Role of septins and the exocyst complex in the function of hydrolytic enzymes responsible for fission yeast cell separation

Cell separation in Schizosaccharomyces pombe is achieved by the concerted action of the Eng1 endo-beta-1,3-glucanase and the Agn1 endo-alpha-1,3-glucanase, which are transported to the septum and localize to a ringlike structure that surrounds the septum. The requirements for the correct localization of both hydrolases as a ring were analyzed using green fluorescent protein fusion proteins. Targeting to the septum required a functional exocyst, because both proteins failed to localize correctly in sec8-1 or exo70delta mutants, suggesting that Agn1 and Eng1 might be two of the cargo proteins present in the vesicles that accumulate in exocyst mutants. Septins and Mid2 were also required for correct formation of a ring. In their absence, Eng1 and Agn1 were found in a disk-like structure that spanned the septum, rather than in a ring. Even though septin and mid2delta mutants have a cell separation defect, the septum and the distribution of linear beta-1,3-glucans were normal in these cells, suggesting that mislocalization of Eng1 and Agn1 might be the reason underlying the failure to separate efficiently. Thus, one of the functions of the septin ring would be to act as a positional marker for the localization of hydrolytic proteins to the medial region.     

Characterization of the CaENG1 Gene Encoding an Endo-1,3-β-Glucanase Involved in Cell Separation in Candida albicans

The Candida albicans CaENG1 gene encoding an endo-1,3-β-glucanase was cloned by screening a genomic library with a DNA probe obtained by polymerase chain reaction using synthetic oligonucleotides designed according to conserved regions found between two Saccharomyces cerevisiae endo-1,3-β-glucanases (Eng1p and Eng2p). The gene contains a 3435-bp open reading frame (ORF), capable of encoding a protein of 1145 amino acids (124,157 Da), that contains no introns. Comparison of the ScEng1p sequence with partial C. albicans genomic sequences revealed the presence of a second protein with sequence similarity (the product of the Ca20C1.22c ORF, which was named CaENG2). Disruption of the CaENG1 gene in C. albicans had no dramatic effects on the growth rate of the strains, but it resulted in the formation of chains of cells, suggesting that the protein is involved in cell separation. Expression of CaENG1 in S. cerevisiae cells afforded a 12-fold increase in the 1,3-β-glucanase activity detected in culture supernatants, showing that the protein has similar enzymatic activity to that of the S. cerevisiae Eng1p. In addition, when the C. albicans protein was expressed under its native promoter in S. cerevisiae eng1 mutant cells, it was able to complement the separation defect of this mutant, indicating that these two proteins are true functional homologues.     

β(1,3)-Glucanases from Geotrichum lactis: activity on its own nascent and preformed β(1,3)-glucan

Abstract Actively growing mycelium of Geotrichum lactis contains at least three β(1,3)-glucanase activities. Two of the activities have been characterized as exo- and the third as endo-hydrolytic. The action of the activities on β(1,3)-glucan synthesized in vitro by the β(1,3)-glucan synthase system from G. lactis has been studied. One of the exo-β-glucanases and the endo-β-glucanase were active on this β(1,3)-glucan and the degradation rates were higher on nascent than on preformed β(1,3)-glucan.     

Regulation of nitrate reduction in a cyanobacterium Phormidium uncinatum: Distinctive modes of ammonium-repression of nitrate and nitrite reductases

Abstract A 5.8 kbp DNA fragment from Clostridium cellulovorans (ATCC 35296) containing endo-β-1,4-glucanase (1,4-β- d -glucan glucanohydrolase, carboxymethylcellulase, CMCase; EC 3.2.1.4) gene, engD was cloned in Escherichia coli . The clone harboring a subcloned 3.8 kb fragment in plasmid, pEQ52V, produced an enzyme that showed both endo-β-1,4-glucanase activity as well as cellobiosidase activity. Zymograms with the engD encoded enzyme with carboxymethyl-cellulose as the substrate indicated that the molecular mass of the active protein was 50 000.     

