAM (sd N-CAM, 140K; ld N-CAM, 180K) by comparing the expression of each after transfection of the appropriate complementary DNAs into polarized epithelial cells. We find that isoforms with alternative modes of membrane association are targeted to different surfaces of polarized epithelial cells: ssd N-CAM is expressed on the apical surface, whereas sd and ld N-CAM are expressed on the basolateral surface. These results suggest that the different isoforms of N-CAM determine their own diverse cellular destinations. They also support the hypothesis that the GPI anchor acts as an apical targeting signal in epithelia.     

Immunostaining of skeletal and cardiac muscle surface membrane with antibody against Duchenne muscular dystrophy peptide

Duchenne muscular dystrophy (DMD) is a debilitating X-linked muscle disease. We have used sequence information from complementary DNA clones, derived from the gene that is deleted in DMD patients, to generate an antiserum that stains the surface membrane of intact human and mouse skeletal muscle, but not that of DMD patients and mdx mice. Here we identify the protein reacting with this antiserum as a single component of relative molecular mass 210,000 (Mr = 210K) that fractionates with a low-ionic strength extract of intact human and mouse skeletal muscle. It is therefore distinct from the 400 K protein found in the heavy microsomal fraction of normal muscle and identified as a putative product of the DMD gene. We also analyse further the disease specificity of the antiserum. Positive staining is seen in normal controls, and in samples from patients with a wide range of muscular dystrophies other than DMD. Becker muscular dystrophy, which is allelically related to DMD, was the only other exception, and gave a sporadic staining pattern. The demonstration of a specific defect in the surface membrane of DMD muscle fibres substantiates the hypothesis that membrane lesions may initiate muscle degradation in DMD.     

Interaction of elongation factors EF-G and EF-Tu with a conserved loop in 23S RNA

The elongation factors EF-Tu and EF-G interact with ribosomes during protein synthesis: EF-Tu presents incoming aminoacyl transfer RNA to the programmed ribosome as an EF-Tu-GTP-tRNA ternary complex and EF-G promotes translocation of peptidyl-tRNA and its associated messenger RNA from the A to the P site after peptidyl transfer. Both events are accompanied by ribosome-dependent GTP hydrolysis. Here we use chemical probes to investigate the possible interaction of these factors with ribosomal RNA in E. coli ribosomes. We observe EF-G-dependent footprints in vitro and in vivo around position 1,067 in domain II of 23S rRNA, and in the loop around position 2,660 in domain VI.EF-Tu gives an overlapping footprint in vitro at positions 2,655 and 2,661, but shows no effect at position 1,067. The 1,067 region is the site of interaction of the antibiotic thiostrepton, which prevents formation of the EF-G-GTP-ribosome complex and is a site for interaction with the GTPase-related protein L11 (ref. 3). The universally conserved loop in the 2,660 region is the site of attack by the RNA-directed cytotoxins alpha-sarcin and ricin, whose effects abolish translation and include the loss of elongation factor-dependent functions in eukaryotic ribosomes.     

Gene structure and extracellular secretion of Neisseria gonorrhoeae IgA protease

Several human bacterial pathogens, including the Gram-negative diplococcus Neisseria gonorrhoeae, produce extracellular proteases that are specific for human immunoglobulin IgA1. Immunoglobulin A (IgA) proteases have been studied extensively and the genes of some species cloned in Escherichia coli, but their role in pathogenesis remains unclear. Recently we derived a DNA fragment of 5 kilobases (kb) from N. gonorrhoeae MS11 directing extracellular active enzyme in E. coli. Although the mature enzyme of strain MS11 was shown to have a relative molecular mass of 106,000 (Mr 106K) in gels, the DNA sequence of this cloned fragment reveals a single gene coding for a 169K precursor of IgA protease. The precursor contains three functional domains, the amino-terminal leader which is assumed to initiate the inner membrane transport of the precursor, the protease, and a carboxyl-terminal 'helper' domain apparently required for extracellular secretion (excretion). Based on the structural features of the precursor, we propose a model in which the helper serves as a pore for excretion of the protease domain through the outer membrane. IgA protease acquires an active conformation as its extracellular transport proceeds and is released as a proform from the membrane-bound helper by autoproteolysis. The soluble proform further matures into the 106 K IgA protease and a small stable alpha-protein.     

