Old and new pathways of protein export in chloroplasts and bacteria

Targeting of chloroplast proteins to the thylakoid membrane is analogous to bacterial secretion, and much of what we know has been learned from secretory mechanisms in Escherichia coli. However, chloroplasts also use a delta pH-dependent pathway to target thylakoid proteins, at least some of which are folded before transport. Previously, this pathway seemed to have no cognate in bacteria, but recent results have shown that the HCF106 gene in maize encodes a component of this pathway and has bacterial homologues. This delta pH-dependent pathway might be an ancient conserved mechanism for protein translocation that evolved before the endosymbiotic origin of plastids and mitochondria.     

Alcohol intake of P rats is regulated by muscarinic receptors in the pedunculopontine nucleus and VTA

The CtpA protein in the cyanobacterium Synechocystis 6803 is a C-terminal processing protease that is essential for the assembly of the manganese cluster of the photosystem II complex. When fused to different chloroplast-targeting transit peptides, CtpA can be imported into isolated spinach chloroplasts and is subsequently translocated into the thylakoid lumen. Thylakoid transport is mediated by the cyanobacterial signal peptide which demonstrates that the protein transport machinery in thylakoid membranes is functionally conserved between chloroplasts and cyanobacteria. Transport of CtpA across spinach thylakoid membranes is affected by both nigericin and sodium azide indicating that the SecA protein and a transthylakoidal proton gradient are involved in this process. Saturation of the Sec-dependent thylakoid transport route by high concentrations of the precursor of the 33-kDa subunit of the oxygen-evolving system leads to a strongly reduced rate of thylakoid translocation of CtpA which demonstrates transport by the Sec pathway. However, thylakoid transport of CtpA is affected also by excess amounts of the 23-kDa subunit of the oxygen-evolving system, though to a lesser extent. This suggests that the cyanobacterial protein is capable of also interacing with components of the deltapH-dependent route and that transport of a protein across the thylakoid membrane may not always be restricted to a single pathway.     

Co-translational protein targeting catalyzed by the Escherichia coli signal recognition particle and its receptor

The Ffh-4.5S ribonucleoprotein particle (RNP) and FtsY from Escherichia coli are homologous to essential components of the mammalian signal recognition particle (SRP) and SRP receptor, respectively. The ability of these E. coli components to function in a bona fide co-translational targeting pathway remains unclear. Here we demonstrate that the Ffh-4.5S RNP and FtsY can efficiently replace their mammalian counterparts in targeting nascent secretory proteins to microsomal membranes in vitro. Targeting in the heterologous system requires a hydrophobic signal sequence, utilizes GTP and, moreover, occurs co-translationally. Unlike mammalian SRP, however, the Ffh-4.5S RNP is unable to arrest translational elongation, which results in a narrow time window for the ribosome nascent chain to interact productively with the membrane-bound translocation machinery. The highly negatively charged N-terminal domain of FtsY, which is a conserved feature among prokaryotic SRP receptor homologs, is important for translocation and acts to localize the protein to the membrane. Our data illustrate the extreme functional conservation between prokaryotic and eukaryotic SRP and SRP receptors and suggest that the basic mechanism of co-translational protein targeting is conserved between bacteria and mammals.     

Overlapping functions of components of a bacterial Sec-independent protein export pathway

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.     

Lysine 106 of the putative catalytic ATP-binding site of the Bacillus subtilis SecA protein is required for functional complementation of Escherichia coli secA mutants in vivo

The SecA protein is a major component of the cellular machinery that mediates the translocation of proteins across the Escherichia coli plasma membrane. The secA gene from Bacillus subtilis was cloned and expressed in E. coli under the control of the lac or trc promoter. The temperature-sensitive growth and secretion defects of various E. coli secA mutants were complemented by the B. subtilis SecA protein, provided the protein was expressed at moderate levels. Under overproduction conditions, no complementation was observed. One of the main features of the SecA protein is the translocation ATPase activity which, together with the protonmotive force, drives the movement of proteins across the plasma membrane. A putative ATP-binding motif can be identified in the SecA protein resembling the consensus Walker A type motif. Replacement of a lysine residue at position 106, which corresponds to an invariable amino acid residue, in the consensus motif by asparagine (K106N) resulted in the loss of the ability of the B. subtilis SecA protein to complement the growth and secretion defects of E. coli secA mutants. In addition, the presence of the K106N SecA protein interfered with protein translocation, most likely at an ATP-requiring step. We conclude that lysine 106 is part of the catalytic ATP-binding site of the B. subtilis SecA protein, which is required for protein translocation in vivo.     

