RHO GTPases in the control of cell morphology, cell polarity, and actin localization in fission yeast.

The fission yeast Schizosaccharomyces pombe undergoes morphogenetic changes during both vegetative and sexual cell cycles that require asymmetric cell growth and actin cytoskeleton reorganisations. Different complex signal transduction pathways participate in S. pombe morphogenesis. The Rho family of GTPases are present in all eukaryotic cells, from yeast to mammals, and their role as key regulators in the signalling pathways that control actin organisation and morphogenetic processes is well known. In this review, we will briefly summarize the role of the Rho GTPases in the establishment and maintenance of cell polarity and growth of S. pombe. As in other fungi, S. pombe morphogenesis is closely related to cell wall biosynthesis, and Rho GTPases are critical modulators of this process. They provide the coordinated regulation of cell wall biosynthetic enzymes and actin organisation required to maintain cell integrity and polarised growth.

Schizosaccharomyces pombe protein kinase C homologues, pck1p and pck2p, are targets of rho1p and rho2p and differentially regulate cell integrity.

Schizosaccharomyces pombe rho1(+) is required for maintenance of cell integrity and polarization of the actin cytoskeleton. However, no other effector besides the (1,3)beta-D-glucan synthase enzyme has been identified in S. pombe. We have further investigated if rho1(+ )signalling could be also mediated by the two protein kinase C homologues, pck1p and pck2p. We show in this study that both kinases interact with rho1p and rho2p only when bound to GTP, as most GTPase effectors do. Interestingly, the interaction was mapped in a different part of the proteins than in Saccharomyces cerevisiae Pkc1p. Thus, active rho1p binds to the amino-terminal region of the pcks where two HR1 motifs are located, and binding to the GTPase dramatically stabilizes the kinases. Detailed biochemical analysis suggests that pck2p is more important in the regulation of the enzyme (1-3)beta-D-glucan synthase. Thus, overexpression of pck2(+), but not pck1(+), caused a general increase in cell wall biosynthesis, mainly in beta-glucan, and (1-3)beta-D-glucan synthase activity was considerably augmented. When this activity was separated into soluble and membrane fractions and reconstituted, the increase caused by pck2(+) overexpression was exclusively detected in the membrane component. We also show that both protein kinase C homologues are required for the maintenance of cell integrity. pck1delta and pck2delta strains present a number of defects related to the cell wall, indicating that this structure might be co-ordinately regulated by both kinases. In addition, pck2p, but not pck1p, seems to be involved in keeping cell polarity. Genetic evidence indicates that both pck1(+) and pck2(+) interact with cps1(+) and gls2(+), two genes similar to S. cerevisiae FKS1 and FKS2 that encode membrane subunits of the (1-3)beta-D-glucan synthase. pck1(+ )also showed a genetic interaction with ras1(+) and ral1(+) suggesting the existence of a functional link between both signalling pathways.

Characterization of the geranylgeranyl transferase type I from Schizosaccharomyces pombe.

The Schizosaccharomyces pombe cwg2+ gene encodes the beta-subunit of geranylgeranyl transferase I (GGTase I), which participates in the post-translational C-terminal modification of several small GTPases, allowing their targeting to the membrane. Using the two-hybrid system, we have identified the cwp1+ gene that encodes the alpha-subunit of the GGTase I. cwp1p interaction with cwg2p was mapped to amino acids 1-244 or 137-294 but was not restricted to amino acids 137-244. The genomic cwp1+ was isolated and sequenced. It has two putative open reading frames of 677 and 218 bp, separated by a 51 bp intron. The predicted amino acid sequence shows significant similarity to GGTase I alpha-subunits from different species. However, complementation of Saccharomyces cerevisiae ram2-1 mutant by overexpressing the cwp1+ gene was not possible. Expression of both cwg2+ and cwp1+ in Escherichia coli allowed 'in vitro' reconstitution of the GGTase I activity. S. pombe cells expressing the mutant enzyme containing the cwg2-1 mutation do not grow at 37 degrees C, but the growth defect can be suppressed by the addition of sorbitol. Actin immunostaining of the cwg2-1 mutant strain grown at 37 degrees C showed an abnormal distribution of actin patches. The cwg2-1 mutation was identified as a guanine to adenine substitution at nucleotide 604 of the coding region, originating the change A202T in the cwg2p. Deletion of the cwg2 gene is lethal; delta cwg2 spores can divide two or three times before losing viability. Most cells have aberrant morphology and septation defects. Overexpression of the rho1G15VC199R double-mutant allele in S. pombe caused loss of polarity but was not lethal and did not render the (1-3)beta-D-glucan synthase activity independent of GTP. Therefore, geranylgeranylation of rho1p is required for the appropriate function of this GTPase.