Quantification of Ca2+-activated K+ channels under hormonal control in pig pancreas acinar cells

Ca2+- and voltage-activated K+ channels are found in many electrically excitable cells and have an important role in regulating electrical activity. Recently, the large K+ channel has been found in the baso-lateral plasma membranes of salivary gland acinar cells, where it may be important in the regulation of salt transport. Using patch-clamp methods to record single-channel currents from excised fragments of baso-lateral acinar cell membranes in combination with current recordings from isolated single acinar cells and two- and three-cell clusters, we have now for the first time characterized the K+ channels quantitatively. In pig pancreatic acini there are 25-60 K+ channels per cell with a maximal single channel conductance of about 200 pS. We have quantified the relationship between internal ionized Ca2+ concentration [( Ca2+]i) membrane potential and open-state probability (p) of the K+ channel. By comparing curves obtained from excised patches relating membrane potential to p, at different levels of [Ca2+]i, with similar curves obtained from intact cells, [Ca2+]i in resting acinar cells was found to be between 10(-8) and 10(-7) M. In microelectrode experiments acetylcholine (ACh), gastrin-cholecystokinin (CCK) as well as bombesin peptides evoked Ca2+-dependent opening of the K+ conductance pathway, resulting in membrane hyperpolarization. The large K+ channel, which is under strict dual control by internal Ca2+ and voltage, may provide a crucial link between hormone-evoked increase in internal Ca2+ concentration and the resulting NaCl-rich fluid secretion.     

Structure of the cell-puncturing device of bacteriophage T4

Bacteriophage T4 has a very efficient mechanism for infecting cells. The key component of this process is the baseplate, located at the end of the phage tail, which regulates the interaction of the tail fibres and the DNA ejection machine. A complex of gene product (gp) 5 (63K) and gp27 (44K), the central part of the baseplate, is required to penetrate the outer cell membrane of Escherichia coli and to disrupt the intermembrane peptidoglycan layer, promoting subsequent entry of phage DNA into the host. We present here a crystal structure of the (gp5-gp27)3 321K complex, determined to 2.9 A resolution and fitted into a cryo-electron microscopy map at 17 A resolution of the baseplate-tail tube assembly. The carboxy-terminal domain of gp5 is a triple-stranded beta-helix that forms an equilateral triangular prism, which acts as a membrane-puncturing needle. The middle lysozyme domain of gp5, situated on the periphery of the prism, serves to digest the peptidoglycan layer. The amino-terminal, antiparallel beta-barrel domain of gp5 is inserted into a cylinder formed by three gp27 monomers, which may serve as a channel for DNA ejection.     

Homology of 54K protein of signal-recognition particle, docking protein and two E. coli proteins with putative GTP-binding domains

Most proteins exported from mammalian cells contain a signal sequence which mediates targeting to and insertion into the membrane of the endoplasmic reticulum (ER). Involved in this process are the signal-recognition particle (SRP) and docking protein (DP), the receptor for SRP in the ER membrane. SRP interacts with the signal sequence on nascent polypeptide chains and retards their further elongation, which resumes only after interaction of the arrested ribosomal complex with the docking protein. SRP is a ribonucleoprotein particle comprising a 7S RNA and six polypeptides with relative molecular masses (Mr) of 9,000 (9K) 14K, 19K, 54K, 68K and 72K (ref. 1). The 9K and 14K proteins are essential for elongation arrest and the 68K-72K heterodimer is required for docking to the ER membrane. The 54K protein binds to the signal sequence when it emerges from the ribosome. Docking protein consists of two polypeptides, a 72K alpha-subunit (DP alpha) and a 30K beta-subunit (DP beta). No components structurally homologous to SRP and docking protein have yet been found in yeast or Escherichia coli. To understand the molecular nature of the interaction between the signal sequence and its receptor(s) we have characterized a complementary DNA coding for the 54K protein of SRP. Significant sequence homology was found to part of DP alpha and two E. coli proteins of unknown function. The homologous region includes a putative GTP-binding domain.     

