Secretion of Cryparin, a Fungal Hydrophobin

Cryparin is a cell-surface-associated hydrophobin of the filamentous ascomycete Cryphonectria parasitica. This protein contains a signal peptide that directs it to the vesicle-mediated secretory pathway. We detected a glycosylated form of cryparin in a secretory vesicle fraction, but secreted forms of this protein are not glycosylated. This glycosylation occurred in the proprotein region, which is cleaved during maturation by a Kex2-like serine protease, leaving a mature form of cryparin that could be isolated from both the cell wall and culture medium. Pulse-chase labeling experiments showed that cryparin was secreted through the cell wall, without being bound, into the culture medium. The secreted protein then binds to the cell walls of C. parasitica, where it remains. Binding of cryparin to the cell wall occurred in submerged culture, presumably because of the lectin-like properties unique to this hydrophobin. Thus, the binding of this hydrophobin to the cell wall is different from that of other hydrophobins which are reported to require a hydrophobic-hydrophilic interface for assembly.     

Secretion of cryparin, a fungal hydrophobin

Cryparin is a cell-surface-associated hydrophobin of the filamentous ascomycete Cryphonectria parasitica. This protein contains a signal peptide that directs it to the vesicle-mediated secretory pathway. We detected a glycosylated form of cryparin in a secretory vesicle fraction, but secreted forms of this protein are not glycosylated. This glycosylation occurred in the proprotein region, which is cleaved during maturation by a Kex2-like serine protease, leaving a mature form of cryparin that could be isolated from both the cell wall and culture medium. Pulse-chase labeling experiments showed that cryparin was secreted through the cell wall, without being bound, into the culture medium. The secreted protein then binds to the cell walls of C. parasitica, where it remains. Binding of cryparin to the cell wall occurred in submerged culture, presumably because of the lectin-like properties unique to this hydrophobin. Thus, the binding of this hydrophobin to the cell wall is different from that of other hydrophobins which are reported to require a hydrophobic-hydrophilic interface for assembly.     

Effect of Inhibition of Deoxyribonucleic Acid and Protein Synthesis on the Direction of Cell Wall Growth in Streptococcus faecalis

Selective inhibition of protein synthesis in Streptococcus faecalis (ATCC 9790) was accompanied by a rapid and severe inhibition of cell division and a reduction of enlargement of cellular surface area. Continued synthesis of cell wall polymers resulted in rapid thickening of the wall to an extent not seen in exponential-phase populations. Thus, the normal direction of wall growth was changed from a preferential feeding out of new wall surface to that of thickening existing cell surfaces. However, the overall manner in which the wall thickened, from nascent septa toward polar regions, was the same in both exponential-phase and inhibited populations. In contrast, selective inhibition of deoxyribonucleic acid (DNA) synthesis using mitomycin C was accompanied by an increase in cellular surface area and by division of about 80% of the cells in random populations. Little or no wall thickening was observed until the synthesis of macromolecules other than DNA was impaired and further cell division ceased. Concomitant inhibition of both DNA and protein synthesis inhibited cell division but permitted an increase in average cell volume. In such doubly inhibited cells, walls thickened less than in cells inhibited for protein synthesis only. On the basis of the results obtained, a model for cell surface enlargement and cell division is presented. The model proposes that: (i) each wall enlargement site is influenced by an individual chromosome replication cycle; (ii) during chromosome replication peripheral surface enlargement would be favored over thickening (or septation); (iii) a signal associated with chromosome termination would favor thickening (and septation) at the expense of surface enlargement; and (iv) a factor or signal related to protein synthesis would be required for one or more of the near terminal stages of cell division or cell separation, or both.     

Peptide Surface Display and Secretion Using Two LPXTG-Containing Surface Proteins from Lactobacillus fermentum BR11

