Microtubules and coated vesicles in guard-cell protoplasts ofAllium cepa L.

Protoplasts were prepared from the guard cells ofA. cepa. Epidermal peels taken from expanding green leaves and largely free of mesophyll were treated with Cellulysin, and protoplasts were harvested after 18 h of digestion. That the protoplasts were derived from guard cells was ascertained from their characteristic vacuolar autofluorescence and from observations showing that all other epidermal cells are killed in the peeling procedure. The protoplasts proved to be a good system with which to view the cell cortex and inner surface of the plasmalemma. The lysis of cells adhering to polylysine-treated, Formvar-coated grids, followed by negative staining in uranyl acetate, showed that many microtubules normally present in ordered arrays in situ remain closely applied to the inner surface of the plasmalemma in protoplasts. In addition, numerous vesiculate elements including coated vesicles and/or pits are present amongst the microtubules. Similar vesicles are evident in thin sections of fixed, embedded guard cells and protoplasts. The significance of these structures in the cell cortex is discussed.     

THE SURFACE CHARGE OF RAT LIVER MITOCHONDRIA AND THEIR MEMBRANES : Clarification of Some Controversies Concerning Mitochondrial Structure

The surface charge of intact mitochondria and submitochondrial particles was examined by the technique of preparative free flow electrophoresis. When submitochondrial preparations obtained by a swelling-contraction procedure were examined with this technique, two fractions were observed. One of these fractions exhibited the same electrophoretic properties as intact mitochondria, which indicated that it was derived from the outer limiting membrane of the mitochondrion. This fraction was found to contain the enzymes monoamine oxidase and rotenone-insensitive NADH-cytochrome c reductase which have been reported to be localized in the outer mitochondrial membrane. The other fraction exhibited an electrophoretic mobility which was different from that of intact mitochondria, and this fraction contained enzymes characteristic of the inner membrane-matrix fraction such as soluble and particulate enzymes of the Krebs cycle. Microsomes exhibited an electrophoretic mobility which was almost identical with that of the outer mitochondrial membrane. In addition to resolving the localization of enzymes in mitochondrial membranes, these data indicate that the outer limiting membrane of the mitochondrion is the sole determinant of the surface charge of mitochondria.     

Interaction of lactoferrin and transferrins with the outer membrane of Bordetella pertussis

Bordetella pertussis was able to grow in vitro under conditions where the only iron present was bound to the iron-binding proteins ovotransferrin, transferrin or lactoferrin. Under these conditions the bacteria produced neither hydroxamate nor phenolate-catecholate siderophores to assist in the procurement of iron. Examination of B. pertussis outer-membrane preparations by SDS-PAGE and immunoblotting showed that the iron-binding protein ovotransferrin was bound directly to the bacterial surface. Assays of the binding of radiolabelled transferrin by the bacteria showed that the association was a specific process and that there was turnover of the bound proteins. Competitive binding assays indicated that lactoferrin could be bound in the same way. It is suggested that B. pertussis obtains iron directly from host iron-binding proteins during infection.     

Siderophore-Mediated Utilization of Iron Bound to Transferrin by Vibrio parahaemolyticus

Vibrio parahaemolyticus produces a structurally novel type of siderophore, termed vibrioferrin, in response to iron-limitation. This study was performed to examine whether vibrioferrin can assimilate iron from human iron-binding proteins for growth. Comparison of the growth rates between V. parahaemolyticus AQ 3354 and its spontaneously arising, vibrioferrin-deficient mutant revealed that vibrioferrin was able to sequester iron from 30% iron-saturated human transferrin for growth, but not from human lactoferrin even if fully saturated with iron. In both strains, iron limitation induced two high-molecular-weight outer membrane proteins with apparent molecular masses of approximately 78 and 83 kDa. Since only the outer membrane fraction including these proteins showed a binding capacity to ferric vibrioferrin complex, either of them may function as its cell surface receptor. These results suggested that the organism might utilize such a source of host iron through the action of vibrioferrin during in vivo survival and proliferation, although its importance in pathogenesis is unknown.     

