The actin-binding protein comitin (p24) is a component of the Golgi apparatus

Comitin (p24) was first identified in Dictyostelium discoideum as a membrane-associated protein which binds in gel overlay assays to G and F actin. To analyze its actin-binding properties we used purified, bacterially expressed comitin and found that it binds to F actin in spin down experiments and increases the viscosity of F actin solutions even under high-salt conditions. Immunofluorescence studies, cell fractionation experiments and EM studies of vesicles precipitated with comitin-specific monoclonal antibodies showed that comitin was present in D. discoideum on: (a) a perinuclear structure with tubular or fibrillary extensions; and (b) on vesicles distributed throughout the cell. In immunofluorescence experiments using comitin antibodies NIH 3T3 fibroblasts showed a similar staining pattern as D. discoideum cells. Using bona fide Golgi markers the perinuclear structure was identified as the Golgi apparatus. The results were supported by an electron microscopic study using cryosections. Based on these data we propose that also in Dictyostelium the stained perinuclear structure is the Golgi apparatus. In vivo the perinuclear structure was found to be attached to the actin and the microtubule network. Alteration of the actin network or depolymerization of the microtubules led to its dispersal into vesicles distributed throughout the cell. These results suggest that the Golgi apparatus in D. discoideum is connected to the actin network by comitin. This protein seems also to be present in mammalian cells.     

Identification of actin-binding protein as the protein linking the membrane skeleton to glycoproteins on platelet plasma membranes

Platelets have previously been shown to contain a membrane skeleton that is composed of actin filaments, actin-binding protein, and three membrane glycoproteins (GP), GP Ib, GP Ia, and a minor glycoprotein of Mr = 250,000. The present study was designed to determine how the membrane glycoproteins were linked to actin filaments. Unstimulated platelets were lysed with Triton X-100, and the membrane skeleton was isolated on sucrose density gradients or by high-speed centrifugation. The association of the membrane glycoproteins with the actin filaments was disrupted when actin-binding protein was hydrolyzed by activity of the Ca2+-dependent protease, which was active in platelet lysates upon addition of Ca2+ in the absence of leupeptin. Similarly, activation of the Ca2+-dependent protease in intact platelets by the addition of a platelet agonist also caused the membrane glycoproteins to dissociate from the membrane skeleton. Affinity-purified actin-binding protein antibodies immunoprecipitated the membrane glycoproteins from platelet lysates in which actin filaments had been removed by DNase I-induced depolymerization and high-speed centrifugation. These results demonstrate that actin-binding protein links actin filaments of the platelet membrane skeleton to three plasma membrane glycoproteins and that filaments are released from their attachment site when actin-binding protein is hydrolyzed by the Ca2+-dependent protease within intact platelets during platelet activation.     

Distribution among tissues and intracellular localization of cofilin, a 21kDa actin-binding protein

Cofilin, a 21kDa actin-binding protein, binds to F-actin in a 1:1 molar ratio of cofilin to actin molecule (Nishida, E., S. Maekawa, and H. Sakai, Biochemistry, 23, 5307-5313, 1984) and is capable of controlling actin polymerization and depolymerization in vitro in a pH-sensitive manner (Yonezawa, N., E. Nishida, and H. Sakai, J. Biol. Chem., 260, 14410-14412, 1985). In this study, immunoblot analysis using monospecific antibodies against cofilin showed that cofilin is ubiquitously distributed in a variety of bovine and rat organs and tissues. Cofilin is also present in various cultured cell lines. Indirect immunofluorescence staining of mouse fibroblastic cells and human epidermoid carcinoma cells indicated that cofilin is distributed nearly uniformly in the cytoplasm and is concentrated in ruffling membranes where F-actin is also concentrated as revealed by staining with rhodamine-phalloin. Stress fiber structures were not strongly stained with the anti-cofilin antibody, although stress fiber staining was sometimes observed near the cell periphery in mouse 3T3 cells. These results suggest that the bulk of cofilin may not be associated with F-actin bundles in vivo.     