Observation and isolation of the amyloplasts ribosomes in cotyledon cells of lotus

Ribosome-like particles have been found in the proplastids in young cotyledon cells of lotus (Nelumbo nucifera Gaertn L.) Following the development of young embryo, some lamellar structures and tubular complex occurred in the plastids in young cotyledon cells, and some ribosomlike particles appeared in the loose region of these membrane system and stroma. About 15–20 d after fertilization, with the further development of plastid, a large number of starch and DNA were synthesized in the plastids, and the plastids contained abundant and clear morphologically ribosomes, some of which presented spiral structure. About 16–18 d after fertilization, amyloplasts were isolated and purified from cotyledon of lotus, and ribosomes bands were obtained by use of sucrose density gradient centrifugation of ribosomes isolated from amyloplasts. RNA and protein contents of ribosomes have also been determined.     

X-ray structure of a voltage-dependent K+ channel

Voltage-dependent K+ channels are members of the family of voltage-dependent cation (K+, Na+ and Ca2+) channels that open and allow ion conduction in response to changes in cell membrane voltage. This form of gating underlies the generation of nerve and muscle action potentials, among other processes. Here we present the structure of KvAP, a voltage-dependent K+ channel from Aeropyrum pernix. We have determined a crystal structure of the full-length channel at a resolution of 3.2 A, and of the isolated voltage-sensor domain at 1.9 A, both in complex with monoclonal Fab fragments. The channel contains a central ion-conduction pore surrounded by voltage sensors, which form what we call 'voltage-sensor paddles'-hydrophobic, cationic, helix-turn-helix structures on the channel's outer perimeter. Flexible hinges suggest that the voltage-sensor paddles move in response to membrane voltage changes, carrying their positive charge across the membrane.     

Differential activation by atrial and brain natriuretic peptides of two different receptor guanylate cyclases

Alpha atrial natriuretic peptide (alpha-ANP) and brain natriuretic peptide are homologous polypeptide hormones involved in the regulation of fluid and electrolyte homeostasis. These two natriuretic peptides apparently share common receptors and stimulate the intracellular production of cyclic GMP as a second messenger. Molecular cloning has defined two types of natriuretic peptide receptors: the ANP-C receptor of relative molecular mass (Mr) 60-70,000 (60-70 K), which is not coupled to cGMP production and may function in the clearance of ANP and the ANP-A receptor of Mr 120-140 K, which is a membrane form of guanylate cyclase in which ligand binding to the extracellular domain activates the cytoplasmic domain of the enzyme. Here we report the cloning and expression of a second human natriuretic peptide-receptor guanylate cyclase, the ANP-B receptor. The ANP-B receptor is preferentially activated by porcine brain natriuretic peptide rather than human alpha-ANP, whereas the ANP-A receptor responds similarly to both natriuretic peptides. These observations may have important implications for our understanding of the central and peripheral control of cardiovascular homeostasis.     

Model for signal sequence recognition from amino-acid sequence of 54K subunit of signal recognition particle

Protein targeting to the endoplasmic reticulum in mammalian cells is catalysed by signal recognition particle (SRP). Cross-linking experiments have shown that the subunit of relative molecular mass 54,000 (Mr 54K; SRP54) interacts directly with signal sequences as they emerge from the ribosome. Here we present the sequence of a complementary DNA clone of SRP54 which predicts a protein that contains a putative GTP-binding domain and an unusually methionine-rich domain. The properties of this latter domain suggest that it contains the signal sequence binding site. A previously uncharacterized Escherichia coli protein has strong homology to both domains. Closely homologous GTP-binding domains are also found in the alpha-subunit of the SRP receptor (SR alpha, docking protein) in the endoplasmic reticulum membrane and in a second E. coli protein, ftsY, which resembles SR alpha. Recent work has shown that SR alpha is a GTP-binding protein and that GTP is required for the release of SRP from the signal sequence and the ribosome on targeting to the endoplasmic reticulum membrane. We propose that SRP54 and SR alpha use GTP in sequential steps of the targeting reaction and that essential features of such a pathway are conserved from bacteria to mammals.     