Coleoptile growth-inducing capacities of exo-β-(1→3)-glucanases from fungi

An exo-β-(1β3)-glucanase derived from Selerotinia libertiana induced growth of Avena sativa coleoptiles and degraded hemicelluloses and β-(1→4):(1→3) mixed linked glucan. However, neither endo-β-(1→4)- nor endo-β-(1→3)-glucanase activity could be detected in the enzyme preparation. Nojirimycin inhibited the glucan degradation caused by the enzyme but glucono-1,5-lactone did not. Another exo-β-(1→3)-glucanase derived from Basidiomycete QM 806 did not induce coleoptile growth and did not degrade the glucan. Growth-inducing properties of exo-β-(1→3)-glucanases are discussed.     

Mapping of the gene for endo-β-1,3-1,4-glucanase of Bacillus subtilis

Abstract An integrating plasmid has been used to mutagenise the gene coding for endo-β-1,3-1,4-glucanase of Bacillus subtilis . The gene, named bgl , has been mapped by PBS-1 transduction to the sacA-pureA region of the B. subtilis chromosome and is closely linked to the hutP 1 locus. The order of markers in this region is sacA 321- thiC 5- bgl - hutP 1- purA 16.     

Effect of 1,3;1,6-β-D-glucan on Infection of Detached Tobacco Leaves with Tobacco Mosaic Virus

Glucan preparations were obtained by transformation of laminaran from the alga Laminaria cichorioides with endo-β-1,3-glucanase from marine mollusks. These preparations, like those of other sources, have an inhibitory effect on tobacco mosaic virus (TMV) infection of detached leaves of local and systemic host tobacco plants.     

Proteomic analysis of β-1,3-glucanase in grape berry tissues

Grape berries are considered recalcitrant materials in proteomic analysis, because berry tissues contain large amounts of secondary metabolites, especially phenolic compounds, which severely interfere with protein extraction and electrophoresis separation. We report hereby a PVPP/TCA-based protein extraction protocol for grape berries. Phenolic compounds in berry extracts were removed with repeated PVPP cleanups, and proteins were recovered with TCA precipitation. Protein resolution in 2-D gels was gradually improved with the increase of PVPP cleanup steps. By the protocol, about 760 protein spots of berry tissues were clearly resolved in 2-D gels with CBB staining. This protocol was also used to analyze β-1,3-glucanase (EC 3.2.1.39) in berry tissues. An anti-synthetic peptide antibody was prepared against 15 amino acid sequence residing on the surface of β-1,3-glucanase molecule. It detected two major spots in 2-D blots of berry extracts. The spots were identified by MALDI-TOF analysis as β-1,3-glucanase. The present study validates that β-1,3-glucanase is present in higher abundance in berry skins than in pulps, and in red berries than in white berries. Therefore, β-1,3-glucanase displays a tissue-specific expression. The preferential accumulation of β-1,3-glucanase in skins may be relevant to berry ripening.     

The multiprotein exocyst complex is essential for cell separation in Schizosaccharomyces pombe

Schizosaccharomyces pombe cells divide by medial fission through the use of an actomyosin-based contractile ring. A mulitlayered division septum is assembled in concert with ring constriction. Finally, cleavage of the inner layer of the division septum results in the liberation of daughter cells. Although numerous studies have focused on actomyosin ring and division septum assembly, little information is available on the mechanism of cell separation. Here we describe a mutant, sec8-1, that is defective in cell separation but not in other aspects of cytokinesis. sec8-1 mutants accumulate about 100-nm vesicles and have reduced secretion of acid phosphatase, suggesting that they are defective in exocytosis. Sec8p is a component of the exocyst complex. Using biochemical methods, we show that Sec8p physically interacts with other members of the exocyst complex, including Sec6p, Sec10p, and Exo70p. These exocyst proteins localize to regions of active exocytosis-at the growing ends of interphase cells and in the medial region of cells undergoing cytokinesis-in an F-actin-dependent and exocytosis-independent manner. Analysis of a number of mutations in various exocyst components has established that these components are essential for cell viability. Interestingly, all exocyst mutants analyzed appear to be able to elongate and to assemble division septa but are defective for cell separation. We therefore propose that the fission yeast exocyst is involved in targeting of enzymes responsible for septum cleavage. We further propose that cell elongation and division septum assembly can continue with minimal levels of exocyst function.     