Evidence for dimer participation and evidence against channel mechanism in A23187-mediated monovalent metal ion transport across phospholipid vesicular membrane

The decay of the pH difference (DeltapH) across soybean phospholipid vesicular membrane by ionophore A23187 (CAL)-mediated H+/M+ exchange (M+ = Li+, Na+, K+, and Cs+) has been studied in the pH range 6-7.6. The DeltapH in these experiments were created by temperature jump. The observed dependence of DeltapH relaxation rate 1/tau on the concentration of CAL, pH, and the choice of M+ in vesicle solutions lead to the following conclusions. 1) The concentrations of dimers and other oligomers of A23187 in the membrane are small compared to the total concentration of A23187 in the membrane, similar to that in chloroform solutions reported in the literature. 2) In the H+ transport cycle leading to DeltapH decay, the A23187-mediated H+ translocation across the membrane is a fast step, and the rate-limiting step is the A23187-mediated M+ translocation. 3) Even though the monomeric Cal-H is the dominant species translocating H+, Cal-M is not the dominant species translocating M+ (even at concentrations higher than [Cal-H]), presumably because its dissociation rate is much higher than its translocation rate. 4) The pH dependence of 1/tau shows that the dimeric species Cal2LiLi, Cal2NaNa, Cal2KH, and Cal2CsH are the dominant species translocating M+. The rate constant associated with their translocation has been estimated to be approximately 5 x 10(3) s-1. With this magnitude for the rate constants, the dimer dissociation constants of these species in the membrane have been estimated to be approximately 4, 1, 0.05, and 0.04 M, respectively. 5) Contrary to the claims made in the literature, the data obtained in the DeltapH decay studies do not favor the channel mechanism for the ion transport in this system. 6) However, they support the hypothesis that the dissociation of the divalent metal ion-A23187 complex is the rate limiting step of A23187-mediated divalent metal ion transport.     

In vivo analyses of interactions between SecE and SecY, core components of the Escherichia coli protein translocation machinery

We have carried out structure-function studies on the cytoplasmic membrane protein, SecE, a component of the Escherichia coli secretion machinery. SecE, along with SecY, form a complex in the cytoplasmic membrane essential for protein translocation. By directed mutagenesis, we altered highly conserved residues of the second cytoplasmic domain (CD2) and of the COOH-terminal periplasmic region (PD2) of SecE. These mutants, as well as previously constructed mutations in the third membrane-spanning segment of SecE (MSS3), were tested for their ability to complement a secE null mutation, for their effects on protein export in vivo, and for their ability to form a stable complex with SecY. Most single mutations at the conserved positions in CD2 caused secretion defects, but had little effect on growth at 37 degrees C. Double mutations in CD2, or the introduction or removal of proline residues, affected growth and protein translocation more severely. Co-immunoprecipitations of SecE and SecY revealed that all mutant proteins, except those altered in PD2, destabilized the SecE-SecY complex. These results suggest that several regions contribute to the formation of a stable SecE-SecY complex but the elimination of a single contact point does not necessarily affect the functionality of the complex.     

A novel EspA-associated surface organelle of enteropathogenic Escherichia coli involved in protein translocation into epithelial cells

Enteropathogenic Escherichia coli (EPEC), like many bacterial pathogens, employ a type III secretion system to deliver effector proteins across the bacterial cell. In EPEC, four proteins are known to be exported by a type III secretion system_EspA, EspB and EspD required for subversion of host cell signal transduction pathways and a translocated intimin receptor (Tir) protein (formerly Hp90) which is tyrosine-phosphorylated following transfer to the host cell to become a receptor for intimin-mediated intimate attachment and 'attaching and effacing' (A/E) lesion formation. The structural basis for protein translocation has yet to be fully elucidated for any type III secretion system. Here, we describe a novel EspA-containing filamentous organelle that is present on the bacterial surface during the early stage of A/E lesion formation, forms a physical bridge between the bacterium and the infected eukaryotic cell surface and is required for the translocation of EspB into infected epithelial cells.     