Dual-function protein in plant defence: seed lectin from Dolichos biflorus (horse gram) exhibits lipoxygenase activity

Plant-pathogen interactions play a vital role in developing resistance to pests. Dolichos biflorus (horse gram), a leguminous pulse crop of the subtropics, exhibits amazing defence against attack by pests/pathogens. Investigations to locate the possible source of the indomitable pest resistance of D. biflorus, which is the richest source of LOX (lipoxygenase) activity, have led to a molecule that exhibits LOX-like functions. The LOX-like activity associated with the molecule, identified by its structure and stability to be a tetrameric lectin, was found to be unusual. The evidence for the lectin protein with LOX activity has come from (i) MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS, (ii) N-terminal sequencing, (iii) partial sequencing of the tryptic fragments of the protein, (iv) amino acid composition, and (v) the presence of an Mn2+ ion. A hydrophobic binding site of the tetrameric lectin, along with the presence of an Mn2+ ion, accounts for the observed LOX like activity. This is the first ever report of a protein exhibiting both haemagglutination and LOX-like activity. The two activities are associated with separate loci on the same protein. LOX activity associated with this molecule adds a new dimension to our understanding of lectin functions. This observation has wide implications for the understanding of plant defence mechanisms against pests and the cellular complexity in plant-pathogen interactions that may lead to the design of transgenics with potential to impart pest resistance to other crops.     

Isolation of a mannose-specific lectin from Escherichia coli and its role in the adherence of the bacteria to epithelial cells.

A high molecular weight protein aggregate, which agglutinates yeast cells, human epithelial cells and mouse lymphocytes, was isolated from extracts of Escherichia coli by differential centrifugation and gel filtration. The agglutination is specifically inhibited by d-mannose and its derivatives, the best inhibitor being p-nitrophenyl α-d-mannoside. Sodium dodecyl sulfate gel electrophoresis showed that the lectin consists of protein subunits with identical M_r of ～36500. The amino acid composition of the purified lectin is different from that reported for the type I pili protein, the K99 antigen and the major outer membrane protein Ia of E. coli. The protein appears to be located on the bacterial surface, and is probably involved in the mannose-specific adherence of E. coli to eukaryotic cells.     

Cholesterol-rich intracellular membranes: a precursor to the plasma membrane

The disposition of newly synthesized sterols in cultured human fibroblasts has been examined in this study. We began by demonstrating that cholesterol mass and exogenously added [3H]cholesterol both are markers for the plasma membrane, perhaps better than 5'-nucleotidase. Cells were incubated with radioactive acetate to label their endogenous sterols biosynthetically, treated with cholesterol oxidase to convert plasma membrane cholesterol to cholestenone, and then homogenized and spun to equilibrium on sucrose gradients. The density gradient profiles of the various organelles were monitored using these markers: plasma membrane, radioactive cholestenone; smooth endoplasmic reticulum, 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase); and Golgi apparatus, galactosyltransferase. The buoyant density profiles of radioactive intracellular cholesterol and lanosterol both had a peak at 1.12 g/cm3, similar to 5'-nucleotidase and galactosyltransferase but not to HMG-CoA reductase. This result suggests that cholesterol biosynthesis is not taken to completion in the endoplasmic reticulum. Digitonin treatment shifted the profiles of both plasma membrane and intracellular cholesterol to higher densities. Pretreatment of intact cells with cholesterol oxidase abolished the digitonin shift of plasma membranes but not the intracellular cholesterol, indicating that these two membrane pools are not entirely physically associated. Because intracellular cholesterol was shifted more than any of the organelle markers, it must reside in a separate membrane. Since digitonin selectively shifts the density of membranes rich in cholesterol, we infer that newly synthesized cholesterol accumulates in such membranes prior to its delivery to the plasma membrane. Taken together, these results suggest that cholesterol may be concentrated for delivery to the plasma membrane by being synthesized from a sterol precursor such as lanosterol in a discrete but undefined intracellular membrane.     