A locus encoding two repetitive proteins that have LPXTG cell wall anchoring signals from Lactobacillus fermentum BR11 has been identified by using an antiserum raised against whole L. fermentum BR11 cells. The first protein, Rlp, is similar to the Rib surface protein from Streptococcus agalactiae, while the other protein, Mlp, is similar to the mucus binding protein Mub from Lactobacillus reuteri. It was shown that multiple copies of mlp exist in the genome of L. fermentum BR11. Regions of Rlp, Mlp, and the previously characterized surface protein BspA were used to surface display or secrete heterologous peptides in L. fermentum. The peptides tested were 10 amino acids of the human cystic fibrosis transmembrane regulator protein and a six-histidine epitope (His₆). The BspA promoter and secretion signal were used in combination with the Rlp cell wall sorting signal to express, export, and covalently anchor the heterologous peptides to the cell wall. Detection of the cell surface protein fusions revealed that Rlp was a significantly better surface display vector than BspA despite having lower cellular levels (0.7 mg per liter for the Rlp fusion compared with 4 mg per liter for the BspA fusion). The mlp promoter and encoded secretion signal were used to express and export large (328-kDa at 10 mg per liter) and small (27-kDa at 0.06 mg per liter) amino-terminal fragments of the Mlp protein fused to the His₆ and CFTR peptides or His₆ peptide, respectively. Therefore, these newly described proteins from L. fermentum BR11 have potential as protein production and targeting vectors.     

Apparent Inhibition of beta-Fructosidase Secretion by Tunicamycin May Be Explained by Breakdown of the Unglycosylated Protein during Secretion

Suspension-cultured carrot (Daucus carota) cells synthesize and secrete β-fructosidase, a glycoprotein with asparagine-linked glycans. Treatment of the cells with tunicamycin completely inhibits the apparent secretion of β-fructosidase as measured by the accumulation of the radioactive protein in the cell wall or the culture medium. In the past, such a result has been interpreted as an inhibition of secretion by tunicamycin, but we suggest another explanation based on the following results. In the presence of tunicamycin, unglycosylated β-fructosidase is synthesized and is associated with an endoplasmic-reticulum-rich microsomal fraction. Pulse-chase experiments show that the unglycosylated β-fructosidase does not remain in the cells and appears to be secreted in the same way as glycosylated β-fructosidase; however, no radioactive, unglycosylated β-fructosidase accumulates extracellularly (cell wall or medium). Protoplasts obtained from carrot cells secrete β-fructosidase protein and activity, and treatment of the protoplasts with tunicamycin results in the synthesis of unglycosylated β-fructosidase. In the presence of tunicamycin, there is no accumulation of β-fructosidase activity or unglycosylated β-fructosidase polypeptide in the protoplast incubation medium. These results are consistent with the interpretation that the glycans of β-fructosidase are necessary for its stability, and that in these suspension-cultured cells, the unglycosylated enzyme is degraded during the last stage(s) of secretion, or immediately after its arrival in the wall.     

Secretion of a nonspecific lipid transfer protein by hepatoma cells in culture

Nonspecific lipid transfer protein (sterol carrier protein2) has previously been proposed to function as (i) a catalyst for intracellular movement of newly synthesized phospholipid, (ii) a cofactor in the biosynthesis and metabolism of cholesterol, and (iii) a cofactor in the feedback inhibition of cholesterol synthesis. Each of these functions is based upon the premise that nonspecific lipid transfer protein (nsLTP) is cytosolic. However, evidence presented in this report suggests that, at least in the case of cultured hepatoma cells, nsLTP is secreted. This conclusion is supported by three observations. First, after culture of hepatoma cells for 10 h, 88% of the nsLTP (as judged by its phosphatidylethanolamine transfer activity) appears in the medium, whereas the cytosolic level of transfer activity remains unchanged. Furthermore, this is accompanied by the appearance in the medium of a polypeptide of Mr 12,200-12,500, which corresponds to the known molecular weight of nsLTP. Finally, it was observed that the appearance of both the activity and the polypeptide in the medium are inhibited by monensin, an inhibitor of secretion. Thus their appearance seems to represent secretion and not simply leakage from the cells. Further evidence that nsLTP does not play an important role in the cytosolic transport of phospholipid and sterol is provided by our observation that hepatoma cells containing a level of nsLTP only 10-15% of that found in liver nevertheless possess near-normal membrane phospholipid compositions and retain the ability to feedback-inhibit cholesterol biosynthesis.     