Differences in cellulose digestive systems among castes in two termite lineages

Abstract.  Termites (Isoptera) are eusocial insects and express polyphenism. Soldiers have specialized morphology for colony defense, but their feeding activity is dependent on other colony members. To determine differences in cellulose degradation between soldier and worker termites, enzymatic activity and cellulase gene expression, as well digestive tract histology, are examined in two phylogenetically distant species. In Hodotermopsis sjostesti (family Termopsidae) , endo-β-1,4-glucanase activity is identified in the salivary glands, whereas β-glucosidase activity is identified in salivary glands and hindgut. The relative expression levels of endo-β-1,4-glucanase genes in soldiers are significantly lower than in workers. Thin sections of salivary gland of workers and soldiers are different in H. sjostedti . In Nasutitermes takasagoensis (family Termitidae), the endo-β-1,4-glucanase activity is restricted to the midgut in four tested castes (i.e. three types of workers and soldier). Examination of activity per termite reveals the highest activity in minor workers and the lowest activity in major workers and soldiers. The β-glucosidase activity is also concentrated on the midgut in all four castes. The relative expression level of the endo-β-1,4-glucanase gene does not correspond with its activity in the midgut. In thin sections prepared from N. takasagoensis , the folds and pulvillus in the gizzards, and cuticle structure of soldiers are less developed compared with the other three worker castes. The differences in digestive system among termite castes in terms of caste development in each species are discussed.     

The anther-specific protein encoded by the Brassica napus and Arabidopsis thaliana A6 gene displays similarity to β-1,3-glucanases

An anther-specific Brassica napus cDNA, A6, and two corresponding Arabidopsis thaliana genes have been isolated. Sequence analyses of A6 revealed similarity to β-1,3-glucanases. The deduced A6 protein differs from other β-1,3-glucanases in the possession of a long C-terminus. Immunoblotting using an antibody raised to the A6 protein detects a temporal 60 kDa protein in B. napus buds, suggesting that the long C-terminal region is present in the mature protein. A6 promoter—GUS and RNase fusions demonstrate that the A6 gene is tapetum-specific and temporally expressed with a peak in activity when the plant normally expresses callase (a complex of endo- and exo-β-1,3-glucanase activities). The sequence similarity of A6 to other β-1,3-glucanases, coupled with the temporal and spatial expression data, suggests that A6 may be part of the callase enzyme complex.     

Enzymic and structural studies on processed proteins from the vacuolar (lutoid-body) fraction of latex of Hevea brasiliensis

The lutoid-body (bottom) fraction of latex from the rubber tree (Hevea brasiliensis) contains a limited number of major proteins. These are the chitin-binding protein hevein, its precursor and C-terminal fragment of the precursor, a basic chitinase/lysozyme, and a β-1,3-glucanase. The content and properties of the latter enzyme differ between lutoid-body fractions from four different rubber clones (cultivars). While the enzyme from clone GT.1 is a glycoprotein with carbohydrate attached to two glycosylation sites, the enzymes from other clones contain little or no carbohydrate. Latex from clone GT.1 has a higher β-1,3-glucanase content than those from the other three clones, but with a significantly lower specific activity. The enzyme exhibits a pH optimum at 4.5, but there is a second one at 6.7. Peptides isolated from β-1,3-glucanase of clone GT.1 showed that the enzyme is heterogeneous at the C-terminus, probably as a result of removal of a vacuolar targeting sequence by an endopeptidase, followed by further removal of C-terminal residues by a carboxypeptidase-like activity. This incomplete digestion can be related to glycosylation at the extreme C-terminus of the mature enzyme. Non-glycosylated Hevea β-1,3-glucanases exhibit less C-terminal heterogeneity. A homologue of the antifungal protein osmotin was isolated from rubber clones which are less susceptible to fungal diseases. Another identified protein is identical to a citrate binding protein (CBP), already sequenced as cDNA, but with cleaved-off N-terminal signal and C-terminal vacuolar targeting peptides. Four C-terminal propeptides of vacuolar proteins in Hevea are positively identified, which is a valuable contribution to previously known examples of this type of processing.     

