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Transposable Elements In Phytopathogenic Verticillium Spp.: Insights Into Genome Evolution And Inter- And Intra-Specific Diversification, Stefan G. Amyotte, Xiaoping Tan, Kayla Pennerman, Maria Del Mar Jimenez-Gasco, Steven J. Klosterman, Li-Jun Ma, Katherine F. Dobinson, Paola Veronese

Li-Jun Ma

Background Verticillium dahliae (Vd) and Verticillium albo-atrum (Va) are cosmopolitan soil fungi causing very disruptive vascular diseases on a wide range of crop plants. To date, no sexual stage has been identified in either microorganism suggesting that somatic mutation is a major force in generating genetic diversity. Whole genome comparative analysis of the recently sequenced strains VdLs.17 and VaMs.102 revealed that non-random insertions of transposable elements (TEs) have contributed to the generation of four lineage-specific (LS) regions in VdLs.17. Results We present here a detailed analysis of Class I retrotransposons and Class II “cut-and-paste” DNA elements detected in the sequenced …

Identification And Expression Analysis Of Methyl Jasmonate Responsive Ests In Paclitaxel Producing Taxus Cuspidata Suspension Culture Cells, Sangram K. Lenka, Nadia Boutaoui, Bibin Paulose, Kham Vongpaseuth, Jennifer Normanly, Susan C. Roberts, Elsbeth L. Walker

Jennifer Normanly

Background Taxol® (paclitaxel) promotes microtubule assembly and stabilization and therefore is a potent chemotherapeutic agent against wide range of cancers. Methyl jasmonate (MJ) elicited Taxus cell cultures provide a sustainable option to meet the growing market demand for paclitaxel. Despite its increasing pharmaceutical importance, the molecular genetics of paclitaxel biosynthesis is not fully elucidated. This study focuses on identification of MJ responsive transcripts in cultured Taxus cells using PCR-based suppression subtractive hybridization (SSH) to identify genes involved in global pathway control. Results Six separate SSH cDNA libraries of paclitaxel-accumulating Taxus cuspidata P991 cell lines were constructed at three different post-elicitation …

Foldeco: A Model For Proteostasis In E. Coli, Evan T. Powers, David L. Powers, Lila Gierasch

Lila Gierasch

To gain insight into the interplay of processes and species that maintain a correctly folded, functional proteome, we have developed a computational model called FoldEco. FoldEco models the cellular proteostasis network of the E. coli cytoplasm, including protein synthesis, degradation, aggregation, chaperone systems, and the folding characteristics of protein clients. We focused on E. coli because much of the needed input information—including mechanisms, rate parameters, and equilibrium coefficients—is available, largely from in vitro experiments; however, FoldEco will shed light on proteostasis in other organisms. FoldEco can generate hypotheses to guide the design of new experiments. Hypothesis generation leads to system-wide …

Contribution Of Taxane Biosynthetic Pathway Gene Expression To Observed Variability In Paclitaxel Accumulation In Taxus Suspension Cultures, Rohan A. Patil, Martin E. Kolewe, Jennifer Normanly, Elsbeth L. Walker, Susan C. Roberts

Jennifer Normanly

Variability in product accumulation is one of the major obstacles limiting the widespread commercialization of plant cell culture technology to supply natural product pharmaceuticals. Despite extensive process engineering efforts, which have led to increased yields, plant cells exhibit variability in productivity that is poorly understood. Elicitation of Taxus cultures with methyl jasmonate (MeJA) induces paclitaxel accumulation, but to varying extents in different cultures. In this work, cultures with different aggregation profiles were established to create predictable differences in paclitaxel accumulation upon MeJA elicitation. Expression of known paclitaxel biosynthetic genes in MeJA-elicited cultures exhibiting both substantial (15-fold) and moderate (2-fold) differences …

Mutations In A Mitochondrial Transcription Termination Factor (Mterf)-Related Protein Enhance Thermotolerance In The Absence Of The Major Molecular Chaperone Hsp101, M. Kim, U. Lee, I. Small, C. Des Francs-Small, Elizabeth Vierling

Elizabeth Vierling

The molecular chaperone heat shock protein101 (HSP101) is required for acquired thermotolerance in plants and other organisms. To identify factors that interact with HSP101 or that are involved in thermotolerance, we screened for extragenic suppressors of a dominant-negative allele of Arabidopsis thaliana HSP101, hot1-4. One suppressor, shot1 (for suppressor of hot1-4 1), encodes a mitochondrial transcription termination factor (mTERF)–related protein, one of 35 Arabidopsis mTERFs about which there is limited functional data. Missense (shot1-1) and T-DNA insertion (shot1-2) mutants suppress the hot1-4 heat-hypersensitive phenotype. Furthermore, shot1-2 suppresses other heat-sensitive mutants, and shot1-2 alone is more heat tolerant than the wild …

Transient Pharmacologic Lowering Of Aβ Production Prior To Deposition Results In Sustained Reduction Of Amyloid Plaque Pathology, Pritam Das, Christophe Verbeeck, Lisa Minter, Paramita Chakrabarty, Kevin Felsenstein, Thomas Kukar, Ghulam Maharvi, Abdul Fauq, Barbara A. Osborne, Todd E. Golde

Barbara A. Osborne

Background: Alzheimer’s disease (AD) is the leading cause of dementia among the elderly. Disease modifying therapies targeting Aβ that are in development have been proposed to be more effective if treatment was initiated prior to significant accumulation of Aβ in the brain, but optimal timing of treatment initiation has not been clearly established in the clinic. We compared the efficacy of transient pharmacologic reduction of brain Aβ with a γ-secretase inhibitor (GSI ) for 1–3 months (M) treatment windows in APP Tg2576 mice and subsequent aging of the mice to either 15M or 18M. Results: These data show that reducing …

Arabidopsis And Tobacco Superman Regulate Hormone Signalling And Mediate Cell Proliferation And Differentiation, C. Nibau, V. S. Di Stilio, H. M. Wu, Alice Cheung

Alice Cheung

Arabidopsis thaliana superman (SUP) plays an important role during flower development by maintaining the boundary between stamens and carpels in the inner two whorls. It was proposed that SUP maintains this boundary by regulating cell proliferation in both whorls, as loss-of-function superman mutants produce more stamens at the expense of carpels. However, the cellular mechanism that underlies SUP function remains unknown. Here Arabidopsis or tobacco (Nicotiana tabacum) SUP was overexpressed in tobacco plants to substantiate SUP's role as a regulator of cell proliferation and boundary definition and provide evidence that its biological role may be mediated via hormonal changes. It …

Comparative Genomics Yields Insights Into Niche Adaptation Of Plant Vascular Wilt Pathogens, Steven J. Klosterman, Krishna V. Subbarao, Seogchan Kang, Paola Veronese, Scott E. Gold, Bart P. H. J. Thomma, Zehua Chen, Bernard Henrissat, Yong-Hwan Lee, Jongsun Park, Dez J. Barbara, Maria D. Garcia-Pedrajas, Amy Anchieta, Ronnie De Jonge, Parthasarathy Santhanam, Karunakaran Maruthachalam, Zahi Atallah, Stefan G. Amyotte, Zahi Paz, Patrik Inderbitzin, Ryan J. Hayes, David I. Heiman, Sarah Young, Qiandong Zeng, Reinhard Engels, James Galagan, Christina A. Cuomo, Katherine F. Dobinson, Li-Jun Ma

Li-Jun Ma

The vascular wilt fungi Verticillium dahliae and V. albo-atrum infect over 200 plant species, causing billions of dollars in annual crop losses. The characteristic wilt symptoms are a result of colonization and proliferation of the pathogens in the xylem vessels, which undergo fluctuations in osmolarity. To gain insights into the mechanisms that confer the organisms' pathogenicity and enable them to proliferate in the unique ecological niche of the plant vascular system, we sequenced the genomes of V. dahliae and V. albo-atrum and compared them to each other, and to the genome of Fusarium oxysporum, another fungal wilt pathogen. Our analyses …

A Highway For War And Peace: The Secretory Pathway In Plant-Microbe Interactions, Dong Wang, X Dong

Dong Wang

Secretion of proteins and other molecules is the primary means by which a cell interacts with its surroundings. The overall organization of the secretory system is remarkably conserved among eukaryotes, and many of the components have been investigated in detail in animal models. Plant cells, because of their sessile lifestyle, are uniquely reliant on the secretory pathway to respond to changes in their environments, either abiotic, such as the absence of nutrients, or biotic, such as the presence of predators or pathogens. In particular, most plant pathogens are extracellular, which demands a robust and efficient host secretory system directed at …

Role Of The Extensin Superfamily In Primary Cell Wall Architecture, Derek T.A. Lamport, Marcia J. Kieliszewski, Yuning Chen, Maura Cannon

Maura Cannon

No abstract provided.

Rac/Rop Gtpases And Auxin Signaling, H. M. Wu, O. Hazak, Alice Cheung, S. Yalovsky

Alice Cheung

Auxin functions as a key morphogen in regulating plant growth and development. Studies on auxin-regulated gene expression and on the mechanism of polar auxin transport and its asymmetric distribution within tissues have provided the basis for realizing the molecular mechanisms underlying auxin function. In eukaryotes, members of the Ras and Rho subfamilies of the Ras superfamily of small GTPases function as molecular switches in many signaling cascades that regulate growth and development. Plants do not have Ras proteins, but they contain Rho-like small G proteins called RACs or ROPs that, like fungal and metazoan Rhos, are regulators of cell polarity …

Protein Folding In The Cell: Challenges And Progress, Anne Gershenson, Lial M. Gierasch

Anne Gershenson

It is hard to imagine a more extreme contrast than that between the dilute solutions used for in vitro studies of protein folding and the crowded, compartmentalized, sticky, spatially inhomogeneous interior of a cell. This review highlights recent research exploring protein folding in the cell with a focus on issues that are generally not relevant to in vitro studies of protein folding, such as macromolecular crowding, hindered diffusion, co-translational folding, molecular chaperones, and evolutionary pressures. The technical obstacles that must be overcome to characterize protein folding in the cell are driving methodological advances, and we draw attention to several examples, …

Pollen Tubes Lacking A Pair Of K+ Transporters Fail To Target Ovules In Arabidopsis, Y. Lu, S. Chanroj, L. Zulkifli, M. A. Johnson, N. Uozumi, Alice Cheung, H. Sze

Alice Cheung

Flowering plant reproduction requires precise delivery of the sperm cells to the ovule by a pollen tube. Guidance signals from female cells are being identified; however, how pollen responds to those cues is largely unknown. Here, we show that two predicted cation/proton exchangers (CHX) in Arabidopsis thaliana, CHX21 and CHX23, are essential for pollen tube guidance. Male fertility was unchanged in single chx21 or chx23 mutants. However, fertility was impaired in chx21 chx23 double mutant pollen. Wild-type pistils pollinated with a limited number of single and double mutant pollen producing 62% fewer seeds than those pollinated with chx23 single mutant …

Segmental Isotopic Labeling Of The Hsp70 Molecular Chaperone Dnak Using Expressed Protein Ligation, Eugenia M. Clerico, Anastasia Zhuravleva, Robert G. Smock, Lila Gierasch

Lila Gierasch

Introducing biophysical labels into specific regions of large and dynamic multidomain proteins greatly facilitates mechanistic analysis. Ligation of expressed domains that are labeled in a desired manner before assembly of the intact molecular machine provides such a strategy. We have elaborated an experimental route using expressed protein ligation (EPL) to create an Hsp70 molecular chaperone (in this case the E. coli Hsp70, DnaK) where only one of the two constituent domains was labeled, in this case with NMR active isotopes, allowing visualization of the single domain in the context of the two domain protein. Several technical obstacles were overcome, including …

The Sv40 Late Protein Vp4 Is A Viroporin That Forms Pores To Disrupt Membranes For Viral Release, Smita Raghava, Kristen M. Giorda, F. B. Romano, Alejandro P. Heuck, Daniel Hebert

Alejandro P. Heuck

Nonenveloped viruses are generally released by the timely lysis of the host cell by a poorly understood process. For the nonenveloped virus SV40, virions assemble in the nucleus and then must be released from the host cell without being encapsulated by cellular membranes. This process appears to involve the well-controlled insertion of viral proteins into host cellular membranes rendering them permeable to large molecules. VP4 is a newly identified SV40 gene product that is expressed at late times during the viral life cycle that corresponds to the time of cell lysis. To investigate the role of this late expressed protein …

Coexpressed D1- And D2-Like Dopamine Receptors Antagonistically Modulate Acetylcholine Release In Caenorhabditis Elegans., Andrew T. Allen, Kathryn N. Maher, Khursheed A. Wani, Katherine E. Betts, Daniel Chase

Daniel Chase

Dopamine acts through two classes of G protein-coupled receptor (D1-like and D2-like) to modulate neuron activity in the brain. While subtypes of D1- and D2-like receptors are coexpressed in many neurons of the mammalian brain, it is unclear how signaling by these coexpressed receptors interacts to modulate the activity of the neuron in which they are expressed. D1- and D2-like dopamine receptors are also coexpressed in the cholinergic ventral-cord motor neurons of Caenorhabditis elegans. To begin to understand how coexpressed dopamine receptors interact to modulate neuron activity, we performed a genetic screen in C. elegans and isolated mutants defective in …

The Transition From Closed To Open Conformation Of Treponema Pallidum Outer Membrane-Associated Lipoprotein Tp0453 Involves Membrane Sensing And Integration By Two Amphipathic Helices, Amit Luthra, Guangyu Zhu, Daniel C. Desrosiers, Christian H. Eggers, Vishwaroop Mulay, Arvind Anand, Fiona A. Mcarthur, Fabian B. Romano, Melissa J. Caimano, Alejandro P. Heuck, Michael G. Malkowski, Justin D. Radolf

Alejandro P. Heuck

The molecular architecture and composition of the outer membrane (OM) of Treponema pallidum (Tp), the noncultivable agent of venereal syphilis, differ considerably from those of typical Gram-negative bacteria. Several years ago we described TP0453, the only lipoprotein associated with the inner leaflet of the Tp OM. Whereas polypeptides of other treponemal lipoproteins are hydrophilic, non-lipidated TP0453 can integrate into membranes, a property attributed to its multiple amphipathic helices (AHs). Furthermore, membrane integration of the TP0453 polypeptide was found to increase membrane permeability, suggesting the molecule functions in a porin-like manner. To better understand the mechanism of membrane integration of TP0453 …

Efficient Isolation Of Pseudomonas Aeruginosa Type Iii Secretion Translocators And Assembly Of Heteromeric Transmembrane Pores In Model Membranes, Fabian B. Romano, Kyle C. Rossi, Christos G. Sava, Andreas Holzenburg, Eugenia M. Clerico, Alejandro P. Heuck

Alejandro P. Heuck

Translocation of bacterial toxins or effectors into host cells using the type III secretion (T3S) system is a conserved mechanism shared by many Gram-negative pathogens. Pseudomonas aeruginosa injects different proteins across the plasma membrane of target cells, altering the normal metabolism of the host. Protein translocation presumably occurs through a proteinaceous transmembrane pore formed by two T3S secreted protein translocators, PopB and PopD. Unfolded translocators are secreted through the T3S needle prior to insertion into the target membrane. Purified PopB and PopD form pores in model membranes. However, their tendency to form heterogeneous aggregates in solution had hampered the analysis …

Feronia Receptor-Like Kinase Regulates Rho Gtpase Signaling Of Root Hair Development, Q. Duan, D. Kita, C. Li, Alice Cheung, H. M. Wu

Alice Cheung

Plant RHO GTPases (RAC/ROPs) mediate multiple extracellular signals ranging from hormone to stress and regulate diverse cellular processes important for polarized cell growth, differentiation, development, reproduction, and responses to the environment. They shuttle between the GDP-bound inactive state and the GTP-bound activated state and their activation is predominantly mediated by a family of guanine nucleotide exchange factors (GEFs) referred to as ROPGEFs. Using the Arabidopsis ROPGEF1 as bait, we identified members of a receptor-like kinase (RLK) family as potential upstream regulators for RAC/ROP signaling. NADPH oxidase-derived reactive oxygen species (ROS) are emerging as important regulators for growth and development and …

An Interdomain Sector Mediating Allostery In Hsp70 Molecular Chaperones, Robert G. Smock, Olivier Rivoire, William P. Russ, Joanna F. Swain, Stanislas Leibler, Rama Ranganathan, Lila Gierasch

Lila Gierasch

Allosteric coupling between protein domains is fundamental to many cellular processes. For example, Hsp70 molecular chaperones use ATP binding by their actin-like N-terminal ATPase domain to control substrate interactions in their C-terminal substrate-binding domain, a reaction that is critical for protein folding in cells. Here, we generalize the statistical coupling analysis to simultaneously evaluate co-evolution between protein residues and functional divergence between sequences in protein sub-families. Applying this method in the Hsp70/110 protein family, we identify a sparse but structurally contiguous group of co-evolving residues called a ‘sector’, which is an attribute of the allosteric Hsp70 sub-family that links the …

Role Of Hsp70 Atpase Domain Intrinsic Dynamics And Sequence Evolution In Enabling Its Functional Interactions With Nefs, Ying Liu, Lila Gierasch, Ivet Bahar

Lila Gierasch

12 Hide Figures Abstract Author Summary Introduction Materials and Methods Results Discussion Supporting Information Acknowledgments Author Contributions References Reader Comments (0) Figures Abstract Catalysis of ADP-ATP exchange by nucleotide exchange factors (NEFs) is central to the activity of Hsp70 molecular chaperones. Yet, the mechanism of interaction of this family of chaperones with NEFs is not well understood in the context of the sequence evolution and structural dynamics of Hsp70 ATPase domains. We studied the interactions of Hsp70 ATPase domains with four different NEFs on the basis of the evolutionary trace and co-evolution of the ATPase domain sequence, combined with elastic …

Molecular Basis For Distinct Pathways For Protein Import Into Arabidopsis Chloroplasts, Danny Schnell, C. Rounds, H. Inoue

Danny Schnell

The translocons at the outer envelope membrane of chloroplasts (TOCs) initiate the import of thousands of nucleus-encoded proteins into the organelle. The identification of structurally and functionally distinct TOC complexes has led to the hypothesis that the translocons constitute different import pathways that are required to coordinate the import of sets of proteins whose expression varies in response to organelle biogenesis and physiological adaptation. To test this hypothesis, we examined the molecular basis for distinct TOC pathways by analyzing the functional diversification among the Toc159 family of TOC receptors. We demonstrate that the N-terminal A-domains of the Toc159 receptors regulate …

Determinants For Stop-Transfer And Post-Import Pathways For Protein Targeting To The Chloroplast Inner Envelope Membrane, Antonio A. B. Viana, Ming Li, Danny Schnell

Danny Schnell

he inner envelope membrane (IEM) of the chloroplast plays key roles in controlling metabolite transport between the organelle and cytoplasm and is a major site of lipid and membrane synthesis within the organelle. IEM biogenesis requires the import and integration of nucleus-encoded membrane proteins. Previous reports have led to the conclusion that membrane proteins are inserted into the IEM during protein import from the cytoplasm via a stop-transfer mechanism or are completely imported into the stroma and then inserted into the IEM in a post-import mechanism. In this study, we examined the determinants for each pathway by comparing the targeting …

Systematic Discovery Of Regulatory Motifs In Fusarium Graminearum By Comparing Four Fusarium Genomes, Lokesh Kumar, Andrew Breakspear, Corby Kistler, Li-Jun Ma, Xiaohui Xie

Li-Jun Ma

Background

Fusarium graminearum (Fg), a major fungal pathogen of cultivated cereals, is responsible for billions of dollars in agriculture losses. There is a growing interest in understanding the transcriptional regulation of this organism, especially the regulation of genes underlying its pathogenicity. The generation of whole genome sequence assemblies for Fg and three closely related Fusarium species provides a unique opportunity for such a study.

Results

Applying comparative genomics approaches, we developed a computational pipeline to systematically discover evolutionarily conserved regulatory motifs in the promoter, downstream and the intronic regions of Fg genes, based on the multiple alignments of sequenced Fusarium …

The 1.9 Å Structure Of Human Α-N-Acetylgalactosaminidase The Molecular Basis Of Schindler And Kanzaki Diseases, Nathaniel E. Clark, Scott Garman

Scott Garman

alpha-N-acetylgalactosaminidase (alpha-NAGAL; E.C. 3.2.1.49) is a lysosomal exoglycosidase that cleaves terminal alpha-N-acetylgalactosamine residues from glycopeptides and glycolipids. In humans, a deficiency of alpha-NAGAL activity results in the lysosomal storage disorders Schindler disease and Kanzaki disease. To better understand the molecular defects in the diseases, we determined the crystal structure of human alpha-NAGAL after expressing wild-type and glycosylation-deficient glycoproteins in recombinant insect cell expression systems. We measured the enzymatic parameters of our purified wild-type and mutant enzymes, establishing their enzymatic equivalence. To investigate the binding specificity and catalytic mechanism of the human alpha-NAGAL enzyme, we determined three crystallographic complexes with different …

Myosin Xi Is Essential For Tip Growth In Physcomitrella Patens, L Vidali, Gm Burkart, Rc Augustine, E Kerdavid, E Tuzel, M Bezanilla

Magdalena Bezanilla

Class XI myosins are plant specific and responsible for cytoplasmic streaming. Because of the large number of myosin XI genes in angiosperms, it has been difficult to determine their precise role, particularly with respect to tip growth. The moss Physcomitrella patens provides an ideal system to study myosin XI function. P. patens has only two myosin XI genes, and these genes encode proteins that are 94% identical to each other. To determine their role in tip growth, we used RNA interference to specifically silence each myosin XI gene using 5′ untranslated region sequences. We discovered that the two myosin XI …

Toc Receptor Dimerization Participates In The Initiation Of Membrane Translocation During Protein Import Into Chloroplasts, Jeonghwa Lee, Fei Wang Wang, Danny Schnell

Danny Schnell

The post-translational import of nucleus-encoded preproteins into chloroplasts occurs through multimeric translocons in the outer (Toc) and inner (Tic) membranes. The high fidelity of the protein import process is maintained by specific recognition of the transit peptide of preproteins by the coordinate activities of two homologous GTPase Toc receptors, Toc34 and Toc159. Structural and biochemical studies suggest that dimerization of the Toc receptors functions as a component of the mechanism to control access of preproteins to the membrane translocation channel of the translocon. We show that specific mutations that disrupted receptor dimerization in vitro reduced the rate of protein import …

Sinorhizobium Meliloti Cpdr1 Is Critical For Co-Ordinating Cell Cycle Progression And The Symbiotic Chronic Infection, H. Kobayashi, N. J. Kobayashi, Peter Chien, L. A. Simmons, G. C. Walker

Peter Chien

ATP-driven proteolysis plays a major role in regulating the bacterial cell cycle, development and stress responses. In the nitro -fixing symbiosis with host plants, Sinorhizobium meliloti undergoes a profound cellular differentiation, including endoreduplication of the ome. The regulatory mechanisms governing the alterations of the S. meliloti cell cycle in planta are largely unknown. Here, we report the characterization of two cpdR homologues, cpdR1 and cpdR2, of S. meliloti that encode single-domain response regulators. In Caulobacter crescentus, CpdR controls the polar localization of the ClpXP protease, thereby mediating the regulated proteolysis of key protein(s), such as CtrA, involved in cell cycle …

Genomic Analysis Of The Basal Lineage Fungus Rhizopus Oryzae Reveals A Whole-Genome Duplication, Li-Jun Ma, Ashraf S. Ibrahim, Christopher Skory, Manfred G. Grabherr, Gertraud Burger, Margi Butler, Marek Elias, Alexander Idnurm, B. Franz Lang, Teruo Sone, Ayumi Abe, Sarah E. Calvo, Luis M. Corrochano, Reinhard Engels, Jianmin Fu, Wilhelm Hansberg, Jung-Mi Kim, Chinnappa D. Kodira, Michael J. Koehrsen, Bo Liu, Diego Miranda-Saavedra, Sinead O'Leary, Lucila Ortiz-Castellanos, Russell Poulter, Julio Rodriguez-Romero, José Ruiz-Herrera, Yao-Qing Shen, Qiandong Zeng, James Galagan, Bruce W. Birren, Christina A. Cuomo, Brian L. Wickes

Li-Jun Ma

Rhizopus oryzae is the primary cause of mucormycosis, an emerging, life-threatening infection characterized by rapid angioinvasive growth with an overall mortality rate that exceeds 50%. As a representative of the paraphyletic basal group of the fungal kingdom called “zygomycetes,” R. oryzae is also used as a model to study fungal evolution. Here we report the genome sequence of R. oryzae strain 99–880, isolated from a fatal case of mucormycosis. The highly repetitive 45.3 Mb genome assembly contains abundant transposable elements (TEs), comprising approximately 20% of the genome. We predicted 13,895 protein-coding genes not overlapping TEs, many of which are paralogous …

Fluorescence Correlation Spectroscopy Of Phosphatidylinositolspecific Phospholipase C Monitors The Interplay Of Substrate And Activator Lipid Binding, Mingming Pu, Mary F. Roberts, Anne Gershenson

Anne Gershenson

Phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes simultaneously interact with the substrate, PI, and with non-substrate lipids such as phosphatidylcholine (PC). For Bacillus thuringiensis PI-PLC these interactions are synergistic with maximal catalytic activity observed at low to moderate mole fractions of PC (XPC) and maximal binding occurring at low mole fractions of anionic lipids. It has been proposed that residues in α helix B help modulate membrane binding and that dimerization on the membrane surface both increases affinity for PC and activates PI-PLC yielding the observed PI/PC synergy. Vesicle binding and activity measurements using a variety of PI-PLC mutants support many aspects …