Secretion marker proteins and cell-wall polysaccharides move through different secretory pathways

The building up of the cell wall is tightly dependent on the functionality of the secretory pathway. Syntaxins as well as other SNARE proteins play important roles during vesicle secretion and fusion. We have compared the secretion of newly synthesised cell-wall polysaccharides to that of secretory marker proteins such as secreted green-fluorescent protein (secGFP) and secreted rat preputial β-glucuronidase (secRGUS) in leaf protoplasts and roots of wild-type and transgenic Nicotiana tabacum plants, overexpressing a syntaxin homologue NtSyr1 (Sp1) and its soluble variant Sp2 that interferes specifically with Sp1 function, affecting post-Golgi transport. In protoplasts transiently transformed with secGFP and Sp1, no variation was observed in the pattern of fluorescence with respect to control; on the contrary, GFP fluorescence accumulate within the cells in protoplasts co-transformed with secGFP and Sp2. Sp2 reduced the percentage of marker protein secretion to 53% as quantified with secRGUS. In protoplasts obtained from leaves of wild-type and transformed tobacco plants expressing Sp1, Sp2 and Sp1 plus Sp2, no remarkable differences in the percentage of newly synthesised polysaccharides incorporated into the regenerating cell walls were observed. The same results were confirmed in roots of whole transformed seedlings. Tests with cytochalasin D (CD) showed a marked decrease in the amount of newly synthesised polysaccharides into the wall and a simultaneous sharp increase in membrane-associated polysaccharides. SecRGUS secretion was also inhibited by CD. The data indicate that marker proteins and matrix polysaccharides, as well as cellulose synthase complexes, are secreted through the involvement of different secretory machineries. Maria Rosaria Leucci and Gian-Pietro Di Sansebastiano contribute equally to this work. Giuseppe Dalessandro and Gabriella Piro wish to dedicate this work to the memory of Professor Don Northcote.     

Inhibition of cell wall autolysis and auxin-induced elongation of Cicer arietinum epicotyls by β-galactosidase antibodies

Polyclonal antibodies were raised in response to βIII-galactosidase purified from cell wall of Cicer arietinum epicotyls. The antibody preparation generated, bound to βIII protein giving a major protein band in the zone corresponding to M_r 45 000, the molecular mass previously estimated for βIII-galactosidase. These antibodies clearly suppress autolytic reactions in isolated walls of Cicer arietinum epicotyl segments, while the preimmune serum had no effect on autolytic reaction. The results strongly support the idea that the autolytic degradation of the cell wall is carried out by the βIII-galactosidase.
The antibodies against β-galactosidase were also able to inhibit cell wall hydrolysis mediated by both total cell wall protein extracted by LiCl and cell wall hydrolysis mediated by βIII-galactosidase.
Since autolysis is thought to be related to the process of cell wall loosening, the effects of the antibodies against the autolytic enzyme was also tested on epicotyl growth. β-galactosidase antibodies consistently inhibited IAA-induced elongation.     

Bacitracin Inhibits the Synthesis of Lipid-linked Saccharides and Glycoproteins in Plants

The particulate enzyme fraction from mung bean (Phaseolus aureus) seedlings catalyzes the incorporation of mannose from GDP-[¹⁴C]mannose into mannosyl-phosphoryl-dolichol and of N-acetylglucosamine from UDP-[³H]N-acetylglucosamine into N-acetylglucosamine-pyrophosphoryl-polyisoprenol. Bacitracin inhibits the transfer of both of these sugars into the lipid-linked saccharides with 50% inhibition being observed at 5 mm bacitracin. This antibiotic did not inhibit the transfer of glucose from UDP-[¹⁴C]glucose into steryl glucosides or the incorporation of glucose into a cell wall glucan. Bacitracin also inhibited the in vivo incorporation of [¹⁴C]mannose into mannosyl-phosphoryl-dolichol and into glycoprotein by carrot (Daucus carota) slices. While bacitracin also inhibited the incorporation of lysine into proteins by these slices, protein synthesis was less sensitive than glycosylation. Thus at 2 mm bacitracin glycosylation was inhibited 92%, while protein synthesis was inhibited only 50%.     

A Gatekeeper Chaperone Complex Directs Translocator Secretion during Type Three Secretion

Many Gram-negative bacteria use Type Three Secretion Systems (T3SS) to deliver effector proteins into host cells. These protein delivery machines are composed of cytosolic components that recognize substrates and generate the force needed for translocation, the secretion conduit, formed by a needle complex and associated membrane spanning basal body, and translocators that form the pore in the target cell. A defined order of secretion in which needle component proteins are secreted first, followed by translocators, and finally effectors, is necessary for this system to be effective. While the secreted effectors vary significantly between organisms, the ∼20 individual protein components that form the T3SS are conserved in many pathogenic bacteria. One such conserved protein, referred to as either a plug or gatekeeper, is necessary to prevent unregulated effector release and to allow efficient translocator secretion. The mechanism by which translocator secretion is promoted while effector release is inhibited by gatekeepers is unknown. We present the structure of the Chlamydial gatekeeper, CopN, bound to a translocator-specific chaperone. The structure identifies a previously unknown interface between gatekeepers and translocator chaperones and reveals that in the gatekeeper-chaperone complex the canonical translocator-binding groove is free to bind translocators. Structure-based mutagenesis of the homologous complex in Shigella reveals that the gatekeeper-chaperone-translocator complex is essential for translocator secretion and for the ordered secretion of translocators prior to effectors.     

