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Precursor protein translocation across the outer chloroplast membrane depends on the action of the Toc complex, containing GTPases as recognizing receptor components. The G domains of the GTPases are known to dimerize. In the dimeric conformation an arginine contacts the phosphate moieties of bound nucleotide in trans. Kinetic studies suggested that the arginine in itself does not act as an arginine finger of a reciprocal GTPase-activating protein (GAP). Here we investigate the specific function of the residue in two GTPase homologues. Arginine to alanine replacement variants have significantly reduced affinities for dimerization compared with wild-type GTPases. The amino acid exchange does not impact on the overall fold and nucleotide binding, as seen in the monomeric x-ray crystallographic structure of the Arabidopsis Toc33 arginine-alanine replacement variant at 2.0A. We probed the catalytic center with the transition state analogue GDP/AlF(x) using NMR and analytical ultracentrifugation. AlF(x) binding depends on the arginine, suggesting the residue can play a role in catalysis despite the non-GAP nature of the homodimer. Two non-exclusive functional models are discussed: 1) the coGAP hypothesis, in which an additional factor activates the GTPase in homodimeric form; and 2) the switch hypothesis, in which a protein, presumably the large Toc159 GTPase, exchanges with one of the homodimeric subunits, leading to activation.
Glucokinase (GK) is a key enzyme of glucose metabolism in liver and pancreatic beta-cells, and small molecule activators of GK (GKAs) are under evaluation for the treatment of type 2 diabetes. In liver, GK activity is controlled by the GK regulatory protein (GKRP), which forms an inhibitory complex with the enzyme. Here, we performed isothermal titration calorimetry and surface plasmon resonance experiments to characterize GK-GKRP binding and to study the influence that physiological and pharmacological effectors of GK have on the protein-protein interaction. In the presence of fructose-6-phosphate, GK-GKRP complex formation displayed a strong entropic driving force opposed by a large positive enthalpy; a negative change in heat capacity was observed (Kd = 45 nm, DeltaH = 15.6 kcal/mol, TDeltaS = 25.7 kcal/mol, DeltaCp = -354 cal mol(-1) K(-1)). With k(off) = 1.3 x 10(-2) s(-1), the complex dissociated quickly. The thermodynamic profile suggested a largely hydrophobic interaction. In addition, effects of pH and buffer demonstrated the coupled uptake of one proton and indicated an ionic contribution to binding. Glucose decreased the binding affinity between GK and GKRP. This decrease was potentiated by an ATP analogue. Prototypical GKAs of the amino-heteroaryl-amide type bound to GK in a glucose-dependent manner and impaired the association of GK with GKRP. This mechanism might contribute to the antidiabetic effects of GKAs.
In this study I analysed past and recent Daphnia populations from Lake Constance and Greifensee. Herefore, I first established a set of microsatellite markers applicable to European Hyalodaphnia species (chapter 1). Primers were also identified for species specific fragment lengths. 32 markers were then available to characterize the resting egg banks of Daphnia galeata and D. hyalina. Chapter 2 presents the reconstruction of the taxonomic composition in these two ecologically different lakes. This part of my work shows that the eutrophication that occurred in both lakes in the mid of the last century has strongly influenced the Daphnia populations. In both lakes Daphnia galeata established and hybridized with the indigenous D. hyalina. Interspecific hybridization resulted in introgression on the mitochondrial and nuclear level. In chapter 3 resting eggs from the sediments of the 1960s, 1970s, 1980s, 1990s and 2000s were characterized with microsatellite markers. The aim was to specify the extent of interspecific hybridization and nuclear introgression assuming that the genetic exchange between both species has an impact on their adaptation to their habitat. In life history experiments D. galeata and D. galeata x hyalina clones hatched from different time periods showed significant differential responses to food quality. Therefore, the question had to be answered how the Daphnia resting egg bank and the planktonic population are connected. In chapter 4 hatching experiments were conducted to bridge this gap of scientific knowledge in the life cycle of cyclic parthenogenetic waterfleas. Only D. galeata individuals were able to establish a clonal lineage after maturity. All observed recombinant individuals did not reproduce at all or firstly went through another sexual phase of reproduction i.e. produced resting eggs. In order to compare the findings of chapter 4 with the taxon composition of the recent planktonic population of Daphnia in Lake Constance, samples were taken over one season (between May 2005 and September 2006). During the season, the taxonomic composition of Daphnia changes severely with D. galeata being most abundant during the warm season and D. hyalina in the cold season. Moreover, some individuals were detected, that did not follow this pattern. With mitochondrial analysis those individuals were identified as mitochondrial introgressants and processed to life history experiments. Significant differences in the somatic growth rate under different temperatures (5°C, 12.5°C and 20°C) were related to the origin of the mitochondrial genome rather than the nuclear taxonomic assignment of the individual.
