Glycerol kinase of Escherichia coli is activated by interaction with the glycerol facilitator.

Glycerol transport is commonly cited as the only example of facilitated diffusion across the Escherichia coli cytoplasmic membrane. Two proteins, the glycerol facilitator and glycerol kinase, are involved in the entry of external glycerol into cellular metabolism. The glycerol facilitator is thought to act as a carrier or to form a selective pore in the cytoplasmic membrane, whereas the kinase traps the glycerol inside the cell as sn-glycerol-3-phosphate. We found that the kinetics of glycerol uptake in a facilitator-minus strain are significantly different from the kinetics of glycerol uptake in the wild type. Free glycerol was not observed inside wild-type cells transporting glycerol, and diffusion of glycerol across the cytoplasmic membrane was not the rate-limiting step for phosphorylation in facilitator-minus mutants. Therefore, the kinetics of glycerol phosphorylation are different, depending on the presence or absence of the facilitator protein. We conclude that there is an interaction between the glycerol facilitator protein and glycerol kinase that stimulates kinase activity, analogous to the hexokinase- and glycerol kinase-porin interactions in mitochondria.     

Cell expansion-mediated organ growth is affected by mutations in three EXIGUA genes

Organ growth depends on two distinct, yet integrated, processes: cell proliferation and post-mitotic cell expansion. Although the regulatory networks of plant cell proliferation during organ growth have begun to be unveiled, the mechanisms regulating post-mitotic cell growth remain mostly unknown. Here, we report the characterization of three EXIGUA (EXI) genes that encode different subunits of the cellulose synthase complex specifically required for secondary cell wall formation. Despite this highly specific role of EXI genes, all the cells within the leaf, even those that do not have secondary walls, display small sizes in the exi mutants. In addition, we found a positive correlation between cell size and the DNA ploidy levels in exi mutant leaves, suggesting that both processes share some regulatory components. Our results are consistent with the hypothesis that the collapsed xylem vessels of the exi mutants hamper water transport throughout the plant, which, in turn, limits the turgor pressure levels required for normal post-mitotic cell expansion during leaf growth.     

Glycerol kinase deficiency: evidence for complexity in a single gene disorder

Glycerol kinase deficiency (GKD) occurs as part of an Xp21 contiguous gene syndrome or as isolated GKD. The isolated form can be either symptomatic with episodic metabolic and central nervous system (CNS) decompensation or asymptomatic with hyperglycerolemia and glyceroluria only. To better understand the pathogenesis of isolated GKD, we sought individuals with point mutations in the GK coding region and measured their GK enzyme activities. We identified six individuals with missense mutations: four (N288D, A305V, M428T, and Q438R) among males who were asymptomatic and two (D198G, R405Q) in individuals who were symptomatic. GK activity measured in lymphoblastoid cell lines or fibroblasts was similar for the symptomatic and the asymptomatic individuals. Mapping of the individuals' missense mutations to the three-dimensional structure of Escherichia coli GK revealed that the symptomatic individuals' mutations are in the same region as a subset of the mutations among the asymptomatic individuals, adjacent to the active-site cleft. We conclude that, like many other disorders, GK genotype does not predict GKD phenotype. We hypothesize that the phenotype of an individual with GKD is a complex trait influenced by additional, independently inherited genes.     

JIL-1 kinase, a member of the male-specific lethal (MSL) complex, is necessary for proper dosage compensation of eye pigmentation in Drosophila

The upregulation of the JIL-1 kinase on the male X chromosome and its association with the male-specific lethal (MSL) complex suggest that JIL-1 may play a role in regulating dosage compensation. To directly test this hypothesis we measured eye pigment levels of mutants in the X-linked white gene in an allelic series of JIL-1 hypomorphic mutants. We show that dosage compensation of w(a) alleles that normally do exhibit dosage compensation was severely impaired in the JIL-1 mutant backgrounds. As a control we also examined a hypomorphic white allele w(e) that fails to dosage compensate in males due to a pogo element insertion. In this case the relative pigment level measured in males as compared to females remained approximately the same even in the most severe JIL-1 hypomorphic background. These results indicate that proper dosage compensation of eye pigment levels in males controlled by X-linked white alleles requires normal JIL-1 function.     