Vacuolar H+-ATPase binding to microfilaments: regulation in response to phosphatidylinositol 3-kinase activity and detailed characterization of the actin-binding site in subunit B

Vacuolar H(+)-ATPase (V-ATPase) binds microfilaments, and that interaction may be mediated by an actin binding domain in subunit B of the enzyme. To test for possible physiologic functions of the actin binding activity of V-ATPase, early responses of resorbing osteoclasts to inhibition of phosphatidylinositol 3-kinase activity by wortmannin and LY294002 were examined. Rapid co-localization between V-ATPase and F-actin was demonstrated by immunocytochemistry, and corresponding association between V-ATPase and F-actin in immunoprecipitations and pelleting assays was detected. This response was reversed as osteoclasts recovered resorptive activity after inhibitors were removed. By expressing and characterizing fusion proteins containing segments of the actin-binding amino-terminal regions of the B subunits of V-ATPase, we mapped the actin-binding site to a 44-amino acid domain. An 11-amino acid segment with a sequence similar to the actin-binding site of human profilin I was detected within this region. 13-Mers containing these profilin-like segments bound actin in fluorescent anisotropy studies and competed with profilin for binding to actin. Using site-directed mutagenesis, the 11-amino acid profilin-like actin-binding motifs (amino acids 49-59 of B1 and 55-65 of B2) were replaced with an 11-amino acid spacer with a sequence based on the homologous sequence from subunit B of Pyrococcus horikoshii, an organism that lacks an actin cytoskeleton. These substitutions eliminated the actin-binding activity of the B subunit fusion proteins. In summary, binding between V-ATPase and F-actin in osteoclasts occurs in response to blocking phosphatidylinositol 3-kinase activity. This response was fully reversible. The actin binding activities of the B subunits of V-ATPase required 11-amino acid actin-binding motifs that are similar in sequence to the actin-binding site of mammalian profilin I.     

Motile areas of leech neurites are rich in microfilaments and two actin-binding proteins: gelsolin and profilin.

Cell motility is produced by changes in the dynamics and organization of actin filaments. The aim of the experiments described here was to test whether growing neurites contain two actin-binding proteins, gelsolin and profilin, that regulate polymerization of actin and affect non-neuronal cell motility. The distribution of gelsolin, profilin and the microfilaments was compared by immunocytochemistry of leech neurons growing in culture. We observed that microfilaments are enriched in the peripheral motile areas of the neurites. Both gelsolin and profilin are also concentrated in these regions. Gelsolin is abundant in filopodia and is associated with single identifiable microfilament bundles in lamellipodia. Profilin is not prominent in filopodia and shows a diffuse staining pattern in lamellipodia. The colocalization of gelsolin and profilin in motile, microfilament-rich areas supports the hypothesis that they synergistically regulate the actin dynamics that underlie neurite growth.     

A sialic acid-specific lectin from the mushroom Paecilomyces Japonica that exhibits hemagglutination activity and cytotoxicity

The mushroom Paecilomyces japonica, grown on the silkworm larvae, has been used in Asia as a nutraceutical, tea, and Chinese medicine. In the present study, a sialic acid-specific lectin has been purified from the mushroom P. japonica using affinity chromatography on a fetuin-agarose column. Electrophoretical analyses indicated that this lectin, designated P. japonica agglutinin (PJA), is an acidic protein with a molecular mass of 16 kDa, and has no intermolecular disulfide bonds. PJA induced hemagglutination activity in human ABO, mouse, rat, and rabbit erythrocytes. This activity was inhibited by sialic acid and sialoglycoproteins, but not by any other carbohydrates. PJA was stable at pH 4.0-8.0, and at temperatures below 55 degrees C. The activity of PJA was independent of EDTA and divalent cations. In addition, PJA exerts cytotoxic effects on the following cancer cell lines: human stomach cancer SNU-1, human pancreas cancer AsPc-1, and human breast cancer MDA-MB-231.     