The findings of this study show that all organisms exposed to rapid ecological changes and their microevolutionary reaction to those.
A highly efficient method for chromosomal integration of cloned DNA into Methanosarcina spp. was developed utilizing the site-specific recombination system from the Streptomyces phage PhiC31. Host strains expressing the PhiC31 integrase gene and carrying an appropriate recombination site can be transformed with non-replicating plasmids carrying the complementary recombination site at efficiencies similar to those obtained with self-replicating vectors. We have also constructed a series of hybrid promoters that combine the highly expressed M. barkeri PmcrB promoter with binding sites for the tetracycline-responsive, bacterial TetR protein. These promoters are tightly regulated by the presence or absence of tetracycline in strains that express the tetRgene. The hybrid promoters can be used in genetic experiments to test gene essentiality by placing a gene of interest under their control. Thus, growth of strains with tetR-regulated essential genes becomes tetracycline-dependent. A series of plasmid vectors that utilize the site-specific recombination system for construction of reporter gene fusions and for tetracycline regulated expression of cloned genes are reported. These vectors were used to test the efficiency of translation at a variety of start codons. Fusions using an ATG start site were the most active, whereas those using GTG and TTG were approximately one half or one fourth as active, respectively. The CTG fusion was 95% less active than the ATG fusion.
Plastids are complex plant organelles fulfilling essential physiological functions, such as photosynthesis and amino acid metabolism. The majority of proteins required for these functions are encoded in the nuclear genome and synthesized on cytosolic ribosomes as precursors, which are subsequently translocated across the outer and inner membrane of the organelle. Their targeting to the organelle is ensured by a so called transit peptide, which is specifically recognized by GTP-dependent receptors Toc159 and Toc34 at the cytosolic side of outer envelope. They cooperatively regulate the insertion of the precursor protein into the channel protein Toc75, thereby initiating the translocation process. Toc34 is regarded as the primary receptor, while Toc159 probably provides the driving force for the insertion. Precursor transfer is achieved by the physical interaction between both receptors in the GTP loaded state. One translocon unit, also called the Toc core complex, is formed by four molecules Toc34, four molecules Toc75 and one molecule Toc159. In the GDP-loaded state, Toc34 preferably forms homodimers, whose physiological function was investigated in the presented study. It could be shown that the dissociation of GDP and therefore the nucleotide exchange are inhibited by the homodimeric state of Toc34. Dissociation of the homodimer is induced by the recognition of a precursor protein, which renders the binding of GTP and subsequent interaction with Toc159 possible. Thus, the homodimeric conformation could reflect an inactive state of the translocon, preventing GTP consumption in the absence of a precursor protein. Both homodimerization as well as heterodimerization of the receptor are regulated by phosphorylation, which could be demonstrated by in vitro and in vivo approaches using atToc33 from Arabidopsis thaliana as a model system. Since the phosphorylated form of Toc34 cannot be assembled with the Toc core complex, it can be concluded that the interactions between GTPase domains not only regulate the transfer of precursor proteins, but also warrant the integrity of the translocon.
