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https://www.readbyqxmd.com/read/28324143/rapid-customised-operon-assembly-by-yeast-recombinational-cloning
#1
Michael A Liu, Johanna J Kenyon, Jason Lee, Peter R Reeves
We have developed a system called the Operon Assembly Protocol (OAP), which takes advantage of the homologous recombination DNA repair pathway in Saccharomyces cerevisiae to assemble full-length operons from a series of overlapping PCR products into a specially engineered yeast-Escherichia coli shuttle vector. This flexible, streamlined system can be used to assemble several operon clones simultaneously, and each clone can be expressed in the same E. coli tester strain to facilitate direct functional comparisons...
March 21, 2017: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/28302728/a-high-and-phosphatidylinositol-4-phosphate-pi4p-dependent-atpase-activity-for-the-drs2p-cdc50p-flippase-after-removal-of-its-n-and-c-terminal-extensions
#2
Hassina Azouaoui, Cédric Montigny, Thibaud Dieudonné, Philippe Champeil, Aurore Jacquot, José Luis Vázquez-Ibar, Pierre Le Maréchal, Jakob Ulstrup, Miriam-Rose Ash, Joseph A Lyons, Poul Nissen, Guillaume Lenoir
P4-ATPases, also known as phospholipid flippases, are responsible for creating and maintaining transbilayer lipid asymmetry in eukaryotic cell membranes. Here, we use limited proteolysis to investigate the role of the N- and C-termini in ATP hydrolysis and auto-inhibition of the yeast flippase Drs2p/Cdc50p. We show that limited proteolysis of the detergent-solubilized and purified yeast flippase may result in more than one order of magnitude increase of its ATPase activity, which remains dependent on phosphatidylinositol-4-phosphate (PI4P), a regulator of this lipid flippase, and specific to a phosphatidylserine substrate...
March 16, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28288918/increased-carbon-dioxide-levels-stimulate-neutrophils-to-produce-microparticles-and-activate-the-nucleotide-binding-domain-like-receptor-3-inflammasome
#3
Stephen R Thom, Veena M Bhopale, JingPing Hu, Ming Yang
We hypothesized that elevations of carbon dioxide (CO2) commonly found in modern buildings will stimulate leukocytes to produce microparticles (MPs) and activate the nucleotide-binding domain-like receptor 3 (NLRP3) inflammasome due to mitochondrial oxidative stress. Human and murine neutrophils generate MPs with high interleukin-1β (IL-1β) content when incubated ex vivo in buffer equilibrated with 0.1-0.4% additional CO2. Enhanced MPs production requires mitochondrial reactive oxygen species production, which is mediated by activities of pyruvate carboxylase and phosphoenolpyruvate carboxykinase...
March 10, 2017: Free Radical Biology & Medicine
https://www.readbyqxmd.com/read/28272479/plasmalogen-biosynthesis-is-spatiotemporally-regulated-by-sensing-plasmalogens-in-the-inner-leaflet-of-plasma-membranes
#4
Masanori Honsho, Yuichi Abe, Yukio Fujiki
Alkenyl ether phospholipids are a major sub-class of ethanolamine- and choline-phospholipids in which a long chain fatty alcohol is attached at the sn-1 position through a vinyl ether bond. Biosynthesis of ethanolamine-containing alkenyl ether phospholipids, plasmalogens, is regulated by modulating the stability of fatty acyl-CoA reductase 1 (Far1) in a manner dependent on the level of cellular plasmalogens. However, precise molecular mechanisms underlying the regulation of plasmalogen synthesis remain poorly understood...
March 8, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28253836/flippant-an-r-package-for-the-automated-analysis-of-fluorescence-based-scramblase-assays
#5
Richard J Cotton, Birgit Ploier, Michael A Goren, Anant K Menon, Johannes Graumann
BACKGROUND: The lipid scrambling activity of protein extracts and purified scramblases is typically measured using a fluorescence-based assay. While the assay has yielded insight into the scramblase activity in crude membrane preparations, functional validation of candidate scramblases, stoichiometry of scramblase complexes as well as ATP-dependence of flippases, data analysis in its context has remained a task involving many manual steps. RESULTS: With the extension package "flippant" to R, a free software environment for statistical computing and graphics, we introduce an integrated solution for the analysis and publication-grade graphical presentation of dithionite scramblase assays and demonstrate its utility in revisiting an originally manual analysis from the publication record, closely reproducing the reported results...
March 3, 2017: BMC Bioinformatics
https://www.readbyqxmd.com/read/28220754/the-ap-2-complex-has-a-specialized-clathrin-independent-role-in-apical-endocytosis-and-polar-growth-in-fungi
#6
Olga Martzoukou, Sotiris Amillis, Amalia Zervakou, Savvas Christoforidis, George Diallinas
Filamentous fungi provide excellent systems for investigating the role of the AP-2 complex in polar growth. Using Aspergillus nidulans, we show that AP-2 has a clathrin-independent essential role in polarity maintenance and growth. This is in line with a sequence analysis showing that the AP-2 β subunit (β2) of higher fungi lacks a clathrin-binding domain, and experiments showing that AP-2 does not co-localize with clathrin. We provide genetic and cellular evidence that AP-2 interacts with endocytic markers SlaB(End4) and SagA(End3) and the lipid flippases DnfA and DnfB in the sub-apical collar region of hyphae...
February 21, 2017: ELife
https://www.readbyqxmd.com/read/28175960/erratum-to-phospholipid-flippases-attenuate-lps-induced-tlr4-signaling-by-mediating-endocytic-retrieval-of-toll-like-receptor-4
#7
Vincent A van der Mark, Mohammed Ghiboub, Casper Marsman, Jing Zhao, Remco van Dijk, Johan K Hiralall, Kam S Ho-Mok, Zoë Castricum, Wouter J de Jonge, Ronald P J Oude Elferink, Coen C Paulusma
No abstract text is available yet for this article.
April 2017: Cellular and Molecular Life Sciences: CMLS
https://www.readbyqxmd.com/read/28167678/ypk1-ypk2-kinases-maintain-rho1-at-the-plasma-membrane-by-flippase-dependent-lipid-remodelling-after-membrane-stresses
#8
Riko Hatakeyama, Keiko Kono, Satoshi Yoshida
The plasma membrane (PM) is frequently challenged by mechanical stresses. In budding yeast, TORC2-Ypk1/Ypk2 kinase cascade plays a critical role in PM stress responses by reorganizing the actin cytoskeleton via Rho1 GTPase. However, the molecular mechanism by which TORC2-Ypk1/Ypk2 regulates Rho1 is not well defined. Here, we found that Ypk1/Ypk2 maintain PM localization of Rho1 under PM stress via spatial reorganization of the lipids including phosphatidylserine (PS). Genetic evidence suggests that this process is mediated by the Lem3-containing lipid flippase...
February 6, 2017: Journal of Cell Science
https://www.readbyqxmd.com/read/28123063/lipid-flippases-promote-antiviral-silencing-and-the-biogenesis-of-viral-and-host-sirnas-in-arabidopsis
#9
Zhongxin Guo, Jinfeng Lu, Xianbing Wang, Binhui Zhan, Wanxiang Li, Shou-Wei Ding
Dicer-mediated processing of virus-specific dsRNA into short interfering RNAs (siRNAs) in plants and animals initiates a specific antiviral defense by RNA interference (RNAi). In this study, we developed a forward genetic screen for the identification of host factors required for antiviral RNAi in Arabidopsis thaliana Using whole-genome sequencing and a computational pipeline, we identified aminophospholipid transporting ATPase 2 (ALA2) and the related ALA1 in the type IV subfamily of P-type ATPases as key components of antiviral RNAi...
February 7, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28118404/slow-phospholipid-exchange-between-a-detergent-solubilized-membrane-protein-and-lipid-detergent-mixed-micelles-brominated-phospholipids-as-tools-to-follow-its-kinetics
#10
Cédric Montigny, Thibaud Dieudonné, Stéphane Orlowski, José Luis Vázquez-Ibar, Carole Gauron, Dominique Georgin, Sten Lund, Marc le Maire, Jesper V Møller, Philippe Champeil, Guillaume Lenoir
Membrane proteins are largely dependent for their function on the phospholipids present in their immediate environment, and when they are solubilized by detergent for further study, residual phospholipids are critical, too. Here, brominated phosphatidylcholine, a phospholipid which behaves as an unsaturated phosphatidylcholine, was used to reveal the kinetics of phospholipid exchange or transfer from detergent mixed micelles to the environment of a detergent-solubilized membrane protein, the paradigmatic P-type ATPase SERCA1a, in which Trp residues can experience fluorescence quenching by bromine atoms present on phospholipid alkyl chains in their immediate environment...
2017: PloS One
https://www.readbyqxmd.com/read/28118222/maintenance-and-regulation-of-asymmetric-phospholipid-distribution-in-human-erythrocyte-membranes-implications-for-erythrocyte-functions
#11
Nobuto Arashiki, Yuichi Takakuwa
PURPOSE OF REVIEW: The article summarizes new insights in the molecular mechanisms for the maintenance and regulation of the asymmetric distribution of phospholipids in human erythrocyte membranes. We focus on phosphatidylserine, which is primarily found in the inner leaflet of the membrane lipid bilayer under low Ca conditions (<1 μmol/l) and is exposed to the outer leaflet under elevated Ca concentrations (>1 μmol/l), when cells become senescent. Clarification of the molecular basis of phosphatidylserine flipping and scrambling is important for addressing long-standing questions regarding phosphatidylserine functions...
January 21, 2017: Current Opinion in Hematology
https://www.readbyqxmd.com/read/28114371/correction-the-o-antigen-flippase-wzk-can-substitute-for-murj-in-peptidoglycan-synthesis-in-helicobacter-pylori-and-escherichia-coli
#12
Wael Elhenawy, Rebecca M Davis, Jutta Fero, Nina R Salama, Mario F Feldman, Natividad Ruiz
[This corrects the article DOI: 10.1371/journal.pone.0161587.].
2017: PloS One
https://www.readbyqxmd.com/read/28109698/microbubble-assisted-ultrasound-induced-transient-phosphatidylserine-translocation
#13
Jean-Michel Escoffre, Marc Derieppe, Bart Lammertink, Clemens Bos, Chrit Moonen
Microbubble-assisted ultrasound (sonopermeabilization) results in reversible permeabilization of the plasma membrane of cells. This method is increasingly used in vivo because of its potential to deliver therapeutic molecules with limited cell damage. Nevertheless, the effects of sonopermeabilization on the plasma membrane remain not fully understood. We investigated the influence of sonopermeabilization on the transverse mobility of phospholipids, especially on phosphatidylserine (PS) externalization. We performed studies using optical imaging with Annexin V and FM1-43 probes to monitor PS externalization of rat glioma C6 cells...
April 2017: Ultrasound in Medicine & Biology
https://www.readbyqxmd.com/read/28069741/tor-complex-2-regulated-protein-kinase-fpk1-stimulates-endocytosis-via-inhibition-of-ark1-prk1-related-protein-kinase-akl1-in-saccharomyces-cerevisiae
#14
Françoise M Roelants, Kristin L Leskoske, Ross T A Pedersen, Alexander Muir, Jeffrey M-H Liu, Gregory C Finnigan, Jeremy Thorner
Depending on the stress, plasma membrane alterations activate or inhibit yeast Target of Rapamycin (TOR) Complex 2, which, in turn, upregulates or downregulates the activity of its essential downstream effector, protein kinase Ypk1. Through phosphorylation of multiple substrates, Ypk1 controls many processes that restore homeostasis. One such substrate is protein kinase Fpk1, which is negatively regulated by Ypk1. Fpk1 phosphorylates and stimulates flippases that translocate aminoglycerophospholipids from the outer to the inner leaflet of the plasma membrane...
January 9, 2017: Molecular and Cellular Biology
https://www.readbyqxmd.com/read/28061020/a-p4-atpase-subunit-of-the-cdc50-family-plays-a-role-in-iron-acquisition-and-virulence-in-cryptococcus-neoformans
#15
Guanggan Hu, Mélissa Caza, Erik Bakkeren, Matthias Kretschmer, Gaurav Bairwa, Ethan Reiner, James Kronstad
The pathogenic fungus Cryptococcus neoformans delivers virulence factors such as capsule polysaccharide to the cell surface to cause disease in vertebrate hosts. In this study, we screened for mutants sensitive to the secretion inhibitor brefeldin A to identify secretory pathway components that contribute to virulence. We identified an ortholog of the cell division control protein 50 (Cdc50) family of the noncatalytic subunit of type IV P-type ATPases (flippases) that establish phospholipid asymmetry in membranes and function in vesicle-mediated trafficking...
January 6, 2017: Cellular Microbiology
https://www.readbyqxmd.com/read/28057802/cfs1p-a-novel-membrane-protein-in-the-pq-loop-family-is-involved-in-phospholipid-flippase-functions-in-yeast
#16
Takaharu Yamamoto, Konomi Fujimura-Kamada, Eno Shioji, Risa Suzuki, Kazuma Tanaka
Type 4 P-type ATPases (P4-ATPases) function as phospholipid flippases, which translocate phospholipids from the exoplasmic leaflet to the cytoplasmic leaflet of the lipid bilayer, to generate and maintain asymmetric distribution of phospholipids at the plasma membrane and endosomal/Golgi membranes. The budding yeast Saccharomyces cerevisiae has four heteromeric flippases (Drs2p, Dnf1p, Dnf2p, and Dnf3p), associated with the Cdc50p family noncatalytic subunit, and one monomeric flippase, Neo1p They have been suggested to function in vesicle formation in membrane trafficking pathways, but details of their mechanisms remain to be clarified...
January 5, 2017: G3: Genes—Genomes—Genetics
https://www.readbyqxmd.com/read/28034798/phospholipid-flippases-dnfa-and-dnfb-exhibit-differential-dynamics-within-the-a-nidulans-spitzenk%C3%A3-rper
#17
Zachary Schultzhaus, Wenhui Zheng, Zonghua Wang, Rosa Mouriño-Pérez, Brian Shaw
The Spitzenkörper is a structure at the apex of growing cells in many filamentous fungi. Ultrastructural studies indicate that the Spitzenkörper is an organized mass of secretory vesicles, with different types of vesicles present in outer and inner layers. Here, we used live-cell imaging to demonstrate that the phospholipid flippases DnfA and DnfB, which preferentially localize to the outer and inner layers, respectively, exhibit different dynamics in the Spitzenkörper of Aspergillus nidulans. Additionally, deletion of dnfA partially destabilized the Spitzenkörper, while the depletion of cdc50, an essential β-subunit of most flippases, had dramatic effects on hyphal tip organization and morphology...
December 26, 2016: Fungal Genetics and Biology: FG & B
https://www.readbyqxmd.com/read/28024149/crystal-structure-of-the-mop-flippase-murj-in-an-inward-facing-conformation
#18
Alvin C Y Kuk, Ellene H Mashalidis, Seok-Yong Lee
Peptidoglycan (PG) protects bacteria from osmotic lysis, and its biogenesis is a key antibiotic target. A central step in PG biosynthesis is flipping of the lipid-linked PG precursor lipid II across the cytoplasmic membrane for subsequent incorporation into PG. MurJ, part of the multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) transporter superfamily, was recently shown to carry out this process. However, understanding of how MurJ flips lipid II, and of how MOP transporters operate in general, remains limited due to a lack of structural information...
February 2017: Nature Structural & Molecular Biology
https://www.readbyqxmd.com/read/28004795/coupled-atpase-adenylate-kinase-activity-in-abc-transporters
#19
Hundeep Kaur, Andrea Lakatos-Karoly, Ramona Vogel, Anne Nöll, Robert Tampé, Clemens Glaubitz
ATP-binding cassette (ABC) transporters, a superfamily of integral membrane proteins, catalyse the translocation of substrates across the cellular membrane by ATP hydrolysis. Here we demonstrate by nucleotide turnover and binding studies based on (31)P solid-state NMR spectroscopy that the ABC exporter and lipid A flippase MsbA can couple ATP hydrolysis to an adenylate kinase activity, where ADP is converted into AMP and ATP. Single-point mutations reveal that both ATPase and adenylate kinase mechanisms are associated with the same conserved motifs of the nucleotide-binding domain...
December 22, 2016: Nature Communications
https://www.readbyqxmd.com/read/27959517/the-ins-and-outs-of-lipid-flip-flop
#20
John S Allhusen, John C Conboy
Our current view of cellular membranes centers on the fluid-mosaic model, which envisions the cellular membrane as a "liquidlike" bilayer of lipids, cholesterol, and proteins that freely diffuse in two dimensions. In stark contrast, the exchange of materials between the leaflets of a bilayer was presumed to be prohibited by the large enthalpic barrier associated with translocating hydrophilic materials, such as a charged lipid headgroup, through the hydrophobic membrane core. This static picture with regard to lipid translocation (or "flip-flop" as it is affectionately known) has been a long-held belief in the study of membrane dynamics...
December 13, 2016: Accounts of Chemical Research
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