The Role of Glucosidase I (Cwh41p) in the Biosynthesis of Cell Wall β-1,6-Glucan Is Indirect

CWH41, a gene involved in the assembly of cell wall β-1,6-glucan, has recently been shown to be the structural gene for Saccharomyces cerevisiae glucosidase I that is responsible for initiating the trimming of terminal α-1,2-glucose residue in the N-glycan processing pathway. To distinguish between a direct or indirect role of Cwh41p in the biosynthesis of β-1,6-glucan, we constructed a double mutant, alg5Δ (lacking dolichol-P-glucose synthase) cwh41Δ, and found that it has the same phenotype as the alg5Δ single mutant. It contains wild-type levels of cell wall β-1,6-glucan, shows moderate underglycosylation of N-linked glycoproteins, and grows at concentrations of Calcofluor White (which interferes with cell wall assembly) that are lethal to cwh41Δ single mutant. The strong genetic interactions of CWH41 with KRE6 and KRE1, two other genes involved in the β-1,6-glucan biosynthetic pathway, disappear in the absence of dolichol-P-glucose synthase (alg5Δ). The triple mutant alg5Δcwh41Δkre6Δ is viable, whereas the double mutant cwh41Δkre6Δ in the same genetic background is not. The severe slow growth phenotype and 75% reduction in cell wall β-1,6-glucan, characteristic of the cwh41Δkre1Δ double mutant, are not observed in the triple mutant alg5Δcwh41Δkre1Δ. Kre6p, a putative Golgi glucan synthase, is unstable in cwh41Δ strains, and its overexpression renders these cells Calcofluor White resistant. These results demonstrate that the role of glucosidase I (Cwh41p) in the biosynthesis of cell wall β-1,6-glucan is indirect and that dolichol-P-glucose is not an intermediate in this pathway.

The making of the architecture of the plant cell wall: How cells exploit geometry

Cell wall deposition is a key process in the formation, growth, and differentiation of plant cells. The most important structural components of the wall are long cellulose microfibrils, which are synthesized by synthases embedded in the plasma membrane. A fundamental question is how the microfibrils become oriented during deposition at the plasma membrane. The current textbook explanation for the orientation mechanism is a guidance system mediated by cortical microtubules. However, too many contraindications are known in secondary cell walls for this to be a universal mechanism, particularly in the case of helicoidal arrangements, which occur in many situations. An additional construction mechanism involves liquid crystalline self-assembly [A. C. Neville (1993) Biology of Fibrous Composites: Development Beyond the Cell Membrane (Cambridge Univ. Press, Cambridge, U.K.)], but the required amount of bulk material that is able to equilibrate thermally is not normally present at any stage of the wall deposition process. Therefore, we have asked whether the complex ordered texture of helicoidal cell walls can be formed in the absence of direct cellular guidance mechanisms. We propose that they can be formed by a mechanism that is based on geometrical considerations. It explains the genesis of the complicated helicoidal texture and shows that the cell has intrinsic, versatile tools for creating a variety of textures. A compelling feature of the model is that local rules generate global order, a typical phenomenon of life.

A reversibly glycosylated polypeptide (RGP1) possibly involved in plant cell wall synthesis: Purification, gene cloning, and trans-Golgi localization

We purified from pea (Pisum sativum) tissue an ≈40 kDa reversibly glycosylated polypeptide (RGP1) that can be glycosylated by UDP-Glc, UDP-Xyl, or UDP-Gal, and isolated a cDNA encoding it, apparently derived from a single-copy gene (Rgp1). Its predicted translation product has 364 aminoacyl residues and molecular mass of 41.5 kDa. RGP1 appears to be a membrane-peripheral protein. Immunogold labeling localizes it specifically to trans-Golgi dictyosomal cisternae. Along with other evidence, this suggests that RGP1 is involved in synthesis of xyloglucan and possibly other hemicelluloses. Corn (Zea mays) contains a biochemically similar and structurally homologous RGP1, which has been thought (it now seems mistakenly) to function in starch synthesis. The expressed sequence database also reveals close homologs of pea Rgp1 in Arabidopsis and rice (Oryza sativa). Rice possesses, in addition, a distinct but homologous sequence (Rgp2). RGP1 provides a polypeptide marker for Golgi membranes that should be useful in plant membrane studies.