Secretion of Ricinus communis glyceraldehyde-3-phosphate dehydrogenase by Escherichia coli

A plasmid, pWEH1, was constructed containing a fusion of the DNA encoding the signal sequence of the Escherichia coli outer-membrane protein A to the 5'-end of a glyceraldehyde-3-phosphate dehydrogenase cDNA from Ricinus communis. When expressed in E. coli, the fusion protein was secreted by the normal membrane-potential-dependent pathway. Processing by signal peptidase was inhibited by low concentrations of phenethyl alcohol. Quantitative cell fractionation was used to show that the mature plant protein was associated with the bacterial outer membrane. The protein could not be released from the membrane by washing with alkaline sodium carbonate. Radioactivity from [U-14C]-palmitate was incorporated into the heterologous protein. These results suggest that the sequence of this normally cytoplasmic enzyme contains a cryptic lipid-modification site, and the combination of a signal sequence plus a lipid-modification sequence results in specific targeting to the bacterial outer membrane.     

变铅青链霉菌内源线性质粒接合转移必需功能区的鉴定

通常细菌间环型质粒在接合转移过程中,单链质粒DNA在质粒内部“oriT”接合转移起始位点发生缺刻.随后,打开的单链质粒DNA通过细胞膜的Ⅳ型分泌系统转移到受体菌中.但是,链霉菌中的接合型线型质粒带有游离3′端,5′端与末端蛋白结合,因而不能以细胞-细胞间方式转移单链缺刻DNA.报道了变铅青链霉菌线型质粒SLP2衍生的环型质粒,与SLP2一样可以高频高效接合转移,并鉴定了接合转移功能区.质粒有效的接合转移功能区包含6个共转录的基因,分别编码一个Tra样的DNA转移酶、胞壁水解酶、2个膜蛋白(可以与ATP结合蛋白相互作用)和一个功能未知的蛋白质.从SalⅠR-/M-向SalⅠR/M宿主转移的质粒频率下降表明,线型和环型的质粒都是以双链的形式转移的.上述研究结果表明SLP2衍生的线型质粒和环型质粒以相似的与细胞膜/胞壁功能相关的机理进行接合转移.     

Experimental inhibition of cell wall formation and of reversion inNadsonia elongata andSchizosaccharomyces pombe protoplasts

Summary The growth, cell wall regeneration, and the reversion of the protoplasts ofNadsonia elongata andSchizosaccbaromyces pombe cultivated in nutrient media containing snail enzyme was studied by light and electron microscopy. The protoplasts grew in the presence of snail enzyme and an incomplete cell wall composed of fibrils was formed on their surface. Thus, the presence of snail enzyme inhibited the completion of cell wall structure and, consequently, the reversion of the protoplasts to normal cells. The transfer of these protoplasts to medium free from snail enzyme led first to the completion of the cell wall and then to the reversion of the protoplasts to normal cells. The reported experiments confirmed that the regeneration of the complete cell wall preceded the protoplast reversion.     

The Fine Structure of Secretion in Hyalophysa chattoni: Formation of the Attachment Peduncle and the Chitinous Phoretic Cyst Wall

ABSTRACT. The settling tomite stage of the apostome Hyalophysa chattoni secretes a phoretic cyst wall composed of chitin, mucopolysaccharides, and protein. Within 1 1/2 h after settling, an electron-dense proteinaceous cyst layer (the outer layer) is formed from secretions originating at the base of the kineties and from the thick pellicular layer between the kineties. The inner cyst layer, composed primarily of chitin (acidic and neutral polysaccharides are also present), is secreted across the entire cell surface. Cyst wall formation is completed within 6 h. The fine structure of endocyst secretion resembles stages in the secretion of chitin by fungi, yeasts, and arthropods. A proteinaceous attachment peduncle is secreted to anchor the cell to a shrimp host and is formed by the release of electron-dense secretory bodies from the cell's ventral surface.     