Effects of iron deficiency on iron binding and internalization into acidic vacuoles in Dunaliella salina

Uptake of iron in the halotolerant alga Dunaliella salina is mediated by a transferrin-like protein (TTf), which binds and internalizes Fe(3+) ions. Recently, we found that iron deficiency induces a large enhancement of iron binding, which is associated with accumulation of three other plasma membrane proteins that associate with TTf. In this study, we characterized the kinetic properties of iron binding and internalization and identified the site of iron internalization. Iron deficiency induces a 4-fold increase in Fe binding, but only 50% enhancement in the rate of iron uptake and also increases the affinity for iron and bicarbonate, a coligand for iron binding. These results indicate that iron deprivation leads to accumulation and modification of iron-binding sites. Iron uptake in iron-sufficient cells is preceded by an apparent time lag, resulting from prebound iron, which can be eliminated by unloading iron-binding sites. Iron is tightly bound to surface-exposed sites and hardly exchanges with medium iron. All bound iron is subsequently internalized. Accumulation of iron inhibits further iron binding and internalization. The vacuolar inhibitor bafilomycin inhibits iron uptake and internalization. Internalized iron was localized by electron microscopy within vacuolar structures that were identified as acidic vacuoles. Iron internalization is accompanied by endocytosis of surface proteins into these acidic vacuoles. A novel kinetic mechanism for iron uptake is proposed, which includes two pools of bound/compartmentalized iron separated by a rate-limiting internalization stage. The major parameter that is modulated by iron deficiency is the iron-binding capacity. We propose that excessive iron binding in iron-deficient cells serves as a temporary reservoir for iron that is subsequently internalized. This mechanism is particularly suitable for organisms that are exposed to large fluctuations in iron availability.     

Mechanisms and regulation of iron and heme utilization in Gram-negative bacteria

Iron and heme are essential nutrients for most pathogenic microorganisms and play a pivotal role in microbial pathogenesis. To survive within the iron-limited environment of the host, bacteria utilize iron-siderophore complexes, iron-binding proteins (transferrin, lactoferrin), free heme and heme bound to hemoproteins (hemoglobin, haptoglobin, hemopexin). A mechanism of iron and heme transport depends on the structures of Gram-negative bacterial membranes. Siderophores, hemophores and outer membrane receptors take part in iron or heme binding. The transport of these ligands across the outer membrane involves outer membrane receptors. The energy for this transport is delivered from the inner membrane by a TonB-ExbB-ExbD complex. The transport across the cytoplasmic membrane involves periplasmic and inner membrane proteins comprising the ABC systems, which utilize the energy derived from ATP hydrolysis. The major regulatory role in iron homeostasis plays a Fur-Fe2+ repressor.     

Lactoferrin receptors in Gram-negative bacteria: Insights into the iron acquisition process

One component of the anti-microbial function of lactoferrin (Lf) is its ability to sequester iron from potential pathogens. To overcome this iron limitation, a number of gram-negative bacterial pathogens have developed a mechanism for acquiring iron directly from this host glycoprotein. This mechanism involves surface receptors capable of specifically binding Lf from the host, removing iron and transporting it across the outer membrane. The iron is then bound by a periplasmic iron-binding protein, FbpA, and transported into the cell via an inner membrane complex comprised of FbpB and FbpC. The receptor has been shown to consist of two proteins, LbpA and LbpB. LbpB is bilobed lipoprotein anchored to the outer membrane via fatty acyl groups attached to the N-terminal cysteine. LbpA is a homologue of siderophore receptors, which consist of an N-terminal plug and a C-terminal beta-barrel region. We propose that the receptor proteins, LbpA and LbpB, induce conformational changes in human Lf (hLf) that lower its affinity for iron that binding by FbpA can drive the transport across the outer membrane, a mechanism shared with transferrin (Tf) receptors. The interaction between the receptor proteins and Lf is quite extensive and has been previously studied by using chimeric proteins comprised of Lf & Tf. In an attempt to evaluate the role of FbpA in the transport process, a series of site-directed mutants of FbpA were prepared and used to replace the wild-type protein in the iron acquisition pathway. The mutations were made in the iron-binding and anion-binding ligands of FbpA and were designed to result in altered binding properties. Protein crystallography of the iron-bound form of the Q58L mutant protein revealed that it was in the open conformation with iron coordinated by Y195 and Y196 from the C-terminal domain but not by the other iron-liganding amino acids from the N-terminal domain, H9 and E57. Replacement of the native FbpA in Neisseria meningitidis with wild-type or mutant Haemophilus influenzae FbpAs resulted in a defect in growth on Tf or Lf, suggesting that there may be a barrier to functional expression of H. influenzae FbpAs in Neisseria meningitidis. Thus mutants of the N. meningitidis FbpA are being prepared to replace wild-type protein in order to test their ability to mediate transport from hLf.     