Quantitative analysis of snoRNA association with pre-ribosomes and release of snR30 by Rok1 helicase
(2008)
In yeast, three small nucleolar RNAs (snoRNAs) are essential for the processing of pre-ribosomal RNA—U3, U14 and snR30—whereas 72 non-essential snoRNAs direct site-specific modification of pre-rRNA. We applied a quantitative screen for alterations in the pre-ribosome association to all 75 yeast snoRNAs in strains depleted of eight putative helicases implicated in 40S subunit synthesis. For the modification-guide snoRNAs, we found no clear evidence for the involvement of these helicases in the association or dissociation of pre-ribosomes. However, the DEAD box helicase Rok1 was required specifically for the release of snR30. Point mutations in motif I, but not in motif III, of the helicase domain of Rok1 impaired the release of snR30, but this was less marked than in strains depleted of Rok1, and resulted in a dominant-negative growth phenotype. Dissociation of U3 and U14 from pre-ribosomes is also dependent on helicases, suggesting that release of the essential snoRNAs might differ mechanistically from release of the modification-guide snoRNAs. Keywords: ribosome biogenesis; RNA helicase; snoRNA
The reggie protein family consists of two homologous members, reggie-1 and reggie-2, also termed flotillin-2 and flotillin-1, respectively, that are ubiquitously expressed and evolutionarily well conserved, suggesting an important but so far ill-defined function. In various cell types, both reggies have been found to be constitutively associated with lipid rafts by means of acylation modifications and oligomerization. Lipid rafts are glycosphingolipid- and cholesterol-rich membrane microdomains which have been implicated in several cellular processes including membrane transport and signal transduction through growth factor receptors. However, the molecular details of these processes are still poorly understood. With the observation that reggies colocalize with activated glycosylphosphatidylinositolanchored proteins (GPI-APs) and Fyn kinase in rafts, a role for these proteins in signaling events has been suggested. In agreement with that, we have previously shown that reggie-1 becomes multiply tyrosine phosphorylated by Src kinases in response to epidermal growth factor (EGF) stimulation, pointing to a function for reggie-1 in growth factor signaling. Furthermore, overexpression of reggie-1 enhances spreading on fibronectin substrate in a tyrosine-dependent manner, thus revealing a role for reggie-1 in regulation of actin cytoskeleton through growth factor receptors. Due to the similarity shared by reggie proteins at amino acid level and to their ability to form hetero-oligomeric complexes, the first aim of this study was to analyze the putative tyrosine phosphorylation of reggie-2 in growth factor stimulated cells. Similarly to reggie-1, reggie-2 was found to be multiply tyrosine phosphorylated by Src kinase and to exist in a molecular complex with Src, with the degree of co-immunoprecipitation dependent on the activity of Src. Recent studies from us have also shown that administration of EGF results in the endocytosis of reggie-1 from the plasma membrane into endosomes, which is in line with a proposed role for reggies in membrane trafficking processes. In order to characterize in detail the endocytic mechanism that mediates the uptake of reggie-1, the dependency of reggie-1 endocytosis on clathrin and dynamin was investigated by means of overexpressing a variant form of Eps15 or a dominant negative form of dynamin-2. In either case the translocation of reggie-1 into endosomes in response to EGF was not affected, and this, together with the results that reggie-1 colocalized with cholera toxin (CTX) but not with transferrin receptor (TfnR) during EGF signaling, indicates that reggie-1 is taken up by means of a dynaminindependent, raft-mediated pathway. These findings are very well in line with recent data showing the pathway of entry into cells of reggie-2 as a raft-mediated endocytic pathway. The endocytosis of reggie-2 in response to EGF was also analyzed in this study. Similarly to reggie-1, in growth factor stimulated cells reggie-2 underwent a translocation from the plasma membrane to endosomes where the two reggies were found to colocalize with each other, suggesting that epidermal growth factor signaling might trigger the endocytosis of reggie oligomers. In