An essential component of a novel bacterial protein export system with homologues in plastids and mitochondria

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.     

Depletion of Escherichia coli 4.5S RNA leads to an increase in the amount of protein elongation factor EF-G associated with ribosomes

In Escherichia coli, 4.5S RNA is found in complexes with both protein translocation protein, Ffh (a bacterial homolog of mammalian SRP54) and protein synthesis elongation factor G (EF-G). To analyze the function of 4.5S RNA in translation, we initially assessed the sensitivity of the association of 4.5S RNA with the ribosome after treatment with antibiotics that affect various stages of protein synthesis. Fusidic acid and viomycin caused 4.5S RNA to cosediment with the 70S ribosomal fraction, indicating that 4.5S RNA enters the ribosome before ribosomal translocation and release of EF-G-GDP from the ribosome. On the other hand, depletion of 4.5S RNA led to the retention of a significant amount of EF-G on 70S ribosomes. In addition, 4.5S RNA shares a conserved decanucleotide sequence (58GAAGCAGCCA67) motif with the characterized EF-G-binding site at positions 1068-1077 on 23S RNA. We therefore examined by gel mobility-shift assay whether or not mutations in the domain-IV region of 4.5S RNA, including this conserved motif, disturb the binding of EF-G to 23S RNA. Any mutation at the C62, G64 or A67 residues within this motif abolished competition activity. Therefore, we propose that 4.5S RNA is concerned with the mode of association of EF-G with the ribosomes. Moreover, this function depends on the secondary structure of 4.5S RNA as well as a ten-base sequence conserved between the two RNAs.     

A component of the chloroplast protein import apparatus functions in bacteria

Toc36 is a family of 44-kDa envelope polypeptides previously identified as components of the chloroplast protein import apparatus by virtue of their close physical proximity to translocating proteins. An indication of their function thus remains at large. A heterologous in vivo approach for studying the function of Toc36 was developed in this study by introducing a member of Toc36 into E. coli to assess its effect on bacterial protein translocation. The presence of Toc36 enhances the translocation of two bacterial periplasmic proteins in a manner resembling the chloroplast system. Translocation of the two bacterial periplasmic proteins was less sensitive to sodium azide, resembling more the azide-insensitive nature of the chloroplast protein import process. Mutated Toc36 proteins were not capable of causing the same effect as that observed for unaltered Toc36. Toc36 was also capable of complementing bacterial strains with temperature-sensitive secA mutations that affected protein translocation. The combined results provide evidence that Toc36 plays a central role in the chloroplast protein translocation process.     

Mechanical output and electromyographic parameters in males and females during fatiguing knee-extensions

Although extensive research has been carried out on the respiratory and renal effects of intra-abdominal pressure increase, there is limited research with regard to its effects on bacterial translocation. The objective of this study was to discuss whether the high intra-abdominal pressure due to carbon dioxide (CO2) insufflation during laparoscopy leads to bacterial translocation. Eighteen male dogs, 7 of which constituted the control group, were used in the study. Two study groups, in which the intra-abdominal pressure was raised to 15 mm Hg and kept at that level for 30 and 120 minutes, respectively, were set. Blood gases and blood pressure values were observed throughout the experiments. Samples of peritoneal smear, portal vein blood, mesenteric lymph node, liver, spleen, and cecum were examined to detect bacterial translocation. Histopathological examinations of all samples were also carried out. No translocation was detected in the samples of peritoneal smear, portal blood, mesenteric lymph node, liver, or spleen, but in the samples of cecum, bacterial colonization for the second group (p<0.05) and for the third group (p<0.05) was significantly higher compared with the control group. There was a considerable difference between the second and third groups (p<0.05). The changes in the mesenteric lymph nodes were interpreted to be a result of bacterial drainage. Histopathological examination disclosed active changes in the mesenteric lymph nodes in all groups, but there was considerable sinus histiocytosis only in the third group. We conclude that the intraabdominal pressure of 15 mm Hg created by carbon dioxide insufflation does not lead to bacterial translocation but causes intraluminal bacterial colonization in the cecum after 30 minutes and after 2 hours.     