Secretion of cell wall polysaccharides in Vicia root hairs

Root hairs of hairy winter vetch ( Vicia villosa Roth) synthesize and secrete abundant cell wall matrix polysaccharides, making this an excellent system for the study of secretion during tip growth. Roots with newly formed hairs were preserved by cryofixation and freeze substitution. Cryofixed root hairs showed excellent structural and antigenic preservation. Ultrastructural analyses showed numerous vesicles near the tip and a concentration of Golgi bodies in the subapical region of the hair. The distribution of polygalacturonic acid and xyloglucan in the endomembrane system and cell wall were revealed by immunolabeling, using previously characterized monoclonal antibodies. De-esterified polygalacturonic acid was present on the external surface of the cell wall, but was not detected within the cell, although chemical de-esterification revealed abundant antigen in Golgi bodies and secretory vesicles. Methyl-esterified polygalacturonic acid epitopes were detected within the medial and trans cisternae of Golgi bodies, in secretory vesicles, and throughout the wall, indicating that pectin is secreted in a neutral form and may then be de-esterified in muro . Xyloglucan was also detected within the trans cisternae of Golgi bodies, secretory vesicles and throughout the cell wall. Double labeling experiments demonstrated that both polysaccharides occur simultaneously in the same Golgi bodies, and that secretory vesicles containing both polygalacturonic acid and xyloglucan deliver the polysaccharides to the cell wall at the growing tip.     

Comparative compositional analysis of walls with two different morphologies: archetypical versus transfer-cell-like

Summary Abnormally thick cell walls of a clonal maize cell line with the labyrinth wall morphology found in transfer cells were analyzed and compared to the relatively thin and even archetypical walls of a sister cell line. Despite a drastic difference in wall morphology between the transfer and archetypical cell walls, the chemical composition of the walls was essentially the same. There were no major differences in the glycosyl residue composition, in the amount of total lipid, and in the amount of total protein. The amounts of wall material released by chemical extraction of cellulosic, hemicellulosic, and pectic fractions were the same for the two types of walls. There were some differences in the protein profile and in the inorganic ion content between the transfer and archetypical walls. These results indicate that profound changes in wall morphology can be brought about in the absence of gross changes in wall composition and suggest that major changes in time- or place-dependent deposition and/or subtle changes in arrangement of rare wall constituents may be responsible.     

Red Yeast Cell Lysis by Red Yeast Cell Wall Lytic Enzyme and Protease

The purified red yeast cell wall lytic enzyme of Penicillium lilacinum No. 2093 has a potent saccharifying activity against cell walls, but the living cell lytic activity of it is considerably lower than that of the culture filtrate. Therefore, the living cell lytic factors in the culture filtrate were examined. The alkaline protease of Pen. lilacinum played an important role for living cell lysis. The synergistic effect on living cell lysis was also detected, when acid proteases from various origins were combined with the cell wall lytic enzyme. These results indicated that the protein layers of red yeast cell surface inhibited the action of a glycanase,cell wall lytic enzyme, and the protein molecule contributed to retain the rigid structure of the wall.     

Polysaccharide Secretion by the Watermelon Stigma

Secretion of the galactose-containing polysaccharide componentof the watermelon stigma exudate was studied using electronmicroscopy cytochemistry and autoradiography. Polysaccharidelocalization using the thiosemicarbazide–silver proteinatemethod stained the Golgi apparatus and secretory vesicles, thecell wall, wall thickenings and extracellular secretion. Thesame result was obtained at anthesis and at 18 h prior to anthesis,which coincides with the period of maximum secretion. Labellingof stigmas in vivo with D-(1-³H) galactose at 20 h prior toanthesis resulted in different labelling patterns after 2 h(18 h prior to anthesis) and 20 h (anthesis). At 18 h priorto anthesis label was present in the Golgi apparatus and secretoryvesicles, the cell wall and wall thickenings. By anthesis labelhad accumulated in the extracellular secretion in addition tothe Golgi apparatus, secretory vesicles, cell wall and wallthickenings. The results suggest that the polysaccharide componentof the stigma exudate is produced in the Golgi apparatus andsecreted via the cell wall and wall thickenings. Citrullus lanatus Thumb., Matsum and Nakai, watermelon, autoradiography, stigma, secretion, cytochemistry, polysaccharide