Treatment of Mycobacterium tuberculosis with antisense oligonucleotides to glutamine synthetase mRNA inhibits glutamine synthetase activity, formation of the poly-l-glutamate/glutamine cell wall structure, and bacterial replication

New antibiotics to combat the emerging pandemic of drug-resistant strains of Mycobacterium tuberculosis are urgently needed. We have investigated the effects on M. tuberculosis of phosphorothioate-modified antisense oligodeoxyribonucleotides (PS-ODNs) against the mRNA of glutamine synthetase, an enzyme whose export is associated with pathogenicity and with the formation of a poly-l-glutamate/glutamine cell wall structure. Treatment of virulent M. tuberculosis with 10 μM antisense PS-ODNs reduced glutamine synthetase activity and expression by 25–50% depending on whether one, two, or three different PS-ODNs were used and the PS-ODNs' specific target sites on the mRNA. Treatment with PS-ODNs of a recombinant strain of Mycobacterium smegmatis expressing M. tuberculosis glutamine synthetase selectively inhibited the recombinant enzyme but not the endogenous enzyme for which the mRNA transcript was mismatched by 2–4 nt. Treatment of M. tuberculosis with the antisense PS-ODNs also reduced the amount of poly-l-glutamate/glutamine in the cell wall by 24%. Finally, treatment with antisense PS-ODNs reduced M. tuberculosis growth by 0.7 logs (1 PS-ODN) to 1.25 logs (3 PS-ODNs) but had no effect on the growth of M. smegmatis, which does not export glutamine synthetase nor possess the poly-l-glutamate/glutamine (P-l-glx) cell wall structure. The experiments indicate that the antisense PS-ODNs enter the cytoplasm of M. tuberculosis and bind to their cognate targets. Although more potent ODN technology is needed, this study demonstrates the feasibility of using antisense ODNs in the antibiotic armamentarium against M. tuberculosis.

Unexpected homology between inducible cell wall protein QID74 of filamentous fungi and BR3 salivary protein of the insect Chironomus

A gene, qid74, of mycoparasitic filamentous fungus Trichoderma harzianum and its allies encodes a cell wall protein that is induced by replacing glucose in the culture medium with chitin (simulated mycoparasitism conditions). Because no trace of this gene can be detected in related species such as Gibberella fujikuroi and Saccharomyces cerevisiae, the qid74 gene appears to have arisen de novo within the genus Trichoderma. Qid74 protein, 687 residues long, is now seen as highly conserved tandem repeats of the 59-residue-long unit. This unit itself, however, may have arisen as tandem repeats of the shorter 13-residue-long basic unit. Within the genus Trichoderma, the amino acid sequence of Qid74 proteins has been conserved in toto. The most striking is the fact that Qid74 shares 25.3% sequence identity with the carboxyl-terminal half of the 1,572-residue-long BR3 protein of the dipteran insect Chironomus tentans. BR3 protein is secreted by the salivary gland of each aquatic larva of Chironomus to form a tube to house itself. Furthermore, the consensus sequence derived from these 59-residue-long repeating units resembles those of epidermal growth factor-like domains found in divergent invertebrate and vertebrate proteins as to the positions of critical cysteine residues and homology of residues surrounding these cysteines.

Role of the proteasome and NF-κB in streptococcal cell wall-induced polyarthritis

The transcription factor NF-κB activates a number of genes whose protein products are proinflammatory. In quiescent cells, NF-κB exists in a latent form and is activated via a signal-dependent proteolytic mechanism in which the inhibitory protein IκB is degraded by the ubiquitin–proteasome pathway. Consequently, inhibition of the proteasome suppresses activation of NF-κB. This suppression should therefore decrease transcription of many genes encoding proinflammatory proteins and should ultimately have an anti-inflammatory effect. To this end, a series of peptide boronic acid inhibitors of the proteasome, exemplified herein by PS-341, were developed. The proteasome is the large multimeric protease that catalyzes the final proteolytic step of the ubiquitin–proteasome pathway. PS-341, a potent, competitive inhibitor of the proteasome, readily entered cells and inhibited the activation of NF-κB and the subsequent transcription of genes that are regulated by NF-κB. Significantly, PS-341 displayed similar effects in vivo. Oral administration of PS-341 had anti-inflammatory effects in a model of Streptococcal cell wall-induced polyarthritis and liver inflammation in rats. The attenuation of inflammation in this model was associated with an inhibition of IκBα degradation and NF-κB-dependent gene expression. These experiments clearly demonstrate that the ubiquitin–proteasome pathway and NF-κB play important roles in regulating chronic inflammation and that, as predicted, proteasome inhibition has an anti-inflammatory effect.

A family of genes required for maintenance of cell wall integrity and for the stress response in Saccharomyces cerevisiae

The PKC1–MPK1 pathway in yeast functions in the maintenance of cell wall integrity and in the stress response. We have identified a family of genes that are putative regulators of this pathway. WSC1, WSC2, and WSC3 encode predicted integral membrane proteins with a conserved cysteine motif and a WSC1–green fluorescence protein fusion protein localizes to the plasma membrane. Deletion of WSC results in phenotypes similar to mutants in the PKC1–MPK1 pathway and an increase in the activity of MPK1 upon a mild heat treatment is impaired in a wscΔ mutant. Genetic analysis places the function of WSC upstream of PKC1, suggesting that they play a role in its activation. We also find a genetic interaction between WSC and the RAS–cAMP pathway. The RAS–cAMP pathway is required for cell cycle progression and for the heat shock response. Overexpression of WSC suppresses the heat shock sensitivity of a strain in which RAS is hyperactivated and the heat shock sensitivity of a wscΔ strain is rescued by deletion of RAS2. The functional characteristics and cellular localization of WSC suggest that they may mediate intracellular responses to environmental stress in yeast.

A 1.2-Å snapshot of the final step of bacterial cell wall biosynthesis

The cell wall imparts structural strength and shape to bacteria. It is made up of polymeric glycan chains with peptide branches that are cross-linked to form the cell wall. The cross-linking reaction, catalyzed by transpeptidases, is the last step in cell wall biosynthesis. These enzymes are members of the family of penicillin-binding proteins, the targets of β-lactam antibiotics. We report herein the structure of a penicillin-binding protein complexed with a cephalosporin designed to probe the mechanism of the cross-linking reaction catalyzed by transpeptidases. The 1.2-Å resolution x-ray structure of this cephalosporin bound to the active site of the bifunctional serine type d-alanyl-d-alanine carboxypeptidase/transpeptidase (EC 3.4.16.4) from Streptomyces sp. strain R61 reveals how the two peptide strands from the polymeric substrates are sequestered in the active site of a transpeptidase. The structure of this complex provides a snapshot of the enzyme and the bound cell wall components poised for the final and critical cross-linking step of cell wall biosynthesis.

Tracheary Element Differentiation Uses a Novel Mechanism Coordinating Programmed Cell Death and Secondary Cell Wall Synthesis1

Tracheary element differentiation requires strict coordination of secondary cell wall synthesis and programmed cell death (PCD) to produce a functional cell corpse. The execution of cell death involves an influx of Ca2+ into the cell and is manifested by rapid collapse of the large hydrolytic vacuole and cessation of cytoplasmic streaming. This precise means of effecting cell death is a prerequisite for postmortem developmental events, including autolysis and chromatin degradation. A 40-kD serine protease is secreted during secondary cell wall synthesis, which may be the coordinating factor between secondary cell wall synthesis and PCD. Specific proteolysis of the extracellular matrix is necessary and sufficient to trigger Ca2+ influx, vacuole collapse, cell death, and chromatin degradation, suggesting that extracellular proteolysis plays a key regulatory role during PCD. We propose a model in which secondary cell wall synthesis and cell death are coordinated by the concomitant secretion of the 40-kD protease and secondary cell wall precursors. Subsequent cell death is triggered by a critical activity of protease or the arrival of substrate signal precursor corresponding with the completion of a functional secondary cell wall.

Turgor Regulation via Cell Wall Adjustment in White Spruce1

Turgor regulation at reduced water contents was closely associated with changes in the elastic quality of the cell walls of individual needles and shoots of naturally drought-resistant seedlings of white spruce (Picea glauca [Moench] Voss.) and of seedlings of intermediate resistance that had been pretreated with paclobutrazol, a stress-protecting, synthetic plant-growth regulator. Paclobutrazol-treated seedlings showed marked increases in drought resistance, and pressure-volume analysis combined with Chardakov measurements confirmed observations that water stress was ameliorated during prolonged drought. Turgor was maintained in the paclobutrazol-treated and in the naturally resistant drought-stressed seedlings despite water contents near or below the turgor-loss volumes of well-watered controls. The maintenance of turgor in these seedlings was in large part a function of the dynamic process of cell wall adjustment, as reflected by marked reductions in both the saturated and turgor-loss volumes and by large increases in the elastic coefficients of the tissues. Shear and Young's moduli, calculated from pressure-volume curves and the radii and wall thicknesses of mesophyll cells, also confirmed observed changes in the elastic qualities of the cell walls. Elastic coefficients of well-watered, paclobutrazol-treated seedlings were consistently larger than those in well-watered controls and several times larger than the values in untreated plants, which succumbed rapidly to drought. In contrast, untreated seedlings that withstood prolonged drought without wilting displayed elastic coefficients similar to those in seedlings that had been treated with paclobutrazol but that had not been exposed to drought.

Structure of sortase, the transpeptidase that anchors proteins to the cell wall of Staphylococcus aureus

Surface proteins of Gram-positive bacteria play important roles during the pathogenesis of human infections and require sortase for anchoring to the cell-wall envelope. Sortase cleaves surface proteins at the LPXTG motif and catalyzes the formation of an amide bond between the carboxyl group of threonine (T) and the amino group of cell-wall crossbridges. The NMR structure of sortase reveals a unique β-barrel structure, in which the active-site sulfhydryl of cysteine-184 is poised for ionization by histidine-120, presumably enabling the resultant thiolate to attack the LPXTG peptide. Calcium binding near the active site stimulates catalysis, possibly by altering the conformation of a surface loop that recognizes newly translocated polypeptides. The structure suggests a mechanistic relationship to the papain/cathepsin proteases and should facilitate the design of new antiinfective agents.

Changes in Cell Wall Composition during Ripening of Grape Berries1

Cell walls were isolated from the mesocarp of grape (Vitis vinifera L.) berries at developmental stages from before veraison through to the final ripe berry. Fluorescence and light microscopy of intact berries revealed no measurable change in cell wall thickness as the mesocarp cells expanded in the ripening fruit. Isolated walls were analyzed for their protein contents and amino acid compositions, and for changes in the composition and solubility of constituent polysaccharides during development. Increases in protein content after veraison were accompanied by an approximate 3-fold increase in hydroxyproline content. The type I arabinogalactan content of the pectic polysaccharides decreased from approximately 20 mol % of total wall polysaccharides to about 4 mol % of wall polysaccharides during berry development. Galacturonan content increased from 26 to 41 mol % of wall polysaccharides, and the galacturonan appeared to become more soluble as ripening progressed. After an initial decrease in the degree of esterification of pectic polysaccharides, no further changes were observed nor were there large variations in cellulose (30–35 mol % of wall polysaccharides) or xyloglucan (approximately 10 mol % of wall polysaccharides) contents. Overall, the results indicate that no major changes in cell wall polysaccharide composition occurred during softening of ripening grape berries, but that significant modification of specific polysaccharide components were observed, together with large changes in protein composition.