Glycerol kinase, the pacemaker for the dissimilation of glycerol in Escherichia coli

The activity of glycerol kinase is rate-limiting in the metabolism of glycerol by cells of Escherichia coli. A mutant strain producing a glycerol kinase resistant to inhibition by fructose-1,6-diphosphate grows faster than its wild-type parent on glycerol as the sole source of carbon and energy. The amount of intracellular fructose-1,6-diphosphate was determined for wild-type cells growing exponentially on glycerol. The water content of such cells was also determined, allowing calculation of the intracellular concentration of fructose-1,6-diphosphate. This value, 1.7 mm, is adequate to exert substantial inhibition on the wild-type glycerol kinase. The desensitization of glycerol kinase to feedback inhibition also enhances the power of glycerol to exert catabolite repression, both on the enzymes of the glycerol system itself and on those of the lactose system. However, desensitization of glycerol kinase alone does not eliminate the phenomenon of diauxic growth in a glucose-glycerol medium. Biphasic growth in such a medium is abolished if the altered enzyme is produced constitutively. The constitutive production of the mutant kinase at high levels, however, renders the cells vulnerable to glycerol. Thus, when the cells have been grown on a carbon source with a low power for catabolite repression, e.g., succinate, sudden exposure to glycerol leads to overconsumption of the nutrient and cell death.     

The Drosophila Jak kinase hopscotch is required for multiple developmental processes in the eye.

Jak kinases are critical signaling components in hematopoiesis. While a large number of studies have been conducted on the roles of Jak kinases in the hematopoietic cells, much less is known about the requirements for these tyrosine kinases in other tissues. We have used loss of function mutations in the Drosophila Jak kinase Hopscotch (Hop) to determine the role of Hop in eye development. We find that Hop is required for cell proliferation/survival in the eye imaginal disc, for the differentiation of photoreceptor cells, and for the establishment of the equator and of ommatidial polarity. These results indicate that hop activity is required for multiple developmental processes in the eye, both cell-autonomously and nonautonomously.     

Utilisation of glycerol and glycerol 3-phosphate is differently affected by the phosphotransferase system in Bacillus subtilis

Glycerol and glycerol 3-phosphate uptake in Bacillus subtilis does not involve the phosphotransferase system. In spite of this, B. subtilis mutants defective in the general components of the phosphotransferase system, EnzymeI or Hpr, are unable to grow with glycerol as sole carbon and energy source. Here we show that a Hpr mutant can grow on glycerol 3-phosphate and that glycerol 3-phosphate, but not glycerol, can induce glpD encoding glycerol-3-phosphate dehydrogenase. Induction of glpD also requires the glpP gene product which is a regulator of all known glp genes. Thus the phosphotransferase system general components do not interfere with the overall regulation of the glp regulon. Revertants of a Hpr mutant which can grown on glycerol carry mutations closely linked to the glp region at 75 degrees on the B. subtilis chromosomal map. This region contains the glpP, the glpFK and the glpD operons. The glpFK operon encodes the glycerol uptake facilitator (glpF) and glycerol kinase (glpK). The present results demonstrate that one of these genes, or their gene products, is the target for phosphotransferase system control of glycerol utilisation. Furthermore we conclude that utilisation of glycerol and glycerol 3-phosphate is differently affected by the phosphotransferase system in B. subtilis.     

