Light-chain-independent binding of adaptors, AP180, and auxilin to clathrin

Binding of coated vesicle assembly proteins to clathrin causes it to assemble into regular coat structures. The assembly protein fraction of bovine brain coated vesicles comprises AP180, auxilin, and HA1 and HA2 adaptors. Clathrin heavy chains, separated from their light chains, polymerize with unimpaired efficiency when assembly proteins are added. The reassembled coats were purified by sucrose gradient centrifugation and examined for composition by SDS-PAGE and immunoblotting. We found that all four major coat proteins are incorporated in the presence and absence of light chains. Moreover, each of the purified coat proteins is able to associate directly with clathrin heavy chains in preassembled cages as efficiently as with intact clathrin. We conclude that light chains are not essential for the interaction of AP180, auxilin, and HA1 and HA2 with clathrin.     

The Sybtraps: Control of Synaptobrevin Traffic by Synaptophysin, α‐Synuclein and AP‐180

Synaptobrevin II (sybII) is a key fusogenic molecule on synaptic vesicles (SVs) therefore the active maintenance of both its conformation and location in sufficient numbers on this organelle is critical in both mediating and sustaining neurotransmitter release. Recently three proteins have been identified having key roles in the presentation, trafficking and retrieval of sybII during the fusion and endocytosis of SVs. The nerve terminal protein α‐synuclein catalyses sybII entry into SNARE complexes, whereas the monomeric adaptor protein AP‐180 is required for sybII retrieval during SV endocytosis. Overarching these events is the tetraspan SV protein synaptophysin, which is a major sybII interaction partner on the SV. This review will evaluate recent studies to propose working models for the control of sybII traffic by synaptophysin and other Sybtraps (syb II tra fficking p artners ) and suggest how dysfunction in sybII traffic may contribute to human disease.      

Role of the Bullous Pemphigoid Antigen 180 (BP180) in the Assembly of Hemidesmosomes and Cell Adhesion—Reexpression of BP180 in Generalized Atrophic Benign Epidermolysis Bullosa Keratinocytes

Bullous pemphigoid antigen 180 (BP180) is a transmembrane component of hemidesmosomes (HD), cell–substrate attachment complexes in stratified and complex epithelia. To determine the role of BP180 in the assembly of HD and cell adhesion, using SV40 virions we have immortalized BP180-deficient keratinocytes derived from a patient with the inherited skin blistering disorder generalized atrophic benign epidermolysis bullosa (GABEB). The GABEB keratinocytes form HD-like structures, which contain α6β4 integrin and HD1/plectin, but not the bullous pemphigoid antigen 230 (BP230). The expression of integrin subunits by GABEB keratinocytes was comparable to that of an immortalized normal human keratinocyte cell line (NHK), except for α6 and β4, which were less strongly expressed in GABEB cells. In short-term adhesion assays, both GABEB keratinocytes and NHK bound strongly and to a similar extent to laminin-1, laminin-5, fibronectin, and type IV and V collagens, which suggests that BP180 is not involved in promoting the initial adhesion to these ligands. Transfection of GABEB keratinocytes with cDNAs for wild-type or a mutant of BP180 lacking the collagenous extracellular domain resulted in the expression of recombinant BP180 proteins that were correctly polarized at the basal cell surface together with α6β4. In addition, restored synthesis of BP180 affected the subcellular localization of BP230, which was no longer diffusely distributed in the cytoplasm, but was found in HD-like structures. In contrast, a BP180 mutant with a 36-amino-acid deletion from the amino terminus of the cytoplasmic domain failed to localize to HD-like structures. These results demonstrate that a region within the cytoplasmic domain of BP180 is essential for its localization into HD and that BP180 may play a critical role in coordinating the subcellular distribution of BP230.     

Kinematic and Dynamic Characteristics of Pulsating Flow in 180^o Tube

Kinematic and dynamic characteristics of pulsating flow in a model of human aortic arch are obtained by a computational analysis. Three-dimensional flow processes are summarized by pressure distributions on the symmetric plane together with velocity and pressure contours on a few cross sections for systolic acceleration and deceleration. Without considering the effects of aortic tapering and the carotid arteries, the development of tubular boundary layer with centrifugal forces and pulsation are also analyzed for flow separation and backflow during systolic deceleration.     

Densin-180: revised membrane topology, domain structure and phosphorylation status

Densin-180 is a core component of post-synaptic densities, the highly complex molecular assemblies that mediate signaling between neuronal cells. It is a multi-domain scaffold protein characterized by multiple leucine-rich repeat domains plus a single Psd95/Discs large/Zona occludens-1 domain. In its original topology model a single transmembrane segment was proposed with an extracellular N-terminus and an intracellular C-terminus. However, recently discovered in vivo phosphorylation sites are incompatible with this topology. Here, we discuss an all-intracellular and membrane-associated localization of Densin-180 that is consistent with and supported by all the latest experimental data. This revised topology which now includes also a phosphorylation-rich area will have deciding influence on future research involving Densin-180 and its signaling.     

D‐Hillarin,a novel W180‐domain protein,affects cytokinesis through interaction with the septin family member Pnut

By database searches of the Drosophila genome project we have identified D‐hil as the fly member of a novel family of W180‐domain containing proteins. Immunocytochemistry demonstrated that D‐hil is localized to the neuropil of the embryonic CNS, to the cellular cortex of dividing neuroblasts from larval brains, and that it is up‐regulated in the cleavage furrow of S2 cells. We show that D‐hil distribution overlaps extensively with that of the septin family member Pnut. Cross‐immunoprecipitation experiments further indicated that the two proteins may be members of the same protein complex. Analysis of a severe hypomorphic P‐element mutation in the D‐hil locus suggested that D‐hil is a nonessential protein. However, by creating double mutant flies we show that the D‐hil locus acts as a modulator of Pnut function by increasing the level of polyploidy of neuroblasts in Pnut^KG00478/Pnut^KG00478 larval brains. Based on these results we propose that D‐hil may function as a regulator of septin function during cytokinesis in the developing nervous system. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005     

Magnetic circular dichroism of adenine, hypoxanthine, and guanosine 5'-diphosphate to 180 nm

The magnetic circular dichroism (MCD) of adenine, hypoxanthine, and guanosine 5′-diphosphate reveals that, for each species, the uv-absorption band near 200 nm is composed of at least two electronic transitions. The theory of MCD shows that the dipoles of these transitions are not parallel to one another. The transitions are assigned within the framework of current theories of the electronic structure of the purines. Knowledge of the presence of more than one transition within an absorption envelope is important in interpreting the corresponding natural CD of DNA and RNA.     

Cancer and the bromodomains of BAF180

Chromatin remodelling complexes alter the structure of chromatin and have central roles in all DNA-templated activities, including regulation of gene expression and DNA repair. Mutations in subunits of the PBAF (polybromo/Brg1-associated factor) or SWI/SNF-B remodelling complex, including BAF180, are frequently associated with cancer. There are six potential acetyl-lysine-binding BDs (bromodomains) in BAF180, which may function to target the PBAF complex to promoters or sites of DNA repair. In the present review, we discuss what is currently known about the BDs of BAF180 and their potential significance in cancer.     

Structural analysis of the alpha-D-glucan (EPS180) produced by the Lactobacillus reuteri strain 180 glucansucrase GTF180 enzyme

The neutral exopolysaccharide EPS180 produced from sucrose by the glucansucrase GTF180 enzyme from Lactobacillus reuteri 180 was found to be a (1-->3,1-->6)-alpha-D-glucan, with no repeating units present. Based on linkage analysis, periodate oxidation, and 1D/2D 1H and 13C NMR spectroscopy of the intact EPS180, as well as MS and NMR analysis of oligosaccharides obtained by partial acid hydrolysis of EPS180, a composite model, that includes all identified structural features, was formulated as follows: [Formula: see text].     

Author index to volume 180

Fractional extraction and isozyme electrophoresis revealed the presence of small amounts (2.5% of total cellular activity) of mitochondrial creatine kinase (CK) in rabbit fast-twitch muscle. Chronic nerve stimulation resulted in a decrease of extramitochondrial MM-CK to 60% of its normal value but induced an approx. 4-fold increase in mitochondrial CK. This increase occurred in parallel with the rise in enzyme activities of terminal substrate oxidation.     

The ribosome receptor, p180, interacts with kinesin heavy chain, KIF5B

The conventional microtubule-dependent motor protein kinesin consists of heavy and light chains both of which have been documented to bind a variety of potential linker or cargo proteins. In this study we employed a yeast two-hybrid assay to identify additional binding partners of the kinesin heavy chain isoform KIF5B. A human brain cDNA library was screened with a bait corresponding to amino acid residues 814-963 of human KIF5B. This screen identified the ribosome receptor, p180, as a KIF5B-binding protein. The sites of interaction are residues 1294-1413 of p180 and the C-terminal half of the cargo binding-domain of KIF5B (residues 867-907). The KIF5B-binding site in p180 is homologous to the previously determined KIF5B-binding site in kinectin. The interacting regions of p180 and KIF5B consist almost entirely of heptad repeats, suggesting the interaction is a coiled-coil. A role for the kinesin/p180 interaction may include mRNA localization and/or transport of endoplasmic reticulum-derived vesicles.     

Expression, purification of human vasostatin120-180 in Escherichia coli, and its anti-angiogenic characterization

According to codon preference of Escherichia coli, the optimized coding sequence of human vasostatin120-180aa (VAS) was obtained by chemical synthesis and molecular cloning methods. Using PCR and enzyme digestion, the full encoding sequence for VAS was cloned into the E. coli expression vector pALEX and expressed as a GST fusion protein in BL21 (DE3) strain. GST-VAS protein approximately accounted for 45% of the total bacterial proteins. Most of target protein existed in inclusion body. To improve the solubility of GST-VAS, the contribution of low temperature and molecular chaperone co-expression to the solubility of GST-VAS was tested. The results showed that co-expression with chaperons, TF and GroES/GroEL, and low expression temperature cooperatively improved the solubility of GST-VAS from 10 to 85%, and the yield of soluble GST-VAS was sixfold increased. When purified by GST affinity chromatography, 50 mg GST-VAS was obtained with purity over 85% from 1 L culture. Intact VAS was released by enterokinase digestion and further purified by Sephadex G50 gel filtration chromatography. About 7.2 mg intact homogeneous VAS protein was finally produced from 1L bacterial culture. The identity of GST-VAS and VAS was validated by Western blotting analysis. Recombinant VAS protein displayed distinct inhibition of endothelial cell proliferation and anti-angiogenic activity by chick embryo chorioallantoic membrane assay.     

AP180 Couples Protein Retrieval to Clathrin‐Mediated Endocytosis of Synaptic Vesicles

How clathrin‐mediated endocytosis (CME) retrieves vesicle proteins into newly formed synaptic vesicles (SVs) remains a major puzzle. Besides its roles in stimulating clathrin‐coated vesicle formation and regulating SV size, the clathrin assembly protein AP180 has been identified as a key player in retrieving SV proteins. The mechanisms by which AP180 recruits SV proteins are not fully understood. Here, we show that following acute inactivation of AP180 in Drosophila, SV recycling is severely impaired at the larval neuromuscular synapse based on analyses of FM 1‐43 uptake and synaptic ultrastructure. More dramatically, AP180 activity is important to maintain the integrity of SV protein complexes at the plasma membrane during endocytosis. These observations suggest that AP180 normally clusters SV proteins together during recycling. Consistent with this notion, SV protein composition and distribution are altered in AP180 mutant flies. Finally, AP180 co‐immunoprecipitates with SV proteins, including the vesicular glutamate transporter and neuronal synaptobrevin. These results reveal a new mode by which AP180 couples protein retrieval to CME of SVs. AP180 is also genetically linked to Alzheimer's disease. Hence, the findings of this study may provide new mechanistic insight into the role of AP180 dysfunction in Alzheimer's disease.      

180 Relationships Between Water and Sediment Characteristics and Benthic Green Macroalgal Abundance in Yaquina Bay,Oregon: 1999–2002

“Green tides” or blooms of ulvoid green algae are frequent in Yaquina Bay estuary on the central Oregon coast, USA. Measurements of their biomass were made from late spring to early winter in 1999 at six intertidal sites in the estuary, and were continued through the winter of 2002 at two sites that showed the greatest accumulation. The dominant blooming species were the Enteromorpha linza complex, Ulva fenestrata, E. flexuosa, and E. intestinalis. Red and brown algal abundance was negligable. Nutrients in the central channel were monitored, along with incident light and water column absorbence, and sediment temperature. The abundance of benthic amphipods was negatively correlated with the concentration of dissolved sulfides in porewater. However, dissolved sulfide levels were not correlated with green macroalgae biomass. Additional correlations were sought between the variables measured in an effort to suggest or negate major cause and effect relationships. This research is relevant to discerning the effects of eutrophication as a stressor on estuarine processes and could contribute toward an understanding of anthropogenic impacts on biological communities in coastal ecosystems.     

The monomeric clathrin assembly protein, AP180, regulates contractile vacuole size in Dictyostelium discoideum

AP180, one of many assembly proteins and adaptors for clathrin, stimulates the assembly of clathrin lattices on membranes, but its unique contribution to clathrin function remains elusive. In this study we identified the Dictyostelium discoideum ortholog of the adaptor protein AP180 and characterized a mutant strain carrying a deletion in this gene. Imaging GFP-labeled AP180 showed that it localized to punctae at the plasma membrane, the contractile vacuole, and the cytoplasm and associated with clathrin. AP180 null cells did not display defects characteristic of clathrin mutants and continued to localize clathrin punctae on their plasma membrane and within the cytoplasm. However, like clathrin mutants, AP180 mutants, were osmosensitive. When immersed in water, AP180 null cells formed abnormally large contractile vacuoles. Furthermore, the cycle of expansion and contraction for contractile vacuoles in AP80 null cells was twice as long as that of wild-type cells. Taken together, our results suggest that AP180 plays a unique role as a regulator of contractile vacuole morphology and activity in Dictyostelium.