Kinetics of the ATP hydrolysis cycle of the nucleotide-binding domain of Mdl1 studied by a novel site-specific labeling technique

We have recently proposed a "processive clamp" model for the ATP hydrolysis cycle of the nucleotide-binding domain (NBD) of the mitochondrial ABC transporter Mdl1 (Janas, E., Hofacker, M., Chen, M., Gompf, S., van der Does, C., and Tampé, R. (2003) J. Biol. Chem. 278, 26862-26869). In this model, ATP binding to two monomeric NBDs leads to formation of an NBD dimer that, after hydrolysis of both ATPs, dissociates and releases ADP. Here, we set out to follow the association and dissociation of NBDs using a novel minimally invasive site-specific labeling technique, which provides stable and stoichiometric attachment of fluorophores. The association and dissociation kinetics of the E599Q-NBD dimer upon addition and removal of ATP were determined by fluorescence self-quenching. Remarkably, the rate of ATP hydrolysis of the wild type NBD is determined by the rate of NBD dimerization. In the E599QNBD, however, in which the ATP hydrolysis is 250-fold reduced, the ATP hydrolysis reaction controls dimer dissociation and the overall ATPase cycle. These data explain contradicting observations on the rate-limiting step of various ABC proteins and further demonstrate that dimer formation is an important step in the ATP hydrolysis cycle.     

The use of heavy nitrogen in quantitative proteomics experiments in plants

In the growing field of plant systems biology, there is an undisputed need for methods allowing accurate quantitation of proteins and metabolites. As autotrophic organisms, plants can easily metabolize different nitrogen isotopes, resulting in proteins and metabolites with distinct molecular mass that can be separated on a mass spectrometer. In comparative quantitative experiments, treated and untreated samples are differentially labeled by nitrogen isotopes and jointly processed, thereby minimizing sample-to-sample variation. In recent years, heavy nitrogen labeling has become a widely used strategy in quantitative proteomics and novel approaches have been developed for metabolite identification. Here, we present an overview of currently used experimental strategies in heavy nitrogen labeling in plants and provide background on the history and function of this quantitation technique.     

dEHBP1 controls exocytosis and recycling of Delta during asymmetric divisions

Notch signaling governs binary cell fate determination in asymmetrically dividing cells. Through a forward genetic screen we identified the fly homologue of Eps15 homology domain containing protein-binding protein 1 (dEHBP1) as a novel regulator of Notch signaling in asymmetrically dividing cells. dEHBP1 is enriched basally and at the actin-rich interface of pII cells of the external mechanosensory organs, where Notch signaling occurs. Loss of function of dEHBP1 leads to up-regulation of Sanpodo, a regulator of Notch signaling, and aberrant trafficking of the Notch ligand, Delta. Furthermore, Sec15 and Rab11, which have been previously shown to regulate the localization of Delta, physically interact with dEHBP1. We propose that dEHBP1 functions as an adaptor molecule for the exocytosis and recycling of Delta, thereby affecting cell fate decisions in asymmetrically dividing cells.     

Association of the FTO rs9939609 single nucleotide polymorphism with C-reactive protein levels

Adipose tissue is a key factor determining C-reactive protein (CRP) plasma levels. Variation at the fat-mass and obesity-associated (FTO) gene locus has been reported to be associated with increased body fat. We investigated whether the FTO rs9939609 T>A single nucleotide polymorphism might alter CRP levels in a population-based sample of 2,415 participants from a large prospective cohort study. Genotype/phenotype relationships were studied by linear trend analysis stratified by sex. The rs9939609 A-allele was significantly associated with CRP levels in both genders (men, +21%, P = 0.002; women, +14%, P = 0.01 per A-allele). The association was attenuated, but remained statistically significant after additional adjustment for BMI, waist-to-hip ratio, and other potential confounding factors (men, +14%, P = 0.03; women, +12%, P = 0.02; per A-allele). Similar results were obtained when subjects with CRP levels higher then 10 mg/l were excluded. Our data provide preliminary evidence that the FTO rs9939609 T>A polymorphism contributes to variation in plasma CRP levels independently of obesity indices.     

Split-CreERT2: Temporal Control of DNA Recombination Mediated by Split-Cre Protein Fragment Complementation

Background

DNA recombination technologies such as the Cre/LoxP system advance modern biological research by allowing conditional gene regulation in vivo. However, the precise targeting of a particular cell type at a given time point has remained challenging since spatial specificity has so far depended exclusively on the promoter driving Cre recombinase expression. We have recently established split-Cre that allows DNA recombination to be controlled by coincidental activity of two promoters, thereby increasing spatial specificity of Cre-mediated DNA recombination. To allow temporal control of split-Cre-mediated DNA recombination we have now extended split-Cre by fusing split-Cre proteins with the tamoxifen inducible ERT2 domain derived from CreERT2.

Methodology/Principal Findings

In the split-CreERT2 system, Cre-mediated DNA recombination is controlled by two expression cassettes as well as the time of tamoxifen application. By using two independent Cre-dependent reporters in cultured cells, the combination of NCre-ERT2+ERT2-CCre was identified as having the most favorable properties of all constructs tested, showing an induction ratio of about 10 and EC₅₀-values for 4-hydroxy-tamoxifen of 10 nM to 70 nM.

Conclusions/Significance

These characteristics of split-CreERT2 in vitro indicate that split-CreERT2 will be well suited for inducing DNA recombination in living mice harboring LoxP-flanked alleles. In this way, split-CreERT2 will provide a new tool of modern genetics allowing spatial and temporal precise genetic access to cell populations defined by the simultaneous activity of two promoters.     

Ratio‐dependent significance thresholds in reciprocal 15N‐labeling experiments as a robust tool in detection of candidate proteins responding to biological treatment

Metabolic labeling of plant tissues with ¹⁵N has become widely used in plant proteomics. Here, we describe a robust experimental design and data analysis workflow implementing two parallel biological replicate experiments with reciprocal labeling and series of 1:1 control mixtures. Thereby, we are able to unambiguously distinguish (i) inherent biological variation between cultures and (ii) specific responses to a biological treatment. The data analysis workflow is based on first determining the variation between cultures based on ¹⁵N/¹⁴N ratios in independent 1:1 mixtures before biological treatment is applied. In a second step, ratio‐dependent SD is used to define p‐values for significant deviation of protein ratios in the biological experiment from the distribution of protein ratios in the 1:1 mixture. This approach allows defining those proteins showing significant biological response superimposed on the biological variation before treatment. The proposed workflow was applied to a series of experiments, in which changes in composition of detergent resistant membrane domains was analyzed in response to sucrose resupply after carbon starvation. Especially in experiments involving cell culture treatment (starvation) prior to the actual biological stimulus of interest (resupply), a clear distinction between culture to culture variations and biological response is of utmost importance.     

Feedback Inhibition of Ammonium Uptake by a Phospho-Dependent Allosteric Mechanism in Arabidopsis

The acquisition of nutrients requires tight regulation to ensure optimal supply while preventing accumulation to toxic levels. Ammonium transporter/methylamine permease/rhesus (AMT/Mep/Rh) transporters are responsible for ammonium acquisition in bacteria, fungi, and plants. The ammonium transporter AMT1;1 from Arabidopsis thaliana uses a novel regulatory mechanism requiring the productive interaction between a trimer of subunits for function. Allosteric regulation is mediated by a cytosolic C-terminal trans-activation domain, which carries a conserved Thr (T460) in a critical position in the hinge region of the C terminus. When expressed in yeast, mutation of T460 leads to inactivation of the trimeric complex. This study shows that phosphorylation of T460 is triggered by ammonium in a time- and concentration-dependent manner. Neither Gln nor l-methionine sulfoximine–induced ammonium accumulation were effective in inducing phosphorylation, suggesting that roots use either the ammonium transporter itself or another extracellular sensor to measure ammonium concentrations in the rhizosphere. Phosphorylation of T460 in response to an increase in external ammonium correlates with inhibition of ammonium uptake into Arabidopsis roots. Thus, phosphorylation appears to function in a feedback loop restricting ammonium uptake. This novel autoregulatory mechanism is capable of tuning uptake capacity over a wide range of supply levels using an extracellular sensory system, potentially mediated by a transceptor (i.e., transporter and receptor).