Nuclear translocation of activated MAP kinase is developmentally regulated in the developing Drosophila eye

In proneural groups of cells in the morphogenetic furrow of the developing Drosophila eye phosphorylated mitogen activated protein kinase (MAPK) antigen is held in the cytoplasm for hours. We have developed a reagent to detect nuclear MAPK non-antigenically and report our use of this reagent to confirm that MAPK nuclear translocation is regulated by a second mechanism in addition to phosphorylation. This "cytoplasmic hold" of activated MAPK has not been observed in cell culture systems. We also show that MAPK cytoplasmic hold has an essential function in vivo: if it is overcome, developmental patterning in the furrow is disrupted.     

Spermatogenesis in mice is not affected by histone H1.1 deficiency

The linker histone subtype H1.1 belongs to the group of main-type histones and is synthesized in somatic tissues as well as in germ cells during the S phase of the cell cycle. In adult mice the histone gene H1.1 is expressed mainly in thymus, spleen, and testis. The single-copy gene coding for the H1.1 protein was eliminated by homologous recombination in mouse embryonic stem cells. Mice homozygous for the deficient H1.1 gene developed normally until the adult stage without H1.1 mRNA and H1.1 protein. No anatomic abnormalities could be detected. In addition, mice lacking the H1.1 gene were fertile and they showed normal spermatogenesis and testicular morphology.     

Sexual conflict is not counterbalanced by good genes in the laboratory Drosophila melanogaster model system

Sexual conflict theory is based on the observation that females of many species are harmed through their interactions with males. Direct harm to females, however, can potentially be counterbalanced by indirect genetic benefits, where females make up for a reduction in offspring quantity by an increase in offspring quality through a generic increase in offspring fitness (good genes) and/or one restricted to the context of sexual selection (sexy sons). Here, we quantify the magnitude of the good genes mechanism of indirect benefits in a laboratory-adapted population of Drosophila melanogaster. We find that despite high-standing genetic variance for fitness, females gain at most only a modest benefit through the good genes form of indirect benefits--far too little to counterbalance the direct cost of male-induced harm.     

X-linked liver phosphorylase kinase deficiency is associated with mutations in the human liver phosphorylase kinase alpha subunit.

Two Dutch patients with liver phosphorylase kinase (PhK) deficiency were studied for abnormalities in the PhK liver alpha (alpha L) subunit mRNA by reversed-transcribed-PCR (RT-PCR) and RNase protection assays. One patient, belonging to a large Dutch family that expresses X-linked liver PhK deficiency, had a C3614T mutation in the PhK alpha L coding sequence. The C3614T mutation leads to replacement of proline 1205 with leucine, which changes the composition of an amino acid region, containing amino acids 1195-1214 of the PhK alpha L subunit, that is highly conserved in different species. The patient showed normal levels of PhK alpha L mRNA. The second patient, from an unrelated family, was found to have a TCT (bp 419-421) deletion in the PhK alpha L coding sequence, resulting in a phenylalanine 141 deletion. The same deletion was found in the PhK alpha L coding sequence from lymphocytes of the patient's mother, together with a normal PhK alpha L coding sequence. The phenylalanine that is absent in the PhK alpha L coding sequence of the second patient is a highly conserved amino acid between species. Both the C3614T mutation and the TCT (bp 419-421) deletion were not found in a panel of 80 control X chromosomes. On the basis of these results, it is postulated that the mutations found are responsible for liver PhK deficiency in the two patients investigated.     

X-Ray structure of glycerol kinase complexed with an ATP analog implies a novel mechanism for the ATP-dependent glycerol phosphorylation by glycerol kinase.

Glycerol kinase (GK) catalyzes the Mg-ATP-dependent phosphorylation of glycerol which yields glycerol 3-phosphate. The 2.8 A new crystal structure of GK complexed with an ATP analog revealed an unexpected position of the gamma-phosphoryl group, which was 7.2 A distant from the 3-hydroxyl group of glycerol, 5.5 A away from the 3-phosphate of the product (glycerol 3-phosphate) and is stabilized by a beta-hairpin structure. Based on the presented crystal structure and the previously determined structures of GK product complexes, we propose a 3-D model of a nucleophilic in-line transfer mechanism for the ATP-dependent phosphorylation of glycerol by GK.     