Evidence That Auxin-Induced Growth of Tobacco Leaf Tissues Does Not Involve Cell Wall Acidification1

Interveinal strips (10 × 1.5 mm) excised from growing tobacco (Nicotiana tabacum L. cv Xanthi) leaves have an auxin-specific, epinastic growth response that is developmentally regulated and is not the result of ethylene induction (C.P. Keller, E. Van Volkenburgh [1997] Plant Physiol 113: 603–610). We report here that auxin (10 μm naphthalene acetic acid) treatment of strips does not result in plasma membrane hyperpolarization or detectable proton efflux. This result is in contrast to the expected responses elicited by 1 μm fusicoccin (FC) treatment, which in other systems mimics auxin growth promotion through stimulation of the plasma membrane H+-ATPase and resultant acid wall loosening; FC produced both hyperpolarization and proton efflux in leaf strips. FC-induced growth was much more inhibited by a strong neutral buffer than was auxin-induced growth. Measurements of the osmotic concentration of strips suggested that osmotic adjustment plays no role in the auxin-induced growth response. Although cell wall loosening of some form appears to be involved, taken together, our results suggest that auxin-induced growth stimulation of tobacco leaf strips results primarily from a mechanism not involving acid growth.

Rapid Regulation by Acid pH of Cell Wall Adjustment and Leaf Growth in Maize Plants Responding to Reversal of Water Stress1

The role of acid secretion in regulating short-term changes in growth rate and wall extensibility was investigated in emerging first leaves of intact, water-stressed maize (Zea mays L.) seedlings. A novel approach was used to measure leaf responses to injection of water or solutions containing potential regulators of growth. Both leaf elongation and wall extensibility, as measured with a whole-plant creep extensiometer, increased dramatically within minutes of injecting water, 0.5 mm phosphate, or strong (50 mm) buffer solutions with pH ≤ 5.0 into the cell-elongation zone of water-stressed leaves. In contrast, injecting buffer solutions at pH ≥ 5.5 inhibited these fast responses. Solutions containing 0.5 mm orthovanadate or erythrosin B to inhibit wall acidification by plasma membrane H+-ATPases were also inhibitory. Thus, cell wall extensibility and leaf growth in water-stressed plants remained inhibited, despite the increased availability of (injected) water when accompanying increases in acid-induced wall loosening were prevented. However, growth was stimulated when pH 4.5 buffers were included with the vanadate injections. These findings suggest that increasing the availability of water to expanding cells in water-stressed leaves signals rapid increases in outward proton pumping by plasma membrane H+-ATPases. Resultant increases in cell wall extensibility participate in the regulation of water uptake, cell expansion, and leaf growth.

The Boron Requirement and Cell Wall Properties of Growing and Stationary Suspension-Cultured Chenopodium album L. Cells1

Suspension-cultured Chenopodium album L. cells are capable of continuous, long-term growth on a boron-deficient medium. Compared with cultures grown with boron, these cultures contained more enlarged and detached cells, had increased turbidity due to the rupture of a small number of cells, and contained cells with an increased cell wall pore size. These characteristics were reversed by the addition of boric acid (≥7 μm) to the boron-deficient cells. C. album cells grown in the presence of 100 μm boric acid entered the stationary phase when they were not subcultured, and remained viable for at least 3 weeks. The transition from the growth phase to the stationary phase was accompanied by a decrease in the wall pore size. Cells grown without boric acid or with 7 μm boric acid were not able to reduce their wall pore size at the transition to the stationary phase. These cells could not be kept viable in the stationary phase, because they continued to expand and died as a result of wall rupture. The addition of 100 μm boric acid prevented wall rupture and the wall pore size was reduced to normal values. We conclude that boron is required to maintain the normal pore structure of the wall matrix and to mechanically stabilize the wall at growth termination.