Study of β-1,3-glucanase activity during autolysis of Aspergillus nidulans by FPLC ion-exchange chromatography

The behaviour of β-1,3-glucanase activity during Aspergillus nidulans autolysis was studied in a basal medium and in the same medium supplemented with 0.5 g l^-1 of microcrystalline cellulose, laminarin, pectin, seedling of Lycopersicum esculentum extract, chitin and xylan respectively. In any case β-1,3-glucanase activity was detected in the culture fluid before the onset of the autolysis, but afterwards a progressive increase of β-1,3-glucanase activity took place with incubation time. In the media supplemented with pectin and seedling of Lycopersicum esculentum extract higher activity in the first days of autolysis was found. The activity at the end of the studied process by sample was 2.5, 2.1, 2.5, 1.9, 2.2, 2.3 and 2.3 U, and the specific activity 83, 53, 85, 55, 64, 90 and 53 mU mg^-1 of protein for each medium respectively. The β-1,3-glucanase activity in Aspergillus nidulans seems to be related to autolysis and not to the presence of different substances in the culture medium. The behaviour of β-1,3-glucanase activity during the degradative process was followed by FPLC ion-exchange chromatography. Three proteins (I, II, III) with β-1,3-glucanase activity were separated and quantified. These proteins have similar behaviour in all the media. Proteins I and II increase progressively with incubation time but protein III is only present at the first and last days of autolysis.     

Cloning and functional characterization of a complex endo-β-1,3-glucanase from <Emphasis Type="Italic">Paenibacillus</Emphasis> sp.

A β-1,3-glucanase gene, encoding a protein of 1,793 amino acids, was cloned from a strain of Paenibacillus sp. in this study. This large protein, designated as LamA, consists of many putative functional units, which include, from N to C terminus, a leader peptide, three repeats of the S-layer homologous module, a catalytic module of glycoside hydrolase family 16, four repeats of the carbohydrate-binding module of family CBM_4_9, and an analogue of coagulation factor Fa5/8C. Several truncated proteins, composed of the catalytic module with various organizations of the appended modules, were successfully expressed and characterized in this study. Data indicated that the catalytic module specifically hydrolyze β-1,3- and β-1,3–1,4-glucans. Also, laminaritriose was the major product upon endolytic hydrolysis of laminarin. The CBM repeats and Fa5/8C analogue substantially enhanced the hydrolyzing activity of the catalytic module, particularly toward insoluble complex substrates, suggesting their modulating functions in the enzymatic activity of LamA. Carbohydrate-binding assay confirmed the binding capabilities of the CBM repeats and Fa5/8C analogue to β-1,3-, β-1,3–1,4-, and even β-1,4-glucans. These appended modules also enhanced the inhibition effect of the catalytic module on the growth of Candida albicans and Rhizoctonia solani.     

The Putative Exchange Factor Gef3p Interacts with Rho3p GTPase and the Septin Ring during Cytokinesis in Fission Yeast

The small GTP-binding proteins of the Rho family and its regulatory proteins play a central role in cytokinetic actomyosin ring assembly and cytokinesis. Here we show that the fission yeast guanine nucleotide exchange factor Gef3p interacts with Rho3p at the division site. Gef3p contains a putative DH homology domain and a BAR/IMD-like domain. The protein localized to the division site late in mitosis, where it formed a ring that did not constrict with actomyosin ring (cytokinetic actomyosin ring) invagination; instead, it split into a double ring that resembled the septin ring. Gef3p co-localized with septins and Mid2p and required septins and Mid2p for its localization. Gef3p interacts physically with the GTP-bound form of Rho3p. Although Gef3p is not essential for cell separation, the simultaneous disruption of gef3⁺ and Rho3p-interacting proteins, such as Sec8p, an exocyst component, Apm1p, a subunit of the clathrin adaptor complex or For3p, an actin-polymerizing protein, yielded cells with strong defects in septation and polarity respectively. Our results suggest that interactions between septins and Rho-GEFs provide a new targeting mechanism for GTPases in cytokinesis, in this case probably contributing to Rho3p function in vesicle tethering and vesicle trafficking in the later steps of cell separation.