Adipose deficiency and aberrant autophagy in a Drosophila model of MPS VII is corrected by pharmacological stimulators of mTOR

Mucopolysaccharidosis type VII (MPS VII) is a recessively inherited lysosomal storage disorder caused due to β-glucuronidase (β-GUS) enzyme deficiency. Prominent clinical symptoms include hydrops fetalis, musculoskeletal deformities, neurodegeneration and hepatosplenomegaly leading to premature death in most cases. Apart from these, MPS VII is also characterized as adipose storage deficiency disorder although the underlying mechanism of this lean phenotype in the patients or β-GUS-deficient mice still remains a mystery. We addressed this issue using our recently developed Drosophila model of MPS VII (the CG2135^-/- fly), which also exhibited a significant loss of body fat. We report here that the lean phenotype of the CG2135^?/? larvae is due to fewer number of adipocytes, smaller lipid droplets and reduced adipogenesis. Our data further revealed that there is an abnormal accumulation of autophagosomes in the CG2135^?/? larvae due to autophagosome-lysosome fusion defect. Decreased lysosome-mediated turnover also led to attenuated mTOR activity in the CG2135^?/? larvae. Interestingly, treatment of the CG2135^?/? larvae with mTOR stimulators, 3BDO or glucose, led to the restoration of mTOR activity with simultaneous correction of the autophagy defect and adipose storage deficiency. Our finding thus established a hitherto unknown mechanistic link between autophagy dysfunction, mTOR downregulation and reduced adiposity in MPS VII.     

Drosophila eye disc margin is a center for organizing long-range planar polarity

Planar polarity patterning involves long-range signaling and signal transduction. In Drosophila eye, Dishevelled (Dsh) is not only crucial for cell-autonomous transduction of a polarity signal(s) but is also involved in nonautonomous signaling function. To identify the sites for long-range polarity signaling in eye disc, we examined spatial and temporal conditions for nonautonomous Dsh function. Here we show that Dsh and its downstream factor Armadillo (Arm) are required in the border region of eye disc between the peripodial membrane (PM) and the disc proper (DP) for nonautonomous signaling. Conditional misexpression of Dsh or Arm at the posterior margin of the disc was sufficient to induce nonautonomous polarity reversals. A critical time window for the induction of such changes was approximately coincident with the timing of morphogenetic furrow initiation. Our data suggest that the disc margin is an essential site for organizing planar polarity during the initial stage of retinal morphogenesis.     

Enhancement of xylitol production in glycerol kinase disrupted Candida tropicalis by co-expression of three genes involved in glycerol metabolic pathway

Glycerol can be used as a primary carbon source by yeasts, little is known regarding glycerol metabolism in Candida tropicalis. In this study, glycerol kinase gene (gk) was disrupted from xylitol dehydrogenase gene (XYL2) knockout C. tropicalis strain BSXDH-3. The resultant gk knockout C. tropicalis strain was incapable to grow on glycerol. The cells growth on glycerol was resumed by co-expressing Scheffersomyces stipitis gcy1, 2 and 3 genes, which respectively encode NADP⁺-dependent glycerol dehydrogenase 1, 2 and 3, under the control of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter. NADPH-dependent xylitol production was higher in the engineered strain, termed “GK”, than in BSXDH-3. In fermentation experiments using glycerol as co-substrate with xylose, strain GK produced xylitol 0.85 and 1.28 g l^?1 h^?1 at the time periods of 16 and 24 h, respectively, which is 30 and 18 % higher at same time intervals in BSXDH-3. This is the first report of gk gene disruption and co-expression of gcy1, 2 and 3 genes for NADPH regeneration and enhanced xylitol production in C. tropicalis.