addition, colocalization with both the late endosomal marker LAMP3/CD63 and epidermal growth factor receptor (EGFR) was detected, again indicating a function for reggies in signal transduction through growth factor receptors. EGFR has been reported to localize in rafts but, although this association is thought to be functional during EGF stimulation, how segregation of EGFR into rafts modulates its endocytosis and signaling is still under debate. Since reggie oligomers have recently been suggested to define a raft subtype, a further aim of this study was to investigate whether the depletion of reggies by means of small interfering RNA could interfere with the signaling and the trafficking through EGFR. Knockdown of reggie-2 resulted in an altered tyrosine phosphorylation of EGFR in response to EGF, while the degree of ubiquitination was not affected. Less efficient phosphorylation of tyrosine residues, especially of those which are docking sites for Grb2 and Shc, led in turn to an impaired activation of p38 and ERK1/2 MAPKs. Depletion of reggie-2 did not affect the early trafficking of activated EGFRs, with receptors being endocytosed and delivered to late endosomes as efficiently as in control cells. This would be in line with the normal degree of ubiquitination observed for EGFR, as ubiquitin moieties have been proposed to represent sorting tags that ensure receptor endocytosis into early endosomes and its proper intracellular trafficking. On the contrary, after prolonged EGF stimulation, depletion of reggie-2 resulted in a decreased downregulation of both receptor-bound ligand and EGFR, and in their accumulation in intracellular vesicles, thus pointing to a role for reggie-2 in the degradative pathway. Taken all together, these data ndicate that the association of EGFR with reggie-microdomains is likely to be important for proper receptor trafficking and signaling.
The growth of blood vessels is crucial for organ growth in the embryo and repair of wounded tissues in the adult. An imbalance in this process contributes to numerous malignant, inflammatory, ischemic, infectious and immune disorders (Ferrara et al., 2003). Postnatal neovascularization occurs through the recruitment of progenitor cells and angiogenesis. Integrins are heterodimeric cell surface molecules and are the main receptors for extracellular matrix proteins. Regulation of integrin activation is crucial during embryonic development and during adult life. Dysregulation of integrin activity leads to severe diseases. In this study, we have demonstrated that Rap1, a small GTPase regulating integrin activity, and its GEF Epac1 are expressed in both EPC and endothelial cells. Moreover, the pharmacological activator of Epac activates the small GTPase Rap1 in progenitor cells. In parallel the angiogenic growth factors VEGF and bFGF activate Rap1 in endothelial cells. In addition, the regulation of Rap1 activity in EPC and in endothelial cells plays an important role in the regulation of migration and adhesion to matrix proteins, by regulating the activity of different integrins, a mechanism known as integrin inside‐out signaling. Furthermore, regulation of Rap1 activity affects probably indirectly through outside‐in signaling of integrins the activity of several and crucial proteins such PKB/Akt and focal adhesion kinase in endothelial cells. In line with these results, we have demonstrated that Rap1 activity affect angiogenesis, homing of EPC to ischemic tissues and thereby postnatal neovascularization. The understanding how Rap1 regulates integrin activity in endothelial cells is still not completely clear, for example we have demonstrated that the known effectors of Rap1 mediating the increase of integrin activity in T and B cells, such as RAPL and RIAM are, respectively, either not increasing integrin activity or not expressed in endothelial cells. We aim to find the effector of Rap1 promoting integrin activity in endothelial cells and how RAPL regulates integrin functions and angiogenesis. Moreover data from us and others using genetic models and generation of Rap1a or Rap1b deficient mice or deficient for Rap1a and Rap1b led to embryonic lethality suggesting that Rap1 is a key node protein during embryonic development. The development of conditionnal Rap1a/b endothelial/pericytes restricted deficient mice will help us to decipher more precisely the role of Rap1 during vascular development and angiogenesis.
