Phospholipase D1 production of phosphatidic acid at the plasma membrane promotes exocytosis of large dense-core granules at a late stage

Substantial efforts have recently been made to demonstrate the importance of lipids and lipid-modifying enzymes in various membrane trafficking processes, including calcium-regulated exocytosis of hormones and neurotransmitters. Among bioactive lipids, phosphatidic acid (PA) is an attractive candidate to promote membrane fusion through its ability to change membrane topology. To date, however, the biosynthetic pathway, the dynamic location, and actual function of PA in secretory cells remain unknown. Using a short interference RNA strategy on chromaffin and PC12 cells, we demonstrate here that phospholipase D1 is activated in secretagogue-stimulated cells and that it produces PA at the plasma membrane at the secretory granule docking sites. We show that phospholipase D1 activation and PA production represent key events in the exocytotic progression. Membrane capacitance measurements indicate that reduction of endogenous PA impairs the formation of fusion-competent granules. Finally, we show that the PLD1 short interference RNA-mediated inhibition of exocytosis can be rescued by exogenous provision of a lipid that favors the transition of opposed bi-layer membranes to hemifused membranes having the outer leaflets fused. Our findings demonstrate that PA synthesis is required during exocytosis to facilitate a late event in the granule fusion pathway. We propose that the underlying mechanism is related to the ability of PA to alter membrane curvature and promote hemi-fusion.     

Investigating the in vivo activity of the DeaD protein using protein-protein interactions and the translational activity of structured chloramphenicol acetyltransferase mRNAs

Here, we report the use of an in vivo protein-protein interaction detection approach together with focused follow-up experiments to study the function of the DeaD protein in Escherichia coli. In this method, functions are assigned to proteins based on the interactions they make with others in the living cell. The assigned functions are further confirmed using follow-up experiments. The DeaD protein has been characterized in vitro as a putative prokaryotic factor required for the formation of translation initiation complexes on structured mRNAs. Although the RNA helicase activity of DeaD has been demonstrated in vitro, its in vivo activity remains controversial. Here, using a method called sequential peptide affinity (SPA) tagging, we show that DeaD interacts with certain ribosomal proteins as well as a series of other nucleic acid binding proteins. Focused follow-up experiments provide evidence for the mRNA helicase activity of the DeaD protein complex during translation initiation. DeaD overexpression compensates for the reduction of the translation activity caused by a structure placed at the initiation region of a chloramphenicol acetyltransferase gene (cat) used as a reporter. Deletion of the deaD gene, encoding DeaD, abolishes the translation activity of the mRNA with an inhibitory structure at its initiation region. Increasing the growth temperature disrupts RNA secondary structures and bypasses the DeaD requirement. These observations suggest that DeaD is involved in destabilizing mRNA structures during translation initiation. This study also provides further confirmation that large-scale protein-protein interaction data can be suitable to study protein functions in E. coli.     

Spore germination in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>: global gene expression patterns and cell cycle landmarks

Background  

Spore germination in the yeast Saccharomyces cerevisiae is a process in which non-dividing haploid spores re-enter the mitotic cell cycle and resume vegetative growth. To study the signals and pathways underlying spore germination we examined the global changes in gene expression and followed cell-cycle and germination markers during this process.     

What is the role of the helical domain of Gsalpha in the GTPase reaction?

Structural analysis of Gsalpha shows that it is composed of two domains: the ras-like domain (RD) that is conserved in all members of the GTPase superfamily and is homologous to the monomeric G-proteins (e.g., p21ras) and an alpha-helical domain (HD) that is unique to heterotrimeric G-proteins. Little is known about the function of the HD. Recent experiments by Bourne and co-workers, who expressed both the RD and the HD of Gsalpha separately and found that GTP hydrolysis is very slow if only recombinant RD is present but is accelerated when the HD is added, suggest that the HD serves as an intrinsic GTPase-activating protein (GAP). In this work, the GTP hydrolysis in Gsalpha was studied. The results obtained by calculating catalytic effects with and without the HD provide evidence for the role of the HD as a GAP. It is demonstrated that a major part of the catalysis is obtained because of an allosteric influence of the HD on the RD. Structural as well as energetic considerations suggest that the HD confines the RD to a more compact conformation, pushing the phosphate into an orientation where it is further stabilized, thus lowering the overall reaction barrier. The resemblance between the behavior of rasGAP and the HD suggests that the conclusion may be a general conclusion, applicable for all of the G-protein members.     

The ups and downs of biological timers

Background  

The need to execute a sequence of events in an orderly and timely manner is central to many biological processes, including cell cycle progression and cell differentiation. For self-perpetuating systems, such as the cell cycle oscillator, delay times between events are defined by the network of interacting proteins that propagates the system. However, protein levels inside cells are subject to genetic and environmental fluctuations, raising the question of how reliable timing is maintained.     

Respiratory Changes During Seed Germination. Histological Distribution of Respiratory Enzymes and Mobilization of Fat Reserves in Castor Bean Endosperm and Peanut Cotyledons

Germinating peanut cotvledons and germinating castor bean endosperm have been compared with respect to their rates of fat dissimilation and with respect to the anatomical distribution of respiratory activity. The lipid mobilization is much slower in peanut cotyledons than in castor bean endosperm. Light has essentially no effect on either system. As germination progresses, the majority of the succinic dehydrogenase and cytochrome oxidase activities become localized in the vein regions of peanut cotyledons. In the castor bean endosperm these two activities are uniformly distributed throughout the storage parenchyma and increase with germination until the organ becomes soft and visibly senescent.     

Linkage between the <Emphasis Type="Italic">I</Emphasis>-<Emphasis Type="Italic">3</Emphasis> gene for resistance to Fusarium wilt race 3 and increased sensitivity to bacterial spot in tomato

Key message

The negative association between the I - 3 gene and increased sensitivity to bacterial spot is due to linkage drag (not pleiotropy) and may be remedied by reducing the introgression size.

Abstract

Fusarium wilt is one of the most serious diseases of tomato (Solanum lycopersicum L.) throughout the world. There are three races of the pathogen (races 1, 2 and 3), and the deployment of three single, dominant resistance genes corresponding to each of these has been the primary means of controlling the disease. The I-3 gene was introgressed from S. pennellii and confers resistance to race 3. Although I-3 provides effective control, it is negatively associated with several horticultural traits, including increased sensitivity to bacterial spot disease (Xanthomonas spp.). To test the hypothesis that this association is due to linkage with unfavorable alleles rather than to pleiotropy, we used a map-based approach to develop a collection of recombinant inbred lines varying for portions of I-3 introgression. Progeny of recombinants were evaluated for bacterial spot severity in the field for three seasons, and disease severities were compared between I-3 introgression haplotypes for each recombinant. Results indicated that increased sensitivity to bacterial spot is not associated with the I-3 gene, but rather with an upstream region of the introgression. A survey of public and private inbred lines and hybrids indicates that the majority of modern I-3 germplasm contains a similarly sized introgression for which the negative association with bacterial spot likely persists. In light of this, it is expected that the development and utilization of a reduced I-3 introgression will significantly improve breeding efforts for resistance to Fusarium wilt race 3.

     

DNA Crossover Motifs Associated with Epigenetic Modifications Delineate Open Chromatin Regions in Arabidopsis

The rate of crossover, the reciprocal exchanges of homologous chromosomal segments, is not uniform along chromosomes differing between male and female meiocytes. To better understand the factors regulating this variable landscape, we performed a detailed genetic and epigenetic analysis of 737 crossover events in Arabidopsis thaliana. Crossovers were more frequent than expected in promoters. Three DNA motifs enriched in crossover regions and less abundant in crossover-poor pericentric regions were identified. One of these motifs, the CCN repeat, was previously unknown in plants. The A-rich motif was preferentially associated with promoters, while the CCN repeat and the CTT repeat motifs were preferentially associated with genes. Analysis of epigenetic modifications around the motifs showed, in most cases, a specific epigenetic architecture. For example, we show that there is a peak of nucleosome occupancy and of H3K4me3 around the CCN and CTT repeat motifs while nucleosome occupancy was lowest around the A-rich motif. Cytosine methylation levels showed a gradual decrease within ∼2 kb of the three motifs, being lowest at sites where crossover occurred. This landscape was conserved in the decreased DNA methylation1 mutant. In summary, the crossover motifs are associated with epigenetic landscapes corresponding to open chromatin and contributing to the nonuniformity of crossovers in Arabidopsis.