Roles of protein kinase C and actin-binding protein 280 in the regulation of intracellular trafficking of dopamine D3 receptor

D(3) dopamine receptor (D(3)R) is expressed mainly in parts of the brain that control the emotional behaviors. It is believed that the improper regulation of D(3)R is involved in the etiology of schizophrenia. Desensitization of D(3)R is weakly associated with G protein-coupled receptor kinase (GRK)/beta-arrestin-directed internalization. This suggests that there might be an alternative pathway that regulates D(3)R signaling. This report shows that D(3)R undergoes robust protein kinase C (PKC)-dependent sequestration that is accompanied by receptor phosphorylation and the desensitization of signaling. PKC-dependent D(3)R sequestration, which was enhanced by PKC-beta or -delta, was dynamin dependent but independent of GRK, beta-arrestin, or caveolin 1. Site-directed mutagenesis of all possible phosphorylation sites within the intracellular loops of D(3)R identified serine residues at positions 229 and 257 as the critical amino acids responsible for phorbol-12-myristate-13-acetate (PMA)-induced D(3)R phosphorylation, sequestration, and desensitization. In addition, the LxxY endocytosis motif, which is located between residues 252 and 255, was found to play accommodating roles for PMA-induced D(3)R sequestration. A continuous interaction with the actin-binding protein 280 (filamin A), which was previously known to interact with D(3)R, is required for PMA-induced D(3)R sequestration. In conclusion, the PKC-dependent but GRK-/beta-arrestin-independent phosphorylation of D(3)R is the main pathway responsible for the sequestration and desensitization of D(3)R. Filamin A is essential for both the efficient signaling and sequestration of D(3)R.     

Stimulation of actomyosin Mg2+-ATPase activity by a brain microtubule-associated protein fraction. High-molecular-weight actin-binding protein is the stimulating factor

Actomyosin Mg2+-ATPase activity was stimulated by a brain microtubule-associated protein (MAP) fraction. The stimulating activity of the MAP fraction was abolished by boiling and trypsin treatment, suggesting the presence of a protein factor. The factor stimulated actomyosin Mg2+-ATPase activity stoichiometrically by about four times in the optimum conditions (50--75 mM KCl, pH 6.6). The stimulating factor was coprecipitable with actomyosin and was found to be a pair of high-molecular-weight polypeptides (mol wts, 240,000 and 235,000). The polypeptides were not associated with microtubules or myosin, but with fibrous actin. In column chromatographies used for purifying the stimulating factor, the amount of polypeptides coincided with the stimulating activity. Increases in both specific activity and the amount of the paired polypeptides were nearly parallel in the process of the purification. A purified fraction (65% pure with respect to the paired polypeptides) showed a 56-fold increase of the specific stimulating activity as compared with the initial brain supernatant. The two peptides were similar but not identical with filamin and spectrin in terms of electrophoretic mobility. Hence, the pair of polypeptides was identified as an actin-binding protein newly found in brain.     

Developmental changes in protein composition and the actin-binding protein ponticulin in Dictyostelium discoideum plasma membranes purified by an improved method