Our understanding of the impact of recombination, mutation, genetic drift and selection on the evolution of a single gene is still limited. Here we investigate the impact of all of these evolutionary forces at the complementary sex determiner (csd) gene which evolves under a balancing mode of selection. Females are heterozygous at the csd gene and males are hemizygous; diploid males are lethal and occur when csd is homozygous. Rare alleles thus have a selective advantage, are seldom lost by the effect of genetic drift and are maintained over extended periods of time when compared to neutral polymorphisms. Here, we report on the analysis of 17, 19 and 15 csd alleles of Apis cerana, Apis dorsata and Apis mellifera honey bees respectively. We observed great heterogeneity of synonymous (pi S) and nonsynonymous (pi N) polymorphisms across the gene, with a consistent peak in exon 6 and 7. We propose that exons 6 and 7 encode the potential specifying domain (csd-PSD) which has accumulated elevated nucleotide polymorphisms over time by balancing selection. We observed no direct evidence that balancing selection favors the accumulation of nonsynonymous changes at csd-PSD (pi N/pi S ratios are all < 1, ranging from 0.6 to 0.95). We observed an excess of shared nonsynonymous changes, which suggests that strong evolutionary constraints are operating at csd-PSD resulting in the independent accumulation of the same nonsynonymous changes in different alleles across species (convergent evolution). Analysis of a csd-PSD genealogy revealed relatively short average coalescence times (~6 million years), low average synonymous nucleotide diversity (pi S < 0.09) and a lack of trans-specific alleles which substantially contrasts with previously analyzed loci under strong balancing selection. We excluded the possibility of a burst of diversification after population bottlenecking and intragenic recombination as explanatory factors, leaving high turn-over rates as the explanation for this observation. By comparing observed allele richness and average coalescence times with a simplified model of csd-coalescence, we found that small long term population sizes (i.e. Ne <104), but not high mutation rates, can explain short maintenance times, implicating a strong impact of genetic drift on the molecular evolution of highly social honey bees.
Background Drought is the major constraint to increase yield in chickpea (Cicer arietinum). Improving drought tolerance is therefore of outmost importance for breeding. However, the complexity of the trait allowed only marginal progress. A solution to the current stagnation is expected from innovative molecular tools such as transcriptome analyses providing insight into stress-related gene activity, which combined with molecular markers and expression (e)QTL mapping, may accelerate knowledge-based breeding. SuperSAGE, an improved version of the serial analysis of gene expression (SAGE) technique, generating genome-wide, high-quality transcription profiles from any eukaryote, has been employed in the present study. The method produces 26 bp long fragments (26 bp tags) from defined positions in cDNAs, providing sufficient sequence information to unambiguously characterize the mRNAs. Further, SuperSAGE tags may be immediately used to produce microarrays and probes for real-time-PCR, thereby overcoming the lack of genomic tools in non-model organisms. Results We applied SuperSAGE to the analysis of gene expression in chickpea roots in response to drought. To this end, we sequenced 80,238 26 bp tags representing 17,493 unique transcripts (UniTags) from drought-stressed and non-stressed control roots. A total of 7,532 (43%) UniTags were more than 2.7-fold differentially expressed, and 880 (5.0%) were regulated more than 8-fold upon stress. Their large size enabled the unambiguous annotation of 3,858 (22%) UniTags to genes or proteins in public data bases and thus to stress-response processes. We designed a microarray carrying 3,000 of these 26 bp tags. The chip data confirmed 79% of the tag-based results, whereas RT-PCR confirmed the SuperSAGE data in all cases. Conclusion This study represents the most comprehensive analysis of the drought-response transcriptome of chickpea available to date. It demonstrates that – inter alias – signal transduction, transcription regulation, osmolyte accumulation, and ROS scavenging undergo strong transcriptional remodelling in chickpea roots already 6 h after drought stress. Certain transcript isoforms characterizing these processes are potential targets for breeding for drought tolerance. We demonstrate that these can be easily accessed by micro-arrays and RT-PCR assays readily produced downstream of SuperSAGE. Our study proves that SuperSAGE owns potential for molecular breeding also in non-model crops.