The Experts below are selected from a list of 87189 Experts worldwide ranked by ideXlab platform

Matthew K Waldor - One of the best experts on this subject based on the ideXlab platform.

  • genetic analysis of the role of the conserved Inner Membrane protein cvpa in ehec resistance to deoxycholate
    Journal of Bacteriology, 2021
    Co-Authors: Alyson R Warr, Rachel T Giorgio, Matthew K Waldor
    Abstract:

    The function of cvpA, a bacterial gene predicted to encode an Inner Membrane protein, is largely unknown. Early studies in E. coli linked cvpA to Colicin V secretion and recent work revealed that it is required for robust intestinal colonization by diverse enteric pathogens. In enterohemorrhagic E. coli (EHEC), cvpA is required for resistance to the bile salt deoxycholate (DOC). Here, we carried out genome-scale transposon-insertion mutagenesis and spontaneous suppressor analysis to uncover cvpA's genetic interactions and identify common pathways that rescue the sensitivity of a ΔcvpA EHEC mutant to DOC. These screens demonstrated that mutations predicted to activate the σE-mediated extracytoplasmic stress response bypass the ΔcvpA mutant's susceptibility to DOC. Consistent with this idea, we found that deletions in rseA and msbB and direct overexpression of rpoE restored DOC resistance to the ΔcvpA mutant. Analysis of the distribution of CvpA homologs revealed that this Inner Membrane protein is conserved across diverse bacterial phyla, in both enteric and non-enteric bacteria that are not exposed to bile. Together, our findings suggest that CvpA plays a role in cell envelope homeostasis in response to DOC and similar stress stimuli in diverse bacterial species.IMPORTANCE Several enteric pathogens, including Enterohemorrhagic E. coli (EHEC), require CvpA to robustly colonize the intestine. This Inner Membrane protein is also important for secretion of a colicin and EHEC resistance to the bile salt deoxycholate (DOC), but its function is unknown. Genetic analyses carried out here showed that activation of the σE-mediated extracytoplasmic stress response restored the resistance of a cvpA mutant to DOC, suggesting that CvpA plays a role in cell envelope homeostasis. The conservation of CvpA across diverse bacterial phyla suggests that this Membrane protein facilitates cell envelope homeostasis in response to varied cell envelope perturbations.

  • genetic analysis of the role of the conserved Inner Membrane protein cvpa in enterohemorrhagic escherichia coli resistance to deoxycholate
    Journal of Bacteriology, 2020
    Co-Authors: Alyson R Warr, Rachel T Giorgio, Matthew K Waldor
    Abstract:

    ABSTRACT The function of cvpA, a bacterial gene predicted to encode an Inner Membrane protein, is largely unknown. Early studies in Escherichia coli linked cvpA to colicin V secretion, and recent work revealed that it is required for robust intestinal colonization by diverse enteric pathogens. In enterohemorrhagic E. coli (EHEC) strains, cvpA is required for resistance to the bile salt deoxycholate (DOC). Here, we carried out genome scale transposon insertion (TIS) mutagenesis and spontaneous suppressor analysis to uncover the genetic interactions of cvpA and identify common pathways that rescue the sensitivity of a ΔcvpA EHEC mutant to DOC. These screens demonstrated that mutations predicted to activate the σE-mediated extracytoplasmic stress response bypass the ΔcvpA mutant’s susceptibility to DOC. Consistent with this idea, we found that deletions in rseA and msbB and direct overexpression of rpoE restored DOC resistance to the ΔcvpA mutant. Analysis of the distribution of CvpA homologs revealed that this Inner Membrane protein is conserved across diverse bacterial phyla in both enteric and nonenteric bacteria that are not exposed to bile. Together, our findings suggest that CvpA plays a role in cell envelope homeostasis in response to DOC and similar stress stimuli in diverse bacterial species. IMPORTANCE Several enteric pathogens, including enterohemorrhagic E. coli (EHEC) strains, require CvpA to robustly colonize the intestine. This Inner Membrane protein is also important for secretion of a colicin and for EHEC resistance to the bile salt deoxycholate (DOC), but its function is unknown. Genetic analyses carried out here showed that activation of the σE-mediated extracytoplasmic stress response restored the resistance of a cvpA mutant to DOC, suggesting that CvpA plays a role in cell envelope homeostasis. The conservation of CvpA across diverse bacterial phyla suggests that this Membrane protein facilitates cell envelope homeostasis in response to varied cell envelope perturbations.

  • genetic analyses link the conserved Inner Membrane protein cvpa to the σeextracytoplasmic stress response
    bioRxiv, 2020
    Co-Authors: Alyson R Warr, Rachel T Giorgio, Matthew K Waldor
    Abstract:

    The function of cvpA, a bacterial gene predicted to encode an Inner Membrane protein, is largely unknown. Early studies in E. coli linked cvpA to Colicin V secretion and recent work revealed that it is required for robust intestinal colonization by diverse enteric pathogens. In enterohemorrhagic E. coli (EHEC), cvpA is required for resistance to the bile salt deoxycholate (DOC). Here, we carried out genome-scale transposon-insertion mutagenesis and spontaneous suppressor analysis to uncover cvpAs genetic interactions and identify common pathways that rescue the sensitivity of a {Delta}cvpA EHEC mutant to DOC. Collectively, these screens led to the hypothesis that the {Delta}cvpA mutant is impaired in its capacity to activate the {sigma}E-mediated stress response. This idea was supported by showing that mutations that activate {sigma}E, either indirectly or through its direct overexpression, can restore the {Delta}cvpA mutants resistance to DOC. Analysis of the distribution of CvpA homologs revealed that this Inner Membrane protein is conserved across bacterial phyla, in both enteric and non-enteric bacteria that are not exposed to bile. Together, our findings suggest that CvpA may function in triggering activation of the {sigma}E stress response pathway in response to DOC as well as additional stimuli. ImportanceSeveral enteric pathogens, including Enterohemorrhagic E. coli (EHEC), require cvpA to robustly colonize the intestine. This Inner Membrane is also important for secretion of a colicin and EHEC resistance to the bile salt deoxycholate, but its function is unknown. Genetic analyses carried out here suggest that cvpA is required to trigger the {sigma}E stress response pathway in response to deoxycholate. Since CvpA is conserved across diverse bacterial phyla, we propose that this Inner Membrane protein is important for activation of this stress response pathway in response to diverse perturbations of the cell envelope.

  • genetic analyses link the conserved Inner Membrane protein cvpa to the σeextracytoplasmic stress response
    bioRxiv, 2020
    Co-Authors: Alyson R Warr, Rachel T Giorgio, Matthew K Waldor
    Abstract:

    The function of cvpA, a bacterial gene predicted to encode an Inner Membrane protein, is largely unknown. Early studies in E. coli linked cvpA to Colicin V secretion and recent work revealed that it is required for robust intestinal colonization by diverse enteric pathogens. In enterohemorrhagic E. coli (EHEC), cvpA is required for resistance to the bile salt deoxycholate (DOC). Here, we carried out genome-scale transposon-insertion mutagenesis and spontaneous suppressor analysis to uncover cvpA9 genetic interactions and identify common pathways that rescue the sensitivity of a ΔcvpA EHEC mutant to DOC. Collectively, these screens led to the hypothesis that the ΔcvpA mutant is impaired in its capacity to activate the σE-mediated stress response. This idea was supported by showing that mutations that activate σE, either indirectly or through its direct overexpression, can restore the ΔcvpA mutant9s resistance to DOC. Analysis of the distribution of CvpA homologs revealed that this Inner Membrane protein is conserved across bacterial phyla, in both enteric and non-enteric bacteria that are not exposed to bile. Together, our findings suggest that CvpA may function in triggering activation of the σE stress response pathway in response to DOC as well as additional stimuli.

Donald Oliver - One of the best experts on this subject based on the ideXlab platform.

  • integration of seca protein into the escherichia coli Inner Membrane is regulated by its amino terminal atp binding domain
    Molecular Microbiology, 1996
    Co-Authors: Thavamani Rajapandi, Donald Oliver
    Abstract:

    Summary SecA protein, the ATPase promoting translocation of proteins across the Escherichia coli Inner Membrane, contains two ATP-binding domains that differ greatly in their affinity for bound nucleotide. In order to define more precisely the location of the high-affinity nucleotide-binding site, oligonucleotide-directed mutagenesis was used to introduce cysteine residues into the SecA sequence, and a cysteine-specific cleavage reagent was employed to generate defined peptides of SecA protein after photocross-linking with [α-32P]-ATP. This analysis revealed that the nucleotide was cross-linked between amino acid residues 75 and 97 of SecA protein. The biochemical function of the high affinity ATP-binding domain was explored by subcellular fractionation studies which demonstrated that SecA proteins defective in this region were found almost exclusively in their integral Membrane form, while SecA proteins with defects in the low-affinity ATP-domain showed a normal distribution of cytosolic, peripheral and integral Membrane forms. Interestingly, the SecA51(Ts) protein that has a Leu to Pro substitution at amino acid residue 43 bound ATP with high affinity, but its fractionation pattern and translocation ATPase activity were similar to those of proteins with defects in the high-affinity ATP-binding site. These results delimit more precisely the high-affinity ATP-binding domain of SecA, indicate the importance of the early amino-terminal region of SecA protein in the functioning of this domain, and demonstrate the role of this domain in regulating penetration of SecA protein into the Inner Membrane. Our results lead to a simple model for the regulation of a cycle of SecA insertion into, and de-insertion from, the Inner Membrane by the activity of the high-affinity ATP-binding domain.

  • seca protein is exposed to the periplasmic surface of the e coli Inner Membrane in its active state
    Cell, 1994
    Co-Authors: Kim Young Jae, Thavamani Rajapandi, Donald Oliver
    Abstract:

    Summary E. coli cells harboring pCG169 containing the secD secF locus possessed SecA protein almost entirely in an integral Membrane form in which it displayed normal protein translocation activity. These results imply that integral Membrane SecA is the catalytically active form of this enzyme and that products of the secD secF locus regulate SecA association with the Inner Membrane. Protease and biotinylation accessibility studies of right side-out and inside-out Membrane vesicles derived from this strain revealed that SecA was exposed to the periplasmic surface of the Inner Membrane. These studies suggest a model of bacterial protein secretion, whereby insertion of SecA into the Inner Membrane and its association with SecY/E/G promotes assembly of active protein-conducting channels comprised in part of integral Membrane SecA protein, and products of the secD secF locus regulate the channel assembly-disassembly reaction by modulating the SecA insertion-deinsertion step.

  • seca protein autoregulated atpase catalysing preprotein insertion and translocation across the escherichia coli Inner Membrane
    Molecular Microbiology, 1993
    Co-Authors: Donald Oliver
    Abstract:

    Recent insight into the biochemical mechanisms of protein translocation in Escherichia coli indicates that SecA ATPase is required both for the initial binding of preproteins to the Inner Membrane as well as subsequent translocation across this structure. SecA appears to promote these events by direct recognition of the preprotein or preprotein-SecB complex, binding to Inner-Membrane anionic phospholipids, insertion into the Membrane bilayer and association with the preprotein translocator, SecY/SecE. ATP binding appears to control the affinity of SecA for the various components of the system and ATP hydrolysis promotes cycling between its different biochemical states. As a component likely to catalyse a rate-determining step in protein secretion, SecA synthesis is co-ordinated with the activity of the protein export pathway. This form of negative regulation appears to rely on SecA protein binding to its mRNA and repressing translation if conditions of rapid protein secretion prevail within the cell. A precise biochemical scheme for SecA-dependent catalysis of protein export and the details of secA regulation appear to be close at hand. The evolutionary conservation of SecA protein among eubacteria as well as the general requirement for translocation ATPases in other protein secretion systems argues for a mechanistic commonality of all prokaryotic protein export pathways.

Jeremy P Derrick - One of the best experts on this subject based on the ideXlab platform.

  • structure and assembly of an Inner Membrane platform for initiation of type iv pilus biogenesis
    Proceedings of the National Academy of Sciences of the United States of America, 2013
    Co-Authors: Vijaykuma Karuppiah, Richard F Collins, Angela Thistlethwaite, Ya Gao, Jeremy P Derrick
    Abstract:

    Type IV pili are long fibers that are assembled by polymerization of a major pilin protein in the periplasm of a wide range of bacteria and archaea. They play crucial roles in pathogenesis, DNA transformation, and motility, and are capable of rapid retraction, generating powerful motor forces. PilN and PilO are integral Inner Membrane proteins that are essential for type IV pilus formation. Here, we show that PilN and PilO from Thermus thermophilus can be isolated as a complex with PilM, a cytoplasmic protein with structural similarities to the cytoskeletal protein MreB. The crystal structure of the periplasmic portion of PilN forms a homodimer with an extensive, conserved interaction interface. We conducted serial 3D reconstructions by electron microscopy of PilMN, PilMNO, and PilMNO bound to the major pilin protein PilA4, to chart the assembly of the Inner Membrane pilus biogenesis platform. PilN drives the dimerization of the PilMN complex with a stoichiometry of 2:2; binding of two PilO monomers then causes the PilN periplasmic domains to dissociate. Finally, two PilA4 monomers bind to the periplasmic domains of PilN and PilO, to generate a T-shaped complex that is primed for addition of the pilin to the nascent pilus fiber. Docking of structures for PilM, PilN, PilO, and PilA4 into the electron density maps of the transMembrane complexes was used to generate a sequence of molecular structures that chart the initial events in type IV pilus formation, and provide structural information on the early events in this important secretion process.

  • structure of the pilm piln Inner Membrane type iv pilus biogenesis complex from thermus thermophilus
    Journal of Biological Chemistry, 2011
    Co-Authors: Vijaykumar Karuppiah, Jeremy P Derrick
    Abstract:

    Type IV pili are surface-exposed filaments, which extend from a variety of bacterial pathogens and play a major role in pathogenesis, motility, and DNA uptake. Here, we present the crystal structure of a complex between a cytoplasmic component of the type IV pilus biogenesis system from Thermus thermophilus, PilM, in complex with a peptide derived from the cytoplasmic portion of the Inner Membrane protein PilN. PilM also binds ATP, and its structure is most similar to the actin-like protein FtsA. PilN binds in a narrow channel between the 1A and 1C subdomains in PilM; the binding site is well conserved in other Gram-negative bacteria, notably Neisseria meningitidis, Pseudomonas aeruginosa, and Vibrio cholerae. We find no evidence for the catalysis of ATP hydrolysis by PilM; fluorescence data indicate that the protein is likely to be saturated by ATP at physiological concentrations. In addition, binding of the PilN peptide appears to influence the environment of the ATP binding site. This is the first reported structure of a complex between two type IV pilus biogenesis proteins. We propose a model in which PilM binds ATP and then PilN as one of the first steps in the formation of the Inner Membrane platform of the type IV pilus biogenesis complex.

Jodi Nunnari - One of the best experts on this subject based on the ideXlab platform.

  • micos coordinates with respiratory complexes and lipids to establish mitochondrial Inner Membrane architecture
    eLife, 2015
    Co-Authors: Jonathan R Friedman, Arnaud Mourier, Justin Yamada, Michael J Mccaffery, Jodi Nunnari
    Abstract:

    Structures called mitochondria provide energy that cells need to live and grow. To do this, mitochondria convert energy stored within sugars and other carbon-rich compounds into the energy currency of cells, a molecule called adenosine triphosphate (called ATP for short). Defective mitochondria can cause cells to starve and also cause severe human diseases. A double Membrane surrounds each mitochondrion. The outer Membrane allows proteins and other substances to enter, while the Inner Membrane is elaborately folded and contains several groups of proteins—or complexes—including the respiratory complexes that generate ATP. Proper Inner Membrane folding is critically important. The Membrane folds are held in place by structures called cristae junctions, which may also help to restrict proteins to particular areas of the Inner Membrane. A large Inner Membrane complex of proteins known as MICOS is important for organizing the Inner Membrane into folds, although exactly how it does so is not fully understood. MICOS consists of at least six different proteins, most of which are found across yeast and animal species. Friedman et al. have now analyzed how the MICOS complex assembles on the Inner Membrane in yeast cells using a combination of fluorescence and electron microscopy, proteomics and biochemistry. This revealed that in yeast, MICOS is made up of two independent sub-complexes bridged together by a protein called Mic19, which additional experiments suggest controls the number and positions of the cristae junctions that hold the folds of the Inner Membrane in place. As part of the approach to understand MICOS complex organization, Friedman et al. removed the six MICOS proteins from yeast cells. Inside these cells, the Inner mitochondrial Membrane was misfolded. Furthermore, the respiratory complexes did not work normally and as a consequence the cells were unable to grow normally, suggesting that the correct distribution of respiratory complexes in the Inner Membrane is important for ATP production and depends on MICOS. These results indicate that MICOS stabilizes the structure of the Inner Membrane and organizes it into an efficient energy-generating machine. In many human mitochondrial diseases, the Inner Membrane of mitochondria folds incorrectly, in similar ways to the misfolding seen in the yeast cells that did not contain the MICOS complex. Therefore, the MICOS complex may also influence how these diseases develop.

  • a mitochondrial focused genetic interaction map reveals a scaffold like complex required for Inner Membrane organization in mitochondria
    Journal of Cell Biology, 2011
    Co-Authors: Suzanne Hoppins, Sean R Collins, Ann Cassidystone, Rachel M Devay, Laura L Lackner, Benedikt Westermann, Maya Schuldiner, Jonathan S Weissman, Eric Hummel, Jodi Nunnari
    Abstract:

    To broadly explore mitochondrial structure and function as well as the communication of mitochondria with other cellular pathways, we constructed a quantitative, high-density genetic interaction map (the MITO-MAP) in Saccharomyces cerevisiae. The MITO-MAP provides a comprehensive view of mitochondrial function including insights into the activity of uncharacterized mitochondrial proteins and the functional connection between mitochondria and the ER. The MITO-MAP also reveals a large Inner Membrane–associated complex, which we term MitOS for mitochondrial organizing structure, comprised of Fcj1/Mitofilin, a conserved Inner Membrane protein, and five additional components. MitOS physically and functionally interacts with both outer and Inner Membrane components and localizes to extended structures that wrap around the Inner Membrane. We show that MitOS acts in concert with ATP synthase dimers to organize the Inner Membrane and promote normal mitochondrial morphology. We propose that MitOS acts as a conserved mitochondrial skeletal structure that differentiates regions of the Inner Membrane to establish the normal internal architecture of mitochondria.

  • coassembly of mgm1 isoforms requires cardiolipin and mediates mitochondrial Inner Membrane fusion
    Journal of Cell Biology, 2009
    Co-Authors: Rachel M Devay, Suzanne Hoppins, Laura L Lackner, Lenin Dominguezramirez, Henning Stahlberg, Jodi Nunnari
    Abstract:

    Two dynamin-related protein (DRP) families are essential for fusion of the outer and Inner mitochondrial Membranes, Fzo1 (yeast)/Mfn1/Mfn2 (mammals) and Mgm1 (yeast)/Opa1 (mammals), respectively. Fzo1/Mfns possess two medial transMembrane domains, which place their critical GTPase and coiled-coil domains in the cytosol. In contrast, Mgm1/Opa1 are present in cells as long (l) isoforms that are anchored via the N terminus to the Inner Membrane, and short (s) isoforms were predicted to be soluble in the interMembrane space. We addressed the roles of Mgm1 isoforms and how DRPs function in Membrane fusion. Our analysis indicates that in the absence of a Membrane, l- and s-Mgm1 both exist as inactive GTPase monomers, but that together in trans they form a functional dimer in a cardiolipin-dependent manner that is the building block for higher-order assemblies.

  • mitochondrial outer and Inner Membrane fusion requires a modified carrier protein
    Journal of Cell Biology, 2009
    Co-Authors: Suzanne Hoppins, Michael J Mccaffery, Jennifer S Horner, Cheng Song, Jodi Nunnari
    Abstract:

    In yeast, three proteins are essential for mitochondrial fusion. Fzo1 and Mgm1 are conserved guanosine triphosphatases that reside in the outer and Inner Membranes, respectively. At each Membrane, these conserved proteins are required for the distinct steps of Membrane tethering and lipid mixing. The third essential component is Ugo1, an outer Membrane protein in the mitochondrial transport protein family. We show that Ugo1 is a modified member of this family, containing three transMembrane domains and existing as a dimer, a structure that is critical for the fusion function of Ugo1. Our functional analysis of Ugo1 indicates that it is required distinctly for both outer and Inner Membrane fusion after Membrane tethering, indicating that it operates at the lipid-mixing step of fusion. This role is distinct from the fusion dynamin-related proteins and thus demonstrates that at each Membrane, a single fusion protein is not sufficient to drive the lipid-mixing step, but instead, this step requires a more complex assembly of proteins.

  • mitochondrial Inner Membrane fusion and crista maintenance requires the dynamin related gtpase mgm1
    Cell, 2006
    Co-Authors: Shelly Meeusen, Ann Cassidystone, Rachel M Devay, Michael J Mccaffery, Jennifer Block, Sarah M Wayson, Jodi Nunnari
    Abstract:

    Mitochondrial outer- and Inner-Membrane fusion events are coupled in vivo but separable and mechanistically distinct in vitro, indicating that separate fusion machines exist in each Membrane. Outer-Membrane fusion requires trans interactions of the dynamin-related GTPase Fzo1, GTP hydrolysis, and an intact Inner-Membrane proton gradient. Inner-Membrane fusion also requires GTP hydrolysis but distinctly requires an Inner-Membrane electrical potential. The protein machinery responsible for Inner-Membrane fusion is unknown. Here, we show that the conserved interMembrane-space dynamin-related GTPase Mgm1 is required to tether and fuse mitochondrial Inner Membranes. We observe an additional role of Mgm1 in Inner-Membrane dynamics, specifically in the maintenance of crista structures. We present evidence that trans Mgm1 interactions on opposing Inner Membranes function similarly to tether and fuse Inner Membranes as well as maintain crista structures and propose a model for how the mitochondrial dynamins function to facilitate fusion.

Shelley D. Minteer - One of the best experts on this subject based on the ideXlab platform.

  • Mitochondrial Inner Membrane Biomimic for the Investigation of Electron Transport Chain Supercomplex Bioelectrocatalysis
    ACS Catalysis, 2016
    Co-Authors: Lindsey N. Pelster, Shelley D. Minteer
    Abstract:

    Researchers have proposed that the efficiency of the electron transport chain is due to the synergy among complexes I, III, and IV in the Membrane. In this paper, the enzymes of the supercomplex were isolated together, reconstituted into lipids that mimic the Inner Membrane of mitochondria, and immobilized in a tethered lipid bilayer on a gold electrode. The supercomplex enzymes retained their activity with the addition of their substrates and were inhibited by their respective toxins. The bioelectrocatalytic studies indicate the interdependence of the activity of the different complexes in the bioelectrocatalysis of the electron transport chain supercomplex. These fundamental studies provide a starting point to consider the use of supercomplexes and enzyme cascades for bioenergy conversion applications and biosensing through the regulation of the activity by inhibition.

  • Mitochondrial Inner Membrane Biomimic for the Investigation of Electron Transport Chain Supercomplex Bioelectrocatalysis
    2016
    Co-Authors: Lindsey N. Pelster, Shelley D. Minteer
    Abstract:

    Researchers have proposed that the efficiency of the electron transport chain is due to the synergy among complexes I, III, and IV in the Membrane. In this paper, the enzymes of the supercomplex were isolated together, reconstituted into lipids that mimic the Inner Membrane of mitochondria, and immobilized in a tethered lipid bilayer on a gold electrode. The supercomplex enzymes retained their activity with the addition of their substrates and were inhibited by their respective toxins. The bioelectrocatalytic studies indicate the interdependence of the activity of the different complexes in the bioelectrocatalysis of the electron transport chain supercomplex. These fundamental studies provide a starting point to consider the use of supercomplexes and enzyme cascades for bioenergy conversion applications and biosensing through the regulation of the activity by inhibition