We have used a new combination of previously-described methods to obtain a 29-fold purification of plasma membranes from Dictyostelium discoideum. In this procedure, the pellet from a cell lysate is centrifuged through a high-pH sucrose gradient and then through a Renografin gradient. Electron microscopy shows that the resultant "Renografin membranes" are essentially homogeneous. As measured by enzymatic marker assays, contamination with mitochondria, lysosomes, and endoplasmic reticulum is minimal. As assayed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the protein composition of Renografin membranes is similar to that of highly purified membranes isolated using concanavalin A stabilization and detergent extraction. Using Renografin membranes, we have examined developmental changes in the membrane protein composition. In agreement with previous investigations, we observe major changes in lectin-binding glycoproteins and cell-surface-labeled proteins during the first 18 h of D. discoideum development. In contrast to most previous work, which may have employed plasma membranes of lesser purity, we also observe major changes in silver-stained membrane proteins. We conclude that many developmentally regulated proteins, previously thought to be minor membrane constituents, are a larger proportion of the plasma membrane than originally believed. The observed changes in membrane protein composition may correlate with changes in plasma membrane functions during development. For instance, ponticulin, the major salt-sensitive F-actin-binding protein in plasma membranes from vegetative cells, increases at least twofold in plasma membranes during early development when the cells are chemotaxing into large aggregates. The amount of plasma membrane ponticulin then decreases during the pseudoplasmodial stage.     

Shuttling of G protein subunits between the plasma membrane and intracellular membranes

Heterotrimeric G proteins (alphabetagamma) mediate the majority of signaling pathways in mammalian cells. It is long held that G protein function is localized to the plasma membrane. Here we examined the spatiotemporal dynamics of G protein localization using fluorescence recovery after photobleaching, fluorescence loss in photobleaching, and a photoswitchable fluorescent protein, Dronpa. Unexpectedly, G protein subunits shuttle rapidly (t1/2 < 1 min) between the plasma membrane and intracellular membranes. We show that consistent with such shuttling, G proteins constitutively reside in endomembranes. Furthermore, we show that shuttling is inhibited by 2-bromopalmitate. Thus, contrary to present thought, G proteins do not reside permanently on the plasma membrane but are constantly testing the cytoplasmic surfaces of the plasma membrane and endomembranes to maintain G protein pools in intracellular membranes to establish direct communication between receptors and endomembranes.     

Plasma membrane coat and a coated vesicle-associated reticulum of membranes: their structure and possible interrelationship in Chara corallina

Primary fixation with buffered glutaraldehyde plus 2.0 mM CaCl2 and 0.1% tannic acid results in the preservation of certain portions of the plasma membrane coat of Chara when seen with the electron microscope. Such a coat is not observable after fixation with glutaraldehyde alone. The coat appears to be present on all the above ground, vegetative cells of the male plant. Within complex invaginations of the plasma membrane, which are known as charasomes, the coat has two structural components, a central core that is either tubular or solid and a fibrous or granular peripheral region that surrounds the core. The coat material appears to be at least partially derived, via exocytosis, from the contents of single membrane-bound organelles known as glycosomes. Glycosomes seem to originate from within an assemblage of membranes and coated vesicles that can be described, in purely structural terms, as a partially coated reticulum. Such a reticulum is distinguishable from Golgi stacks because the reticulum (a) is not composed of stacked membranes, (b) is extensively involved with large, clearly detailed coated vesicles and coated invaginations, (c) is closely associated with glycosomes, and (d) is only slightly stained by the zinc-iodide- osmium tetraoxide reagent.     

PIGOK: Linking protein identity to gene ontology and function

Here we introduce a computer database that allows for the rapid retrieval of physicochemical properties, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes information about a protein or a list of proteins. We applied PIGOK analyzing Schizosaccharomyces pombe proteins displaying differential expression under oxidative stress and identified their biological functions and pathways. The database is available on the Internet at http://pc4-133.ludwig.ucl.ac.uk/pigok.html.     