NGF binding to the trk tyrosine kinase receptor requires the extracellular immunoglobulin-like domains.

Neurotrophins initiate their biological effects by activating members of the trk tyrosine kinase subfamily. The extracellular region of trk receptors is distinguished by several common structural features, including leucine-rich repeats, clusters of cysteine-rich domains, and two immunoglobulin-like domains. However, the receptor sequences required for ligand binding have not been localized. In order to define the domains involved in NGF binding, a series of chimeric receptors was constructed using cDNA sequences from rat trkA and trkB. The chimeric constructs were expressed after transient transfection in 293 cells and the expression of each receptor was verified by immunoprecipitation and immunoblot analysis. Equilibrium binding of transfected cells revealed that the two IgG domains of trkA are essential for NGF binding. The requirement for the two IgG domains was further confirmed by Scatchard analysis and affinity crosslinking with 125I-NGF. These results indicate that NGF binding is crucially dependent upon interactions with the IgG domains of the trkA receptor.

Domain structure of laccase I from the lignin-degrading basidiomycete PM1 revealed by differential scanning calorimetry.

The application of scanning calorimetry to investigate laccase I from the lignin-degrading basidomycete PM1 (CECT 2971) showed three thermal transitions beneath the overall endotherm following the previous heating of the sample up to 60 degrees C. The thermodynamic parameters of these three transitions satisfy a model of two-state independent unfolding, supporting a three-domain organization of the enzyme. It is shown that the catalytic site of laccase I is located in the domain with the thermally-induced transition at 76 degrees C.

Cloning and DNA sequencing of bgaA, a gene encoding an endo-beta-1,3-1,4-glucanase, from an alkalophilic Bacillus strain (N137).

The gene bgaA encoding an alkaline endo-beta-1,3-1,4-glucanase (lichenase) from an alkalophilic Bacillus sp. strain N137, isolated in our laboratory, was cloned and expressed from its own promoter in Escherichia coli. The nucleotide sequence of a 1,416-bp DNA fragment containing bgaA was determined and revealed an open reading frame of 828 nucleotides. The deduced protein sequence consists of 276 amino acids and has a 31-amino-acid putative signal peptide which is functional in E. coli, in which the BgaA protein is located mainly in the periplasmic space. The lichenase activity of BgaA is stable between pH 6 and 12, it shows optimal activity at a temperature between 60 and 70 degrees C, and it retains 65% of its activity after incubation at 70 degrees C for 1 h. This protein is similar to some other lichenases from Bacillus species such as B. amyloliquefaciens, B. brevis, B. licheniformis, B. macerans, B. polymyxa, and B. subtilis. However, it has a lysine-rich region at the carboxy terminus which is not found in any other published lichenase sequence and might be implicated in the unusual biochemical properties of this enzyme. The location of the mRNA 5' end was determined by primer extension and corresponds to nucleotide 235. A typical Bacillus sigma A promoter precedes the transcription start site.

Characterization and structural analysis of the laccase I gene from the newly isolated ligninolytic basidiomycete PM1 (CECT 2971).

We have isolated and characterized the cDNA and genomic DNA coding for a phenoloxidase, laccase I, previously purified from culture supernatant of the newly isolated ligninolytic basidiomycete PM1 (CECT 2971). A cDNA library from basidiomycete PM1 was constructed, and laccase-encoding cDNAs were identified by screening with antiserum raised against the purified enzyme. The lac1 gene coding for the laccase was identified in a partial genomic library by using the isolated cDNA as a probe. Nucleotide sequence determination of the full-length cDNA revealed an open reading frame of 1,551 bp encoding a polypeptide of 517 amino acid residues with a putative signal peptide of 21 amino acid residues. Ten small introns interrupted the genomic DNA. A single 1.8-kb transcript mRNA was detected by Northern (RNA) blot analysis, and its 5' end maps to a position 51 bp upstream from the site of initiation of protein synthesis. Eukaryotic regulatory sequences, CAAT and TATA, were observed in the 5' flanking region, which also contains sequences similar to those of copper-regulated proteins. Comparative analysis of the predicted amino acid sequence showed that basidiomycete PM1 laccase I had great similarity to the laccases from Coriolus versicolor, Coriolus hirsutus, and Phlebia radiata.

Purification and characterization of a phenoloxidase (laccase) from the lignin-degrading basidiomycete PM1 (CECT 2971).

A new lignin-degrading basidiomycete, strain PM1 (= CECT 2971), was isolated from the wastewater of a paper factory. The major ligninolytic activity detected in the basidiomycete PM1 culture supernatant was a phenoloxidase (laccase). This activity was produced constitutively in defined or complex media and appeared as two protein bands in native gel electrophoresis preparations. No enzyme induction was found after treatment with certain potential laccase inducers. Laccase I was purified to homogeneity by gel filtration chromatography, anion-exchange chromatography, and hydrophobicity chromatography. The enzyme is a monomeric glycoprotein containing 6.5% carbohydrate and having a molecular weight of 64,000. It has an isoelectric point of 3.6, it is stable in a pH range from 3 to 9, and its optimum pH is 4.5. The laccase optimal reaction temperature is 80 degrees C, the laccase is stable for 1 h at 60 degrees C, and its activity increases with temperature. Spectroscopic analysis revealed that the enzyme has four bound copper atoms, a type I copper, a type II copper, and a type III binuclear copper. The amino-terminal sequence of the protein is very similar to the amino-terminal sequences of laccases from Coriolus hirsutus and Phlebia radiata.

Cloning and nucleotide sequence of celA1, and endo-beta-1,4-glucanase-encoding gene from Streptomyces halstedii JM8.

The celA1 gene encoding an endo-beta-1,4-glucanase from a mesophilic actinomycete, strain JM8, identified as Streptomyces halstedii, was cloned and expressed in S. lividans JI66. From the nucleotide sequence of a 1.7-kb DNA fragment we identified an open reading frame of 963 nucleotides encoding a protein of 321 amino acids, starting at TTG (instead of ATG). The Cel1 mature enzyme is a protein of 294 amino acids (after signal peptide cleavage) and can be included in the beta-glycanase family B (N. R. Gilkes, B. Henrissat, D. G. Kilburn, R. C. Miller, Jr., and R. A. J. Warren, Microbiol. Rev. 55:303-315, 1991). The Cel1 enzyme lacks a cellulose-binding domain as predicted by computer analysis of the sequence and confirmed by Avicel binding experiments. The promoter region of celA1 was identified by S1 mapping; the -35 region closely resembles those of housekeeping Streptomyces promoters. Three imperfectly repeated sequences of 15, 15, and 14 nucleotides were found upstream from celA1 [ATTGGGACCGCTTCC-(N85)-ATTGGGACCGCTTCC-(N2)-TGGGAGC GCTCCCA]; The 14-nucleotide sequence has a perfect palindrome identical to that found in several cellulase-encoding genes from Thermomonospora fusca, an alkalophilic Streptomyces strain, and Streptomyces lividans. This sequence has been implicated in the mechanism of induction exerted by cellobiose. Using an internal celA1 probe, we detected similar genes in several other Streptomyces species, most of them cellulase producers.