Formation of domain structure of erythrocyte membrane in Wistar rat fed with CeCl3 per os

To explore the possibility of absorption of lanthanides via digestive duct and their effects on the membrane structure and permeability of erythrocytes,the fine structure of erythrocyte membrane from Wistar rats,fed for 70 days of daily administration per os with 20 mg CeCl3/kg weight,was imaged by means of atomic force microscopy and FT-IR deconvolution spectra.The results show that,although the erythrocytes maintain the intact shape,the change of secondary structure,aggregation and crosslinking of the protein particles of membrane surface and the enlarged lipid regions lead to the domain structure formation.This structure might be responsible for the increasing permeability of erythrocyte membrane.     

The signal sequence of nascent preprolactin interacts with the 54K polypeptide of the signal recognition particle

Hydrophobic signal sequences direct the translocation of nascent secretory proteins and many membrane proteins across the membrane of the endoplasmic reticulum. Initiation of this process involves the signal recognition particle (SRP), which consists of six polypeptide chains and a 7S RNA and interacts with ribosomes carrying nascent secretory polypeptide chains. In the case of aminoterminal, cleavable signal sequences, in the absence of microsomal membranes it exerts a site-specific translational arrest in vitro. The size of the arrested fragment (60-70 amino-acid residues) suggests that elongation stops when the signal sequence has emerged fully from the ribosome. However, a direct interaction between the signal sequence and SRP has not previously been demonstrated and has even been questioned recently. We now show for the first time a direct interaction between the signal sequence of a secretory protein and a component of SRP, the 45K polypeptide (relative molecular mass (Mr) 54,000). This was achieved by means of a new method of affinity labelling which involves the translational incorporation of an amino acid, carrying a photoreactive group, into nascent polypeptides.     

Inositol trisphosphate-dependent periodic activation of a Ca(2+)-activated K+ conductance in glucose-stimulated pancreatic beta-cells.

Glucose-stimulated insulin secretion is associated with the appearance of electrical activity in the pancreatic beta-cell. At intermediate glucose concentrations, beta-cell electrical activity follows a characteristic pattern of slow oscillations in membrane potential on which bursts of action potentials are superimposed. The electrophysiological background of the bursting pattern remains unestablished. Activation of Ca(2+)-activated large-conductance K+ channels (KCa channel) has been implicated in this process but seems unlikely in view of recent evidence demonstrating that the beta-cell electrical activity is unaffected by the specific KCa channel blocker charybdotoxin. Another hypothesis postulates that the bursting arises as a consequence of two components of Ca(2+)-current inactivation. Here we show that activation of a novel Ca(2+)-dependent K+ current in glucose-stimulated beta-cells produces a transient membrane repolarization. This interrupts action potential firing so that action potentials appear in bursts. Spontaneous activity of this current was seen only rarely but could be induced by addition of compounds functionally related to hormones and neurotransmitters present in the intact pancreatic islet. K+ currents of the same type could be evoked by intracellular application of GTP, the effect of which was mediated by mobilization of Ca2+ from inositol 1,4,5-trisphosphate (InsP3)-sensitive intracellular Ca2+ stores. These observations suggest that oscillatory glucose-stimulated electrical activity, which is correlated with pulsatile release of insulin, results from the interaction between the beta-cell and intraislet hormones and neurotransmitters. Our data also provide evidence for a close interplay between ion channels in the plasma membrane and InsP3-induced mobilization of intracellular Ca2+ in an excitable cell.     

Phosphorylation of tubulin enhances its interaction with membranes

Tubulin, the main component of intracellular microtubules, is also a major protein in subcellular membrane preparations and can interact with biological and artificial membranes in vitro. Of particular interest is the association of tubulin with postsynaptic junctional lattices enriched in a polypeptide of relative molecular mass (Mr) 50,000 (50K), recently identified as the major subunit of the calmodulin-dependent protein kinase. Phosphorylation of tubulin with a calmodulin-dependent protein kinase similar to that found in postsynaptic densities inhibits its ability to self-assemble into microtubules in a reversible fashion. This involves the phosphorylation of residues in its 4K carboxy-terminal region, a domain that seems to regulate its self-assembly. The results presented here suggest that the phosphorylation of tubulin with this kinase enhances its ability to interact with membranes. This effect is reversible.     

Crystal structure of the plasma membrane proton pump

A prerequisite for life is the ability to maintain electrochemical imbalances across biomembranes. In all eukaryotes the plasma membrane potential and secondary transport systems are energized by the activity of P-type ATPase membrane proteins: H+-ATPase (the proton pump) in plants and fungi, and Na+,K+-ATPase (the sodium-potassium pump) in animals. The name P-type derives from the fact that these proteins exploit a phosphorylated reaction cycle intermediate of ATP hydrolysis. The plasma membrane proton pumps belong to the type III P-type ATPase subfamily, whereas Na+,K+-ATPase and Ca2+-ATPase are type II. Electron microscopy has revealed the overall shape of proton pumps, however, an atomic structure has been lacking. Here we present the first structure of a P-type proton pump determined by X-ray crystallography. Ten transmembrane helices and three cytoplasmic domains define the functional unit of ATP-coupled proton transport across the plasma membrane, and the structure is locked in a functional state not previously observed in P-type ATPases. The transmembrane domain reveals a large cavity, which is likely to be filled with water, located near the middle of the membrane plane where it is lined by conserved hydrophilic and charged residues. Proton transport against a high membrane potential is readily explained by this structural arrangement.     

Spectroscopic mapping of voltage sensor movement in the Shaker potassium channel

Voltage-gated ion channels underlie the generation of action potentials and trigger neurosecretion and muscle contraction. These channels consist of an inner pore-forming domain, which contains the ion permeation pathway and elements of its gates, together with four voltage-sensing domains, which regulate the gates. To understand the mechanism of voltage sensing it is necessary to define the structure and motion of the S4 segment, the portion of each voltage-sensing domain that moves charged residues across the membrane in response to voltage change. We have addressed this problem by using fluorescence resonance energy transfer as a spectroscopic ruler to determine distances between S4s in the Shaker K+ channel in different gating states. Here we provide evidence consistent with S4 being a tilted helix that twists during activation. We propose that helical twist contributes to the movement of charged side chains across the membrane electric field and that it is involved in coupling voltage sensing to gating.     

Formation of domain structure of erythrocyte membrane in Wistar rat fed with CeCl3 per os

To explore the possibility of absorption of lanthanides via digestive duct and their effects on the membrane structure and permeability of erythrocytes, the fine structure of erythrocyte membrane from Wistar rats, fed for 70 days of daily administrationper os with 20 mg CeCl₃/kg weight, was imaged by means of atomic force microscopy and FT-IR deconvolution spectra. The results show that, although the erythrocytes maintain the intact shape, the change of secondary structure, aggregation and crosslinking of the protein particles of membrane surface and the enlarged lipid regions lead to the domain structure formation. This structure might be responsible for the increasing permeability of erythrocyte membrane.     

Protein targeting and degradation are coupled for elimination of mislocalized proteins

A substantial proportion of the genome encodes membrane proteins that are delivered to the endoplasmic reticulum by dedicated targeting pathways. Membrane proteins that fail targeting must be rapidly degraded to avoid aggregation and disruption of cytosolic protein homeostasis. The mechanisms of mislocalized protein (MLP) degradation are unknown. Here we reconstitute MLP degradation in vitro to identify factors involved in this pathway. We find that nascent membrane proteins tethered to ribosomes are not substrates for ubiquitination unless they are released into the cytosol. Their inappropriate release results in capture by the Bag6 complex, a recently identified ribosome-associating chaperone. Bag6-complex-mediated capture depends on the presence of unprocessed or non-inserted hydrophobic domains that distinguish MLPs from potential cytosolic proteins. A subset of these Bag6 complex 'clients' are transferred to TRC40 for insertion into the membrane, whereas the remainder are rapidly ubiquitinated. Depletion of the Bag6 complex selectively impairs the efficient ubiquitination of MLPs. Thus, by its presence on ribosomes that are synthesizing nascent membrane proteins, the Bag6 complex links targeting and ubiquitination pathways. We propose that such coupling allows the fast tracking of MLPs for degradation without futile engagement of the cytosolic folding machinery.