Characterization of monoclonal antibodies to protoplast membranes of Nicotiana tabacum identified by an enzyme-linked immunosorbent assay

Murine monoclonal antibodies to protoplast membrne antigens were generated using mouse myelomas and spleen cells from mice immunized with Nicotiana tabacum L. leaf protoplasts. For selecting antibody-secreting clones, a sensitive and rapid enzyme-linked immunosorbent assay (ELISA) for monoclonal antibody binding to immobilized cellular membrane preparations or immobilized protoplasts was developed. With intact protoplasts as immobilized antigen, the ELISA is selective for antibodies that bind to plasma-membrane epitopes present on the external surface of protoplasts. Using the membrane ELISA, a total of 24 hybridoma lines were identified that secreted antibodies to plant membrane epitopes. The protoplast ELISA and subsequent immunofluorescence studies identified four hybridoma lines as secreting antibodies which bound to the external surface of protoplasts and cells. The corresponding antigens were not species- or tissue-specific, were periodatesensitive, and were located in membranes which equilibrated broadly throughout a linear sucrose gradient. When protein blots of electrophoretically separated membrane proteins were probed with these antibodies, a band of M_r 14 kilodaltons (kDa) and a smear of bands of M_r 45–120 kDa were labeled. An additional set of three antibodies appeared by immunofluorescence to bind to the plasma membrane of broken but not intact protoplasts and labeled membranes equilibrating at a density of approx. 1.12 kg·l^-1 in a linear sucrose density gradient. These classes of monoclonal antibodies enlarge the library of monoclonal antibodies (Norman et al. 1986, Planta 167, 452–459) available for the study of plant plasma-membrane structure and function.Abbreviations ELISA Enzyme-linked immunosorbent assay - Ig immunoglobulin - kDa kilodalton - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Involvement of outer membrane proteins in enterochelin-mediated iron uptake in Escherichia coli.

Escherichia coli K-12 grown in iron-deficient media contained a large amount of outer membrane proteins O-2a, O-2b, and O-3, while cells grown in iron-supplemented media contained far smaller amounts of these proteins. The iron uptake by the iron-deficient cells was significantly stimulated in the presence of enterochelin, while that by the iron-rich cells was not. The outer membrane isolated from cells grown in the iron-deficient media showed enterochelin-stimulated binding of iron, while the outer membrane from iron-rich cells and cytoplasmic membranes from both types of cells did not show such binding activity. The amount of iron bound by the outer membrane was almost equivalent to the amount of O-2a, O2b, or O-3, irrespective of the amount of these proteins in the outer membrane, which is controlled by the amount of iron in the medium. Small particles rich in these proteins were prepared from cells by EDTA extraction. The particles were active in enterochelin-mediated iron binding and the amount of iron bound was equivalent to the amount of each of these proteins in the particles. Although the outer membrane of E. coli B was as active in iron binding as that of E. coli K-12, it did not possess an appreciable amount of O-2a. Gel electrophoretic analysis revealed that 9-2b and 9-3 were identical with the proteins missing mutants feuB and feuA, respectively.     