Purification and expression of gCap39. An intracellular and secreted Ca2(+)-dependent actin-binding protein enriched in mononuclear phagocytes

A protein of approximately 40 kDa was the major Ca2(+)-binding protein purified by Ca2(+)-dependent hydrophobic affinity chromatography from the cell lysates and conditioned media of RAW macrophages. Other Ca2(+)-binding proteins, including several annexins (calelectrins), S100-like proteins, and calmodulin, were less abundant and preferentially found in the cell lysates. Amino acid sequences of tryptic fragments from the purified 40-kDa protein revealed its identity to gCap39, an actin-binding protein encoded by a cDNA isolated on the basis of its homology with gelsolin. When an expression vector containing the gCap39 coding region was transfected into COS cells, high levels of gCap39 were found in both the cells and conditioned media, whereas annexins were only present in the cells. gCap39 could also be purified from human plasma where it appeared to be a minor component. No signal sequence was detected in the primary structure of gCap39 and the secreted and intracellular forms of gCap39 are of identical size, suggesting that unlike gelsolin, the mechanism of gCap39 secretion may not depend on a signal sequence. The high concentration of gCap39 in macrophages and its constitutive secretion as well as intracellular retention suggest that this protein may have a dual role in macrophage function, namely that of a Ca2(+)- and polyphosphoinositide-regulated intracellular modulator of the cytoskeleton as well as that of a secreted protein involved in the clearance of actin from the extracellular environment.     

Calcium-calmodulin suppresses the filamentous actin-binding activity of a 135-kilodalton actin-bundling protein isolated from lily pollen tubes

We have isolated a 135-kD actin-bundling protein (P-135-ABP) from lily (Lilium longiflorum) pollen tubes and have shown that this protein is responsible for bundling actin filaments in lily pollen tubes (E. Yokota, K. Takahara, T. Shimmen [1998] Plant Physiol 116: 1421-1429). However, only a few thin actin-filament bundles are present in random orientation in the tip region of pollen tubes, where high concentrations of Ca(2+) have also been found. To elucidate the molecular mechanism for the temporal and spatial regulation of actin-filament organization in the tip region of pollen tubes, we explored the possible presence of factors modulating the filamentous actin (F-actin)-binding activity of P-135-ABP. The F-actin-binding activity of P-135-ABP in vitro was appreciably reduced by Ca(2+) and calmodulin (CaM), although neither Ca(2+) alone nor CaM in the presence of low concentrations of Ca(2+) affects the activity of P-135-ABP. A micromolar order of Ca(2+) and CaM were needed to induce the inhibition of the binding activity of P-135-ABP to F-actin. An antagonist for CaM, W-7, cancelled this inhibition. W-5 also alleviated the inhibition effect of Ca(2+)-CaM, however, more weakly than W-7. These results suggest the specific interaction of P-135-ABP with Ca(2+)-CaM. In the presence of both Ca(2+) and CaM, P-135-ABP organized F-actin into thin bundles, instead of the thick bundles observed in the absence of CaM. These results suggest that the inhibition of the P-135-ABP activity by Ca(2+)-CaM is an important regulatory mechanism for organizing actin filaments in the tip region of lily pollen tubes.     

The retinitis pigmentosa-mutated RP2 protein exhibits exonuclease activity and translocates to the nucleus in response to DNA damage

Retinitis pigmentosa (RP) is a genetically heterogeneous disease characterized by degeneration of the retina. Mutations in the RP2 gene are linked to the second most frequent form of X-linked retinitis pigmentosa. RP2 is a plasma membrane-associated protein of unknown function. The N-terminal domain of RP2 shares amino acid sequence similarity to the tubulin-specific chaperone protein co-factor C. The C-terminus consists of a domain with similarity to nucleoside diphosphate kinases (NDKs). Human NDK1, in addition to its role in providing nucleoside triphosphates, has recently been described as a 3' to 5' exonuclease. Here, we show that RP2 is a DNA-binding protein that exhibits exonuclease activity, with a preference for single-stranded or nicked DNA substrates that occur as intermediates of base excision repair pathways. Furthermore, we show that RP2 undergoes re-localization into the nucleus upon treatment of cells with DNA damaging agents inducing oxidative stress, most notably solar simulated light and UVA radiation. The data suggest that RP2 may have previously unrecognized roles as a DNA damage response factor and 3' to 5' exonuclease.