GSP-dependent protein secretion in gram-negative bacteria: the Xcp system of Pseudomonas aeruginosa

Bacteria have evolved several secretory pathways to release proteins into the extracellular medium. In Gram-negative bacteria, the exoproteins cross a cell envelope composed of two successive hydrophobic barriers, the cytoplasmic and outer membranes. In some cases, the protein is translocated in a single step across the cell envelope, directly from the cytoplasm to the extracellular medium. In other cases, outer membrane translocation involves an extension of the signal peptide-dependent pathway for translocation across the cytoplasmic membrane via the Sec machinery. By analogy with the so-called general export pathway (GEP), this latter route, including two separate steps across the inner and the outer membrane, was designated as the general secretory pathway (GSP) and is widely conserved among Gram-negative bacteria. In their great majority, exoproteins use the main terminal branch (MTB) of the GSP, namely the Xcp machinery in Pseudomonas aeruginosa, to reach the extracellular medium. In this review, we will use the P. aeruginosa Xcp system as a basis to discuss multiple aspects of the GSP mechanism, including machinery assembly, exoprotein recognition, energy requirement and pore formation for driving through the outer membrane.     

Regulation of serine biosynthesis in Arabidopsis. Crucial role of plastidic 3-phosphoglycerate dehydrogenase in non-photosynthetic tissues

In plants, Ser is synthesized through a couple of pathways. 3-Phosphoglycerate dehydrogenase (PGDH), the first enzyme that is involved in the phosphorylated pathway of Ser biosynthesis, is responsible for the oxidation of 3-phosphoglycerate to phosphohydroxypyruvate. Here we report the first molecular cloning and characterization of PGDH from Arabidopsis thaliana. Sequence analysis of cDNA and a genomic clone revealed that the PGDH gene is composed of three exons, encoding a 623-amino acid polypeptide (66, 453 Da). The deduced protein, containing three of the most conserved regions in the NAD-dependent 2-hydroxyacid dehydrogenase family, has 38-39% identity to its animal and bacterial counterparts. The presence of an N-terminal signal sequence for translocation into plastids was confirmed by particle-gun bombardment experiments using green fluorescence protein as a reporter protein for subcellular localization. Southern hybridization analysis and restriction fragment length polymorphism mapping indicated that PGDH is a single-copy gene that is mapped to the upper arm of chromosome 1. Northern hybridization analysis indicated preferential expression of PGDH mRNA in root tissues of light-grown plants, suggesting that the phosphorylated pathway of Ser biosynthesis plays an important role in supplying Ser to non-photosynthetic tissues. The recombinant enzyme overproduced in Escherichia coli displayed hyperbolic kinetics with respect to 3-phosphoglycerate and NAD+.     

Drug-combination therapy of rheumatoid arthritis

The cytochromes c are a useful model for the study of the pathways and mechanisms of assembly of the cofactor-containing components of energy transducing membranes. Genetic analyses have identified proteins that are required for the assembly of c-type cytochromes in mitochondria, bacteria and chloroplasts. The components of the pathway operating in fungal and animal mitochondria, i.e. the cytochrome (cyt) c and c1 heme lyases in the intermembrane space, were identified over a decade ago through the study of cytochrome deficiencies in Neurospora crassa and Saccharomyces cerevisiae. More recently, a large number of membrane or membrane-associated components were identified in various alpha- and gamma-proteobacteria as c-type cytochrome assembly factors; they comprise an assembly pathway that is evolutionarily and mechanistically distinct from that in fungal and animal mitochondria. The components function not only in the lyase reaction but also in the delivery and maintenance of the substrates in a state that is suitable for reaction in the bacterial periplasm. Yet a third pathway is required for cytochrome maturation in chloroplasts. Genetic analyses of Chlamydