New advances in coenzyme Q biosynthesis

Summary Coenzyme Q (or ubiquinone) is the product of two distinct biosynthetic pathways: the lipid tail of coenzyme Q is formed via the isoprene biosynthetic pathway, and the quinone ring derives from the metabolism of either shikimic acid or tyrosine. In general, eukaryotic organisms use the classical mevalonate pathway to form isopentenyl- and dimethylallyl-diphosphate, the five carbon building blocks of the polyisoprenoid tail, and prokaryotes use 1-deoxy-D-xylulose-5-phosphate, formed via the Rohmer pathway. The quinone ring precursor is 4-hydroxybenzoic acid, which is formed directly from chorismate inSaccharomyces cerevisiae andEscherichia coli, or from tyrosine in animal cells. Ring modification steps including prenylation, decarboxylation, and successive hydroxylation and methylation steps form the fully substituted benzoquinone ring of coenzyme Q. Many of the genes and polypeptides involved in coenzyme Q biosynthesis have been isolated and characterized by utilizing strains ofE. coli andS. cerevisiae with mutations in theubi andCOQ genes, respectively. This article reviews recent progress in characterizing the biosynthesis of coenzyme Q inE. coli, S. cerevisiae, and other eukaryotic organisms.     

Organelle motility regulated by the cell's environment: dissection of signaling pathways regulating movements of peroxisomes

Summary Chloroplasts and pigment granules are known to be intracellularly translocated upon discrete extracellular stimuli. The machineries transducing these signals inside cells are yet not understood. In studies investigating the motility of peroxisomes, we were able to identify both extracellular and intracellular signaling steps regulating movements of these organelles. Following simultaneous stimulation of CHO cells with both extracellular ATP and lysophosphatidic acid, an arrest of peroxisomes was observed. This block of motility was shown to be dependent on signaling cascades involving heterotrimeric G proteins of the class G_i/G_o, phospholipase C, calcium influx, and activation of protein kinase C as well as of mitogen-activated protein kinase. Cytosolic phospholipase A₂ is a point of convergence for these pathways, resulting in the release of arachidonic acid. This signaling pathway is specific for peroxisomes and does not influence motility of mitochondria, lysosomes, or endosomes. However, since the cytoskeleton and its associated proteins including the motor proteins play an important role in mediating motility of all cell organelles, it may well be that variant signaling cascades exist ensuring specific regulation of each distinct compartment.Abbreviations AA arachidonic acid - ATPS adenosine-5-O-(3-thiotriphosphate) - cAMP cyclic adenosine monophosphate - CaM-PK calmodulin-dependent protein kinase - CLIP cytosolic linker protein - DAG diacylglycerol - DiC₈ 1,2-dioctanoyl-sn-glycerol - GFP green-fluorescent protein - GTPS guanosine-5-O-(3-thiotriphosphate) - IP₃ inositol trisphosphate - LPA lysophosphatidic acid - MAPK mitogen-activated protein kinase - MEK MAPK kinase - PKA protein kinase A - PKC protein kinase C - cPKC classical PKC isoforms - PLA₂ phospholipase A₂ - PLAP PLA₂-activating proteinpeptide - PLC phospholipase C - PP2A protein phosphatase 2A     

From gene to product in yeast: production of fungal cutinase

In the mid-1970s, information technology and recombinant DNA technology were considered as the breakthrough technologies of the final quarter of the 20th century. Now, about 25 years later, information technology has penetrated deeply into our society and nearly everyone uses this technology. Compared to the formidable success of information technology, the progress in the commercialization of recombinant DNA technology is moderate, even when taking into account that all that is related to the technological application of biological sciences needs extensive safety testing. However, there are signs that the speed of this commercialization will increase in the first decade of the 21st century. Moreover, new breakthroughs in our understanding of the complete genetic make up of eukaryotes will contribute to this increase in speed. An important aspect of the commercialization of this technology is the development of cells as factories for the production of valuable and/or useful molecules. Lower eukaryotes, such as yeasts and molds, are the most promising candidates to become the factories of the future, but at present these factories still contains a lot of process lines that may be superfluous under the well controlled conditions in fermentors. On the other hand, the speed and yield of these cellular production lines can be increased by eliminating the rate-determining steps of these process lines. In this contribution to the European Union symposium from Cell to Factory, some steps in the improvement of S. cerevisiae as cell factories for (heterologous) hydrophobic molecules are presented.     

Cell cycle regulation of the microtubular cytoskeleton

The microtubular element of the plant cytoskeleton undergoes dramatic architectural changes in the course of the cell cycle, specifically at the entry into and exit from mitosis. These changes underlie the acquisition of specialized properties and functions involved, for example, in the equal segregation of chromosomes and the correct positioning and formation of the new cell wall. Here we review some of the molecular mechanisms by which the dynamics and the organization of microtubules are regulated and suggest how these mechanisms may be under the control of cell cycle events.     

The yeast polyadenylate-binding protein (PAB1) gene acts as a disease lesion mimic gene when expressed in plants

We have expressed the gene (PAB1) encoding the yeast polyadenylate-binding protein (Pab1p) in tobacco. Plants that accumulate the Pab1p display a range of abnormalities, ranging from a characteristic chlorosis in leaves to a necrosis and large inhibition of growth. The severity of these abnormalities reflects the levels of yeast Pab1p expression in the transgenic plants. In contrast, no obvious differences could be seen in callus cultures between the transgene and vector control. Plants that display PAB-associated abnormalities were resistant to a range of plant pathogens, and had elevated levels of expression of a pathogenesis-related gene. These two properties – impairment of growth and induction of defense responses – indicate that the yeast PAB1 gene can act as a disease lesion mimic gene in plants.     

Osmotic adjustment in triticales grown in presence of NaCl

Growth and Na⁺, K⁺, Cl^-, proteins, sugars and proline concentrations were measured in three triticale genotypes M2A, DF99 and Asseret grown on nutrient solution with or without 75 mM NaCl. In saline conditions, leaf area of the three triticales was reduced by 50 % and dry to fresh mass ratio increased. Total protein concentration was diminished by 10 %. K⁺ concentration decreased whereas Na⁺ and Cl^- accumulated in roots and shoots of salt-stressed plants. This ion accumulation was greater in roots of Asseret than in roots of the other triticales. Soluble sugar concentration increased in M2A and Asseret and decreased in DF99. Proline concentration increased in M2A and DF99 and decreased in Asseret. Osmotic adjustment was essentially realized by Na⁺ and Cl^- uptake. Non-reducing sugars and proline contributed too, but to a lesser extent. This revised version was published online in July 2006 with corrections to the Cover Date.     

Evidence for specific RNA/protein interactions in the differential segment of the W sex chromosome in the amphibian Pleurodeles waltl

Pleurodeles exhibits a ZZ/ZW system of GSD (genotype sex determination). However, the Z and W sex chromosomes appear to be morphologically identical. A short RNA sequence is described that was specifically bound to lampbrush loops in the differential segment of the sexual bivalent IV. The distribution of these labeled loops in experimentally produced ZZ and WW females enabled us to demonstrate that such labeled loops were perfectly correlated with the W chromosome. Therefore, this RNA sequence constitutes an excellent marker for the W differential segment. Furthermore, analysis of the labeled loops under various experimental conditions suggested that their labeling is caused by specific interactions between this RNA sequence and lampbrush loop-associated proteins (RNA/protein interactions). North-western assays revealed that nuclear polypeptide(s) of 65 kDa could be responsible for such binding.     

pEg7, a New Xenopus Protein Required for Mitotic Chromosome Condensation in Egg Extracts

We have isolated a cDNA, Eg7, corresponding to a Xenopus maternal mRNA, which is polyadenylated in mature oocytes and deadenylated in early embryos. This maternal mRNA encodes a protein, pEg7, whose expression is strongly increased during oocyte maturation. The tissue and cell expression pattern of pEg7 indicates that this protein is only readily detected in cultured cells and germ cells. Immunolocalization in Xenopus cultured cells indicates that pEg7 concentrates onto chromosomes during mitosis. A similar localization of pEg7 is observed when sperm chromatin is allowed to form mitotic chromosomes in cytostatic factor-arrested egg extracts. Incubating these extracts with antibodies directed against two distinct parts of pEg7 provokes a strong inhibition of the condensation and resolution of mitotic chromosomes. Biochemical experiments show that pEg7 associates with Xenopus chromosome-associated polypeptides C and E, two components of the 13S condensin.     

PROTEINS IMMUNOLOGICALLY RELATED TO DEHYDRINS IN FUCOID ALGAE

Adult fucoid algae on Atlantic shores have well-characterized, species-specific tolerances to the varying levels of desiccation that occur from the low to high intertidal zones; however, less is known about embryonic tolerances and their mechanistic basis. We investigated this by 1) exposing embryos of Fucus evanescens C. Agardh, F. spiralis L., and F. vesiculosus L. from the Maine shore to osmotic desiccation in hypersaline seawater and 2) examining whether these embryos contain species-specific dehydrins, proteins first identified in higher plants that are hypothesized to confer tolerance to dehydration. Embryonic survival when cultured in hypersaline seawater >100 practical salinity units (psu) correlated with the position of these species in the intertidal zone (F. spiralis > F. vesiculosus > F. evanescens), but all 1-day-old embryos of these species tolerated treatment with 100 psu or lower seawater. Proteins (17–105 kDa) immunologically related to dehydrins were detected on western blots with dehydrin antibodies raised against a synthetic peptide representing the conserved motif of dehydrins in higher plants. These proteins were constitutive and unstable when subjected to prolonged (>15 min) temperatures above 55° C, unlike most higher plant dehydrins, which are inducible and remain soluble at 75°–100° C. The presence of these proteins was species- and stage-specific. Sperm of F. vesiculosus had a characteristic protein of 76 kDa, whereas eggs and embryos (6 h to 3 days old) had a 92-kDa protein. By 1 week of age, expression of the 92-kDa protein decreased, and the 35-kDa protein of adults was present. Embryos of A. nodosum L. and Pelvetia compressa J. Agardh DeToni contained an 85-kDa protein rather than the 92-kDa protein of Fucus embryos (F. distichus L., F. evanescens, F. spiralis, and F. vesiculosus). The 92-kDa protein became more abundant in embryos exposed to hyperosmotic seawater at 50 psu (F. evanescens and F. vesiculosus) or 150 psu (F. spiralis); however, dehydrin-like proteins of some molecular masses decreased in abundance simultaneously. Further characterization of these proteins is required to establish whether they protect embryos against intertidal desiccation.