RESUMO
The Escherichia coli twin arginine translocation (Tat) system mediates Sec-independent export of protein precursors bearing twin arginine signal peptides. The genes tatA, tatB, tatC and tatE code for integral membrane proteins that are components of the Tat pathway. Cells co-overexpressing tatABCDE show an increased rate of export of a signal peptide-defective Tat precursor protein and a complex containing the TatA and TatB proteins can be purified from the membranes of such cells. The purified TatAB complex has an apparent molecular mass of 600 kDa as measured by gel permeation chromatography and, like the membranes of wild-type cells, contains a large molar excess of TatA over TatB. Negative stain electron microscopy of the complex reveals cylindrical structures that may correspond to the Tat protein transport channel.
Assuntos
Proteínas de Transporte/química , Proteínas de Escherichia coli , Escherichia coli/química , Proteínas de Membrana Transportadoras , Sequência de Bases , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/ultraestrutura , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Microscopia Eletrônica , Conformação ProteicaRESUMO
Currently described substrates of the bacterial Tat protein transport system are directed for export by signal peptides containing a pair of invariant arginine residues. The signal peptide of the TtrB subunit of Salmonella enterica tetrathionate reductase contains a single arginine residue but is nevertheless able to mediate Tat pathway transport. This naturally occurring example of a Tat signal peptide lacking a consensus arginine pair expands the range of sequences that can target a protein to the Tat pathway. The possible implications of this finding for the assembly of electron transfer complexes containing Rieske proteins in plant organelles are discussed.
Assuntos
Motivos de Aminoácidos/fisiologia , Oxirredutases/química , Oxirredutases/metabolismo , Sinais Direcionadores de Proteínas/fisiologia , Substituição de Aminoácidos , Arginina/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência Consenso/fisiologia , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oxirredutases/genética , Transporte Proteico/fisiologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Salmonella enterica/enzimologia , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Frações Subcelulares/químicaRESUMO
The Tat system is a recently discovered protein export pathway that serves to translocate folded proteins, often containing redox cofactors, across the bacterial cytoplasmic membrane. Here we report that tat strains are associated with a mutant cell septation phenotype, where chains of up to 10 cells are evident. Mutant strains are also hypersensitive to hydrophobic drugs and to lysis by lysozyme in the absence of EDTA, and they leak periplasmic enzymes, characteristics that are consistent with an outer membrane defect. Both phenotypes are similar to those displayed by strains carrying point mutations in the lpxC (envA) gene. The phenotype was not replicated by mutations affecting synthesis and/or activity of all known or predicted Tat substrates.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Divisão Celular/genética , Membrana Celular/fisiologia , Proteínas de Escherichia coli , Escherichia coli/fisiologia , Proteínas de Membrana Transportadoras , Mutação , Amidoidrolases/genética , Antibacterianos/farmacologia , Bacteriófago P1/fisiologia , Divisão Celular/fisiologia , Membrana Celular/ultraestrutura , Detergentes/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/ultraestrutura , Microscopia Eletrônica , Muramidase/farmacologia , Periplasma/fisiologia , Transporte Proteico , Ribonucleases/metabolismoRESUMO
The Tat system is a recently discovered bacterial protein transport pathway that functions primarily in the biosynthesis of proteins containing redox active cofactors. Analogous transport systems are found in plant organelles. Remarkably and uniquely the Tat system functions to transported a diverse range of folded proteins across a biological membrane, a feat that must be achieved without rendering the membrane freely permeable to protons and other ions. Here we review the operation of the bacterial Tat system and propose a model for the structural organisation of the Tat preprotein translocase.
Assuntos
Adenosina Trifosfatases/metabolismo , Bactérias/metabolismo , Proteínas de Bactérias , Proteínas de Transporte/metabolismo , Proteínas de Escherichia coli , Proteínas de Membrana Transportadoras , Transporte Proteico , Adenosina Trifosfatases/química , Bactérias/genética , Proteínas de Transporte/química , Transporte de Elétrons , Metabolismo Energético , Escherichia coli , Genes Reguladores , Membranas Intracelulares/química , Membranas Intracelulares/metabolismo , Canais de Translocação SEC , Proteínas SecARESUMO
In Escherichia coli a subset of periplasmic proteins is exported through the Tat pathway to which substrates are directed by an NH(2)-terminal signal peptide containing a consensus SRRXFLK "twin arginine" motif. The importance of the individual amino acids of the consensus motif for in vivo Tat transport has been assessed by site-directed mutagenesis of the signal peptide of the Tat substrate pre-SufI. Although the invariant arginine residues are crucial for efficient export, we find that slow transport of SufI is still possible if a single arginine is conservatively substituted by a lysine residue. Thus, in at least one signal peptide context there is no absolute dependence of Tat transport on the arginine pair. The consensus phenylalanine residue was found to be a critical determinant for efficient export but could be functionally substituted by leucine, another amino acid with a highly hydrophobic side chain. Unexpectedly, the consensus lysine residue was found to retard Tat transport. These observations and others suggest that the sequence conservation of the Tat consensus motif is a reflection of the functional importance of the consensus residues. Tat signal peptides characteristically have positively charged carboxyl-terminal regions. However, changing the sign of this charge does not affect export of SufI.
Assuntos
Arginina/análise , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras , Substituição de Aminoácidos , Arginina/genética , Sequência Consenso , Lisina/análise , Lisina/genética , Mutagênese Sítio-Dirigida , Serina/análise , Serina/genéticaRESUMO
The Escherichia coli Tat system mediates Sec-independent export of protein precursors bearing twin arginine signal peptides. Genes known to be involved in this process include tatA, tatB, and tatC that form an operon with a fourth gene, tatD. The tatD gene product has two homologues in E. coli coded by the unlinked ycfH and yjjV genes. An E. coli strain with in-frame chromosomal deletions in all three of tatD, ycfH, and yjjV exhibits no significant defect in the cellular location of five cofactor-containing enzymes that are synthesized with twin arginine signal peptides. Neither these mutations nor overproduction of the TatD protein cause any discernible effect on the export kinetics of an additional E. coli Tat pathway substrate. It is concluded that proteins of the TatD family have no obligate involvement in protein export by the Tat system. TatD is shown to be a cytoplasmic protein. TatD binds to immobilized Ni(2+) or Zn(2+) affinity columns and exhibits magnesium-dependent DNase activity. Features of the tatA operon that may control TatD expression are discussed.
Assuntos
Citoplasma/metabolismo , Desoxirribonucleases/metabolismo , Proteínas de Escherichia coli/metabolismo , Exonucleases/metabolismo , Produtos do Gene tat/metabolismo , Transporte Biológico , DNA Bacteriano , Escherichia coli/genética , Escherichia coli/metabolismo , Cinética , Dados de Sequência Molecular , Precursores de Proteínas/metabolismoRESUMO
In Escherichia coli, transmembrane translocation of proteins can proceed by a number of routes. A subset of periplasmic proteins are exported via the Tat pathway to which proteins are directed by N-terminal "transfer peptides" bearing the consensus (S/T)RRXFLK "twin-arginine" motif. The Tat system involves the integral membrane proteins TatA, TatB, TatC, and TatE. Of these, TatA, TatB, and TatE are homologues of the Hcf106 component of the DeltapH-dependent protein import system of plant thylakoids. Deletion of the tatB gene alone is sufficient to block the export of seven endogenous Tat substrates, including hydrogenase-2. Complementation analysis indicates that while TatA and TatE are functionally interchangeable, the TatB protein is functionally distinct. This conclusion is supported by the observation that Helicobacter pylori tatA will complement an E. coli tatA mutant, but not a tatB mutant. Analysis of Tat component stability in various tat deletion backgrounds shows that TatC is rapidly degraded in the absence of TatB suggesting that TatC complexes, and is stabilized by, TatB.
Assuntos
Escherichia coli/metabolismo , Produtos do Gene tat/metabolismo , Alelos , Sequência de Bases , Transporte Biológico , Produtos do Gene tat/genética , Hidrogenase/metabolismo , Dados de Sequência Molecular , MutaçãoRESUMO
We describe the identification of two Escherichia coli genes required for the export of cofactor-containing periplasmic proteins, synthesized with signal peptides containing a twin arginine motif. Both gene products are homologous to the maize HCF106 protein required for the translocation of a subset of lumenal proteins across the thylakoid membrane. Disruption of either gene affects the export of a range of such proteins, and a complete block is observed when both genes are inactivated. The Sec protein export pathway was unaffected, indicating the involvement of the gene products in a novel export system. The accumulation of active cofactor-containing proteins in the cytoplasm of the mutant strains suggests a role for the gene products in the translocation of folded proteins. One of the two HCF106 homologues is encoded by the first gene of a four cistron operon, tatABCD, and the second by an unlinked gene, tatE. A mutation previously assigned to the hcf106 homologue encoded at the tatABCD locus, mttA, lies instead in the tatB gene.
Assuntos
Proteínas de Bactérias/fisiologia , Escherichia coli/fisiologia , Genes Bacterianos , Proteínas de Plantas , Proteínas de Bactérias/genética , Sequência de Bases , Transporte Biológico , Clonagem Molecular , Citoplasma/metabolismo , DNA Bacteriano , Ativação Enzimática , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Formiato Desidrogenases/metabolismo , Hidrogenase/metabolismo , Proteínas de Membrana/química , Dados de Sequência Molecular , Mutagênese , Oxirredutases N-Desmetilantes/genética , Oxirredutases N-Desmetilantes/metabolismo , Fenótipo , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Proteins are transported across the bacterial plasma membrane and the chloroplast thylakoid membrane by means of protein translocases that recognize N-terminal targeting signals in their cognate substrates. Transport of many of these proteins involves the well defined Sec apparatus that operates in both membranes. We describe here the identification of a novel component of a bacterial Sec-independent translocase. The system probably functions in a similar manner to a Sec-independent translocase in the thylakoid membrane, and substrates for both systems bear a characteristic twin-arginine motif in the targeting peptide. The translocase component is encoded in Escherichia coli by an unassigned reading frame, yigU, disruption of which blocks the export of at least five twin-Arg-containing precursor proteins that are predicted to bind redox cofactors, and hence fold, prior to translocation. The Sec pathway remains unaffected in the deletion strain. The gene has been designated tatC (for twin-arginine translocation), and we show that homologous genes are present in a range of bacteria, plastids, and mitochondria. These findings suggest a central role for TatC-type proteins in the translocation of tightly folded proteins across a spectrum of biological membranes.
Assuntos
Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Mitocôndrias/metabolismo , Proteínas de Plantas , Plastídeos/metabolismo , Formiato Desidrogenases/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fases de Leitura Aberta , Oxirredutases N-Desmetilantes/metabolismo , Sinais Direcionadores de Proteínas/metabolismoRESUMO
The large managed care customer represents concentrated purchasing power and product access to a large patient population. Contracting will continue to grow, and a large portion of product purchases will be under the agreements negotiated, implemented, and supported by the pharmaceutical company's national accounts department. The national accounts department will need to receive support from the organization if it is to do the job successfully.