Fine-structural and chemical analyses on inner and outer sheath of the cyanobacteriumGloeothece sp. PCC 6909

Cells of the unicellular cyanobacteriumGloeothece sp. PCC 6909 are surrounded by an inner (enclosing 1–2 cells) and an outer (enclosing cell groups) sheath. Using conventional Epon-embedding in combination with ruthenium-red staining, the inner and outer sheaths appeared similar and displayed multiple bands of electron-dense subunits. However, embedding in Nanoplast resin to avoid shrinkage led to the detection of two distinct zones (inner and outer zone) each with several distinct layers. The zone delimited by the electron-dense thick inner sheath layer, and the zone enclosed by the thin electron-dense outer sheath layer, are composed of a homogeneous material of little electron-contrast. Whereas the outer zone appears to be of even contrast, the inner zone is characterized by a distinct electron-transparent layer. Element distribution analysis revealed that the electron-transparent layer contained relatively large amounts of sulfur, carbon, and oxygen but only little nitrogen.Inner and outer sheath fractions were isolated by differential mechanical cell breakage and centrifugation. The outer sheath fraction was less hydrated than the inner one. The two fractions differed little in their contents of uronic acids, carbohydrate and protein, although the outer sheath fraction contained less sulfate. A soluble polysaccharide with a chemical composition similar to that of inner and outer sheath fractions was also obtained from the culture supernatant.     

Analysis of binding of biotinylated protoplast-release-inducing protein that induces release of gametic protoplasts in the Closterium peracerosum-strigosum-littorale complex

A protoplast-release-inducing protein (PR-IP) which is released from mating-type plus (mt⁺) cells and induces the release of gametic protoplasts from matingtype minus (mt⁻) cells of Closterium was biotinylated and then used to examine the interaction of this protein with mt⁻ cells. The protoplast-release-inducing activity of PR-IP was not altered after the biotinylation. When mt⁻ cells that had been pre-cultured for 24 h were incubated with biotinylated PR-IP for 6 h in nitrogen-deficient medium that contained 1% (w/v) bovine serum albumin, and then washed with the same medium, only a 19-kDa polypeptide, the smaller subunit of PR-IP, was detected in cells by the avidin and biotinylated horseradish-peroxidase macromolecular complex system. The amount of bound 19-kDa polypeptide increased with increasing doses of PR-IP and reached a maximum at around 10 nM, reflecting the protoplast-release-inducing activity. From a Scatchard plot, the dissociation constant of the polypeptide was calculated to be 2.7 · 10⁻⁸ M. The binding of the polypeptide proceeded only after an appropriate period of pre-culture in the light, and the polypeptide was competitively displaced by non-biotinylated PR-IP. From these results, it appears that the PR-IP induces the release of protoplasts from mt⁻ cells by binding of a polypeptide of relative molecular mass 19000 to the receptor on the cell surface in a manner analogous to the binding of peptide hormones in animals.     

Localization of Acid and Alkaline Phosphatases in Staphylococcus aureus

The localization of acid and alkaline phosphatases in Staphylococcus aureus was studied by fractionation of cells after treatment with the L-11 enzyme and by electron microscopic histochemistry. The two enzyme activities were located in distinctly different positions at the surface of the cells. Acid phosphatase appeared to be localized around the cell membrane of the bacteria, because the enzyme was recovered exclusively in the membrane fraction and because deposition of lead phosphate was detected by electron microscopic histochemistry on the inner surface of the cell membrane of intact bacteria and spheroplasts. The highest specific activity of alkaline phosphatase was also associated with the membrane fraction. However, on electron microscopic histochemistry of intact cells, the deposition of lead phosphate was only seen on the outer surface of the cell wall.     

Monoclonal antibodies directed against protoplasts of soybean cells

Splenocytes, derived from mice that had been immunized with protoplasts prepared from suspension cultures of root cells of Glycine max (L.) Merr. (SB-1 cell line), were fused with a murine myeloma cell line. The resulting hybridoma cultures were screened for the production of antibodies directed against the soybean protoplasts and were then cloned. One monoclonal antibody, designated MVS-1, was found to bind to the outer surface of the plasma membrane on the basis of several criteria: (a) agglutination of the protoplasts; (b) binding of fluorescence-labeled immunoglobulin on protoplasts yielding a ring staining pattern with prominent intensity at the edges; and (c) saturable binding by protoplasts of ¹²⁵I-labeled Antibody MVS-1. The antigenic target of Antibody MVS-1, identified by immunoblotting techniques, contained a polypeptide of relative molecular mass (M_r) approx. 400000 under both reducing and non-reducing conditions. When the antigenic target of Antibody MVS-1 was chromatographed in potassium phosphate buffer, the position of elution corresponded to that of a high-molecular-weight species (M_r 400000). These results provide the protein characterization required for the analysis of the mobility of Antibody MVS-1 bound to the plasma membrane of SB-1 cells.Abbreviations D diffusion coefficient - M_r relative molecular mass - PBS phosphate-buffered saline (8.00 g NaCl, 1.15 g Na₂HPO₄, 0.20 g NaH₂PO₄ per 1 L, pH 7.2) - TPBS phosphate-buffered saline containing 0.5% Tween-20 - TX-100, TX-114 Triton X-100, X-114 - SDS sodium dodecyl sulfate     

Cell cycle related changes in the quantity of TMV virions bound to protoplasts ofNicotiana sylvestris

Summary Attachment of virions of tobacco mosaic virus to protoplasts isolated from dividing suspension cultured cells ofNicotiana sylvestris was estimated using quantitative autoradiography of individual protoplasts. Additionally, the position of each protoplast in the cell cycle was assessed by Feulgen microspectrophotometry. At pH 5.6, after preincubation with 4 g 1^–1 poly-L-ornithine, protoplasts in the G₁ and G₂ phases bound more virions than protoplasts in the S-phase. The possibility that such differential binding was caused by cyclical variation in the net charge on the protoplast membrane has been investigated. It was found that S-phase protoplasts ofN. sylvestris can be separarated from protoplasts of other cycle stages by partition in aqueous, two-phase, immiscible polymer systems, presumably because they differ in charge. Also, electrophoretic studies suggest that G₁ phase protoplasts bear higher surface charge than some non-G₁ protoplasts.     

Physicochemical roles of soluble metal cations in the outer membrane of Escherichia coli K-12

Atomic absorption spectroscopy of isolated native and EDTA-modified (lipopolysaccharide-depleted) outer membrane revealed trace amounts of potassium, manganese, and iron (1.0-7.0 nmol/mg dry weight outer membrane). Sodium, magnesium, and calcium were approximately one order of magnitude more plentiful, but EDTA-modified outer membrane was deficient in calcium. When metal-binding assays were conducted to find the binding capacity of native and EDTA-modified outer membrane, potassium bound poorly compared with sodium. However, there was no difference in the binding of these ions between the OM preparations. In contrast, reduced amounts of magnesium, calcium, manganese, and iron III bound to the EDTA-modified OM. Partitioning of intact cells in a biphasic dextran-polyethyleneglycol system indicated that the reduced lipopolysaccharide content of the EDTA-modified outer membrane increased the hydrophobicity of the cell surface. Exposure of control and EDTA-treated cells to divalent metal salt solutions before phase partitioning also increased cell surface hydrophobicity. Freeze-etching showed that sodium ions had no effect on the membrane fractures observed in control cells, but with EDTA-treated cells, this cation increased the occurrence of small outer membrane fractures (plateaus) which are characteristic of EDTA treatment. Both magnesium and manganese increased the frequency of outer membrane cleavage in control cells, whereas calcium did not. In contrast, all three divalent metallic ions increased the frequency and extent of cleavage in the outer membrane of EDTA-treated cells.     

Concanavalin A binding sites on the plasma membrane of leek stem protoplasts

The binding of concanavalin A (con A) to leek (Allium porrum L.) stem protoplasts has been investigated using sequential treatment with con A and haemocyanin and using con A covalently linked to ferritin. Prefixed protoplasts were evenly labelled. Unfixed protoplasts showed a clustered distribution of label. Low temperature and lanthanum reduced the clustering. Bound con A was lost from unfixed protoplasts incubated for 5 h after treatment, but con A/haemocyanin was not bound to nascent wall materials. Prefixed protoplasts treated with wall-removing enzymes before labelling showed only a small reduction of con A binding. These results indicate that con A is bound to plasma membrane components, but that binding is reduced by competition of nascent wall precursors.Abbreviations con A concanavalin A - con A-H sequential treatment with con A and haemocyanin - con A-F con A covalently linked to ferritin     

Specific binding of a Verticillium dahliae phytotoxin to protoplasts of cotton, Gossypium hirsutum

Summary The occurrence of specific, high-affinity binding sites for a protein-lipopolysaccharide (PLP) phytotoxin purified from culture filtrates of a virulent Vertidllium dahliae isolate has been demonstrated in cotton protoplasts. Binding of the ¹²⁵I-radiolabelled PLP-complex to protoplasts from cotyledon tissue was saturable and with an affinity (K_d = 17.3 nM) comparable with the concentration required for biological activity. A single class of binding site, accessible at the surface of the intact protoplasts, was found and the maximal number of binding sites were estimated as 2.41 × 10^–16 moles per protoplast. The binding affinity to protoplasts proved near identical to that found with purified plasma membrane fractions from roots. When cultivars exhibiting resistance or susceptibility towards the pathogen were compared, no significant differences were found in the affinity of binding, but five times as many binding sites per protoplast and sixteen times as many binding sites per mg membrane protein were found in the resistant cultivar.Abbreviations PLP protein-lipopolysaccharide - k_d dissociation constant - B_max maximal number of binding sites - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

The role of the synergistic phosphate anion in iron transport by the periplasmic iron-binding protein from Haemophilus influenzae

The acquisition of iron from transferrin by Gram-negative bacterial pathogens is dependent on a periplasmic ferric-ion-binding protein, FbpA. FbpA shuttles iron from the outer membrane to an inner membrane transport complex. A bound phosphate anion completes the iron co-ordination shell of FbpA and kinetic studies demonstrate that the anion plays a critical role in iron binding and release in vitro. The present study was initiated to directly address the hypothesis that the synergistic anion is required for transport of iron in intact cells. A series of site-directed mutants in the anion-binding amino acids of the Haemophilus influenzae FbpA (Gln-58, Asn-175 and Asn-193) were prepared to provide proteins defective in binding of the phosphate anion. Crystal structures of various mutants have revealed that alteration of the C-terminal domain ligands (Asn-175 or Asn-193) but not the N-terminal domain ligand (Gln-58) abrogated binding of the phosphate anion. The mutant proteins were introduced into H. influenzae to evaluate their ability to mediate iron transport. All of the single site-directed mutants (Q58L, N175L and N193L) were capable of mediating iron acquisition from transferrin and from limiting concentrations of ferric citrate. The results suggest that the transport of iron by FbpA is not dependent on binding of phosphate in the synergistic anion-binding site.     

Surface coat transformation and capsule formation by Leuconostoc mesenteroides NCDO 523 in the presence of sucrose

When Leuconostoc mesenteroides NCDO 523 was grown in MRS browth, electron microscopy of cells fixed in the presence of ruthenium red showed that the cell wall was covered with a thin layer of filamentous material. When MRS-grown cells were resuspended in the same medium supplemented with 3.6% sucrose, this surface coat doubled in thickness and a number of radial thickenings appeared within it. After 3 h the filamentous component of the surface coat had disappeared leaving only the radial projections. The progressive accumulation of polymer to produce a capsule visible by light microscopy was observed in only about 20% of the population. In this minority of cells, a dense globular dextran composed of fibrillar and particulate elements was always produced in the initial stages of synthesis. After 18 h, the dextran capsule was generally composed of an inner globular and outer fibrillar layer. It appeared that the outer layer was derived from the globular dextran of the capsule by a process of dispersion.     

Reduction of extracellular potassium ferricyanide by transmembrane NADH: (acceptor) oxidoreductase of human erythrocytes

Reduction of extracellular ferricyanide by intact erythrocytes proceeds by a membrane bound, NADH-dependent reaction. It is depressed by a glycolysis inhibitor and a non penetrable sulfhydryl reagent, and activated by dehydroascorbate. Dehydroascorbate activation cannot be accounted for by release of reducing equivalents from the cells. It is concluded that the observed reaction is brought about by transmembrane NADH-acceptor oxidoreductase with donor binding at the inner and acceptor binding at the outer cell surface.