Expression of bovine annexin A4 in E. coli rescues cytokinesis blocked by beta-lactam antibiotics.

Treatment of bacteria with beta-lactam antibiotics can impair the process of cytokinesis, the final step in cell division, leading to the formation of a filamentous form of the bacteria. The expression of a mammalian calcium-dependent, membrane-binding protein, bovine annexin A4, in E. coli was found to reverse the inhibitory effects on cytokinesis of the beta-lactam antibiotics ampicillin, piperacillin, and cephalexin. This novel activity of the annexin was blocked by mutation of calcium binding sites in the annexin, indicating roles for calcium binding to the annexin and the binding of the annexin to membranes in restoring cytokinesis. The filamentous form of the bacteria has been reported to be more resistant to phagocytosis by cells of the immune system in eukaryotic hosts. Therefore, expression of annexins in pathogenic bacteria, by promoting the breakdown of the bacterial filaments, might serve as an adjuvant to enhance the efficacy of beta-lactam antibiotics.

Expression of Metazoan Annexins in Yeast Provides Protection Against Deleterious Effects of the Biofuel Isobutanol.

The ability of microorganisms to produce biofuels by fermentation is adversely affected by the perturbing effects of the hydrophobic biofuel on plasma membrane structure. It is demonstrated here that heterologous expression of metazoan, calcium-dependent, membrane-binding proteins of the annexin class can reduce deleterious effects of isobutanol on Saccharomyces cerevisiae viability and complex membrane functions. Therefore, expression of annexins in industrial strains of yeast or bacteria may prove beneficial in biofuel production.

Quaternary structure of the small amino acid transporter OprG from Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic human pathogen that causes nosocomial infections. The P. aeruginosa outer membrane contains specific porins that enable substrate uptake, with the outer membrane protein OprG facilitating transport of small, uncharged amino acids. However, the pore size of an eight-stranded ß-barrel monomer of OprG is too narrow to accommodate even the smallest transported amino acid, glycine, raising the question of how OprG facilitates amino acid uptake. Pro-92 of OprG is critically important for amino acid transport, with a P92A substitution inhibiting transport and the NMR structure of this variant revealing that this substitution produces structural changes in the barrel rim and restricts loop motions. OprG may assemble into oligomers in the outer membrane (OM) whose subunit interfaces could form a transport channel. Here, we explored the contributions of the oligomeric state and the extracellular loops to OprG's function. Using chemical cross-linking to determine the oligomeric structures of both WT and P92A OprG in native outer membranes and atomic force microscopy, and single-molecule fluorescence of the purified proteins reconstituted into lipid bilayers, we found that both protein variants form oligomers, supporting the notion that subunit interfaces in the oligomer could provide a pathway for amino acid transport. Furthermore, performing transport assays with loop-deleted OprG variants, we found that these variants also can transport small amino acids, indicating that the loops are not solely responsible for substrate transport. We propose that OprG functions as an oligomer and that conformational changes in the barrel-loop region might be crucial for its activity.

Assembly of high molecular weight complexes of lipin on a supported lipid bilayer observed by atomic force microscopy.

Lipins are phosphatidic acid phosphatases involved in the biosynthesis of triacylglycerols and phospholipids. They are associated with the endoplasmic reticulum but can also travel into the nucleus and alter gene expression. Previous studies indicate lipins in solution form high molecular weight complexes, possibly tetramers. This study was undertaken to determine if lipins form complexes on membranes as well. Murine lipin 1b was applied to a supported bilayer of phosphatidylcholine, phosphatidylserine, and cholesterol and examined by atomic force microscopy (AFM) over time. Lipin on bare mica appeared as a symmetric particle with a volume consistent with the size of a monomer. On the bilayer, lipin initially bound as asymmetric, curved particles that sometimes assembled into circular structures with an open center. Subsequently, lipin assemblies grew into large, symmetric particles with an average volume 12 times that of the monomer. Over time, some of the lipin assemblies were removed from the bilayer by the AFM probe leaving behind "footprints" composed of complex patterns that may reflect the substructure of the lipin assemblies. The lipin complexes appeared very flat, with a diameter 20 times their height. The footprints had a similar diameter, providing confirmation of the extensive deformation of the protein under the AFM probe. The ability of lipin to form large complexes on membranes may have significant implications for the local concentrations of the product, diacylglycerol, formed during hydrolysis of phosphatidic acid and for cooperative hormonal regulation of lipin activity through phosphorylation of one or more monomers in the complexes.

Protection of the membrane permeability barrier by annexins.

Biological membranes are exposed to a number of chemical and physical stresses that may alter the structure of the lipid bilayer in such a way that the permeability barrier to hydrophilic molecules and ions is degraded. These stresses include amphiphilic molecules involved in metabolism and signaling, highly charged polyamines, membrane-permeating peptides, and mechanical and osmotic stresses. As annexins are known to bind to lipid headgroups in the presence of calcium and increase the order of the bilayer lipids, this study addressed whether this activity of annexins provides a potential benefit to the membrane by protecting the bilayer against disruptions of this nature or can promote restoration of the permeability barrier after damage by such agents. The release of carboxyfluorescein from large unilamellar vesicles composed of lipids characteristically present in the inner leaflet of cell membranes (phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, and cholesterol) was used to measure membrane permeability. It was determined that in the presence of calcium, annexin A5 reduced the level of baseline leakage from vesicles and reduced or reversed damage due to arachidonic acid, lysophosphatidic acid, lysophosphatidylcholine, diacylglycerol, monoacylglycerol, spermidine, amyloid-ß, amylin, and osmotic shock. Annexin A6 was also able to provide membrane protection in many but not all of these cases. In a cell, it is likely annexins would move to sites of breakdown of the permeability barrier because of the calcium-dependent promotion of the binding of annexins to membranes at sites of calcium entry. Because of the fundamental importance to life of maintaining the permeability barrier of the cell membrane, it is proposed here that this property of annexins may represent a critical, primordial activity that explains their great evolutionary conservation and abundant expression in most cells.

Isolation of chromaffin granules.

Adrenal medullary chromaffin granules (dense core secretory vesicles) have been a valuable model system for the study of the proteins and membrane components involved in the process of exocytosis. Because of the abundance of chromaffin granules in a readily available tissue source, bovine adrenal medullae, and their unique sedimentation properties, it is possible to obtain large quantities of highly purified granules and granule membranes in a short period of time. Two protocols are presented here for the isolation of chromaffin granules: a basic protocol based on differential centrifugation in an iso-osmotic medium that yields intact chromaffin granules, and an alternate protocol based on sedimentation through a density step gradient that provides a greater yield of more highly purified chromaffin granules. Since in the latter case the granules cannot be returned to a medium of physiological osmolarity without lysis after purification on the step gradient, the alternate protocol is more useful to obtain the granule membranes or contents for further study.

Organization and synergistic binding of copine I and annexin A1 on supported lipid bilayers observed by atomic force microscopy.

The transduction of signals across the plasma membrane of cells after receptor activation frequently involves the assembly of interacting protein molecules on the cytoplasmic face of the membrane. However, the structural organization and dynamics of the formation of such complexes has not been well defined. In this study atomic force microscopy was used to monitor the assemblies formed in vitro by two classes of calcium-dependent, membrane-binding proteins that participate in the formation of signaling complexes on membranes - the annexins and the copines. When applied to supported lipid bilayers composed of 25% brain phosphatidylserine and 75% dioleyl phosphatidylcholine in the presence of 1 mM Ca(2+) both human annexin A1 and human copine I bound only to specialized domains that appeared to be 0.5 to 1.0 nm lower than the rest of the bilayer. These domains may be enriched in phosphatidylserine and have a more disordered structure allowing probe penetration. Confinement of the binding of the proteins to these domains may be important in the process of concentrating other signaling proteins bound to the copine or annexin. The binding of the annexin promoted the growth of the domains and created additional binding space for the copine. This may reflect a general ability of annexins to alter membrane structure in such a way that C2 domain-containing proteins like copine can bind. Copine I formed a reticular lattice composed of linear elements approximately 45 nm long on the specialized domains. This lattice might provide a scaffold for the assembly and interaction of copine target proteins in signaling complexes.

Novel protein ligands of the annexin A7 N-terminal region suggest pro-beta helices engage one another with high specificity.

The N-terminal regions of annexins A7 (synexin) and A11 consist of an extended series of short sequence repeats rich in tyrosine, proline, and glycine that provide binding sites for other proteins that may be recruited to membranes by the annexins and that may modulate the calcium and membrane binding activities of the annexin core domains. In this study two new ligands for the annexin A7 N terminal region were identified by yeast two hybrid screening: the TNFalpha receptor regulatory protein SODD (Suppressor Of Death Domains) and KIAA0280, a protein of unknown function. Strikingly, the sites of interaction of these proteins with the annexin also contain sequence repeats similar to those present in the N-termini of annexins A7 and A11. It was also found that the annexin A7 N-terminal region interacts with itself in the two hybrid assay. These results suggest that sequence repeats of this nature form novel structures, called YP pro-beta helices, that are characterized by an ability to interact with one another. Specificity of interactions between the pro-beta helices in different proteins may be encoded by the variations of residues and lengths of the sequence repeats.

Interactions of annexins with the mu subunits of the clathrin assembly proteins.

A number of biochemical and genetic studies have suggested that certain annexins play important roles in the endocytic pathway, possibly involving the generation, localization, or fusion of endocytic compartments. In a yeast two-hybrid screen for proteins that interact with the N-terminal domain of annexin A2 we identified the mu2 subunit of the clathrin assembly protein complex AP-2. The interaction depended upon two copies of a Yxx phi amino acid sequence motif (Y = tyrosine, x = variable residue, phi = bulky, hydrophobic residue) in the annexin that is also characteristic of the binding site for mu2 on the cytoplasmic domains of transmembrane receptors. The interaction between mu2 and full-length annexin A2 was demonstrated in vitro to be direct, to require calcium, and to be functional in the sense that annexin A2 was able to recruit the mu2 to immobilized lipids. Examination of other annexins and mu subunits demonstrated that annexin A2 also binds the mu1 subunit of the AP-1 complex, that annexin A6 binds mu1 and mu2, and that annexin A1 binds only mu1. We propose that annexins can "masquerade" as transmembrane receptors when they are attached to membranes in the presence of calcium and that they might therefore function to initiate calcium-regulated coated pit formation at the cell surface or on intracellular organelles.

Annexins: linking Ca2+ signalling to membrane dynamics.

Eukaryotic cells contain various Ca(2+)-effector proteins that mediate cellular responses to changes in intracellular Ca(2+) levels. A unique class of these proteins - annexins - can bind to certain membrane phospholipids in a Ca(2+)-dependent manner, providing a link between Ca(2+) signalling and membrane functions. By forming networks on the membrane surface, annexins can function as organizers of membrane domains and membrane-recruitment platforms for proteins with which they interact. These and related properties enable annexins to participate in several otherwise unrelated events that range from membrane dynamics to cell differentiation and migration.

The tricalbin C2 domains: lipid-binding properties of a novel, synaptotagmin-like yeast protein family.

The tricalbins are a recently discovered family of Saccharomyces cerevisae proteins containing a predicted N-terminal transmembrane domain and at least three C2 domains. They are thought to be yeast homologues of synaptotagmin, a hypothesis supported by structural similarities and past studies that implicated tricalbins in processes of membrane trafficking and sorting. We expressed and purified constructs consisting of single tricalbin C2 domains, and assayed their ability to bind lipids in response to calcium. Protein-lipid overlay assays indicated that the C-terminal C2 domains (C2C) of tricalbins 1 and 3 bind numerous species of acidic phospholipid, including phosphatidylserine and several phosphoinositides, and the amount of protein bound was greatly enhanced in the presence of 1 mM calcium. Sedimentation assays using mixed phosphatidylserine/phosphatidylcholine (PS/PC) vesicles confirmed that the C2C domains of tricalbin 1 and 3 bind membranes in a calcium-responsive manner and showed that they are more sensitive to calcium than the C2A domain of synaptotagmin I. Both assays revealed that all of the C2 domains of tricalbin 2 are insensitive to calcium. Fluorimetric assays exploiting the position of naturally occurring tryptophans in tricalbin 1 C2C and tricalbin 3 C2C confirmed that these domains are capable of binding calcium and that this is coupled to the binding of acidic phospholipid. Combining this with past protein-protein interaction data, we theorize that the calcium-insensitive tricalbin 2 mediates the creation of hetero-oligomeric tricalbin complexes in which tricalbin 1 or 3 or both supply a calcium-dependent membrane binding activity.

Calcium-dependent regulation of tumour necrosis factor-alpha receptor signalling by copine.

The role of copines in regulating signalling from the TNF-alpha (tumour necrosis factor-alpha) receptor was probed by the expression of a copine dominant-negative construct in HEK293 (human embryonic kidney 293) cells. The construct was found to reduce activation of the transcription factor NF-kappaB (nuclear factor-kappaB) by TNF-alpha. The introduction of calcium into HEK293 cells either through the activation of muscarinic cholinergic receptors or through the application of the ionophore A23187 was found to enhance TNF-alpha-dependent activation of NF-kappaB. This effect of calcium was completely blocked by the copine dominant-negative construct. TNF-alpha was found to greatly enhance the expression of endogenous copine I, and the responsiveness of the TNF-alpha signalling pathway to muscarinic stimulation increased in parallel with the increased copine I expression. The copine dominant-negative construct also inhibited the TNF-alpha-dependent degradation of IkappaB, a regulator of NF-kappaB. All of the effects of the dominant-negative construct could be reversed by overexpression of full-length copine I, suggesting that the construct acts specifically through competitive inhibition of copine. One of the identified targets of copine I is the NEDD8-conjugating enzyme UBC12 (ubiquitin C12), that promotes the degradation of IkappaB through the ubiquitin ligase enzyme complex SCF(betaTrCP). Therefore the copine dominant-negative construct might inhibit TNF-alpha signalling by dysregulation or mislocalization of UBC12. Based on these results, a hypothesis is presented for possible roles of copines in regulating other signalling pathways in animals, plants and protozoa.

Identification of targets for calcium signaling through the copine family of proteins. Characterization of a coiled-coil copine-binding motif.

We provide evidence that copines, members of a ubiquitous family of calcium-dependent, membrane-binding proteins, may represent a universal transduction pathway for calcium signaling because we find copines are capable of interacting with a wide variety of "target" proteins including MEK1, protein phosphatase 5, and the CDC42-regulated kinase, that are themselves components of intracellular signaling pathways. The copine target proteins were identified by yeast two-hybrid screening and the interactions were verified in vitro using purified proteins. In the majority of cases the copine binds to a domain of the target protein that is predicted to form a characteristic coiled-coil. A consensus sequence for the coiled-coil copine-binding site was derived and found to have predictive value for identifying new copine targets. We also show that interaction with copines may result in recruitment of target proteins to membrane surfaces and regulation of the enzymatic activities of target proteins.

Structural and dynamic changes in human annexin VI induced by a phosphorylation-mimicking mutation, T356D.

Phosphorylation of some members of the annexin family of proteins may play a significant role in controlling their calcium-dependent interactions with membranes. Recent electron microscopic studies of annexin VI revealed that the protein's two core domains exhibit a great degree of flexibility and are able to undergo a relative conformational change that could potentially initiate contacts between membranes [Avila-Sakar, A. J., et al. (2000) J. Struct. Biol. 130, 54-62]. To assess the possibility of a regulatory role of phosphorylation in this behavior, the crystal structure of a phosphorylation-mimicking mutant (T356D in the flexible connector region of human annexin VI) was determined to 2.65 A resolution. When the mutant is compared to the wild-type annexin VI, subtle differences are seen at the site of the mutation, while larger changes are evident in one of the calcium-binding loops and in the presence of five calcium ions. Furthermore, biochemical studies provide evidence for additional conformational differences between the T356D and wild-type solution structures. Fluorescence emission and acrylamide quenching suggest a higher level of solvent exposure of Trp-343 in the connector region of T356D in the presence of calcium. Comparisons of retardation coefficients in native gel electrophoresis reveal that T356D has a more extended shape, while proteolytic studies show a greater accessibility of a trypsin cleavage site inside the linker region, indicating a conformation more open than the wild-type form. These data provide insights into a possible regulatory mechanism leading to a higher degree of flexibility and possibly a higher calcium binding affinity of annexin VI upon phosphorylation.

Purine composition of the crystalline cytoplasmic inclusions of Paramecium tetraurelia.

Crystalline cytoplasmic inclusions were isolated by differential centrifugation from mass cultures of Paramecium tetraurelia feeding on Klebsiella pneumonia. Physical and chemical measurements of intact and solubilized crystals determined that they consist primarily of guanine and hypoxanthine with traces of xanthine. Crystals from the mutant sombre consist primarily of xanthine, suggesting there is a disorder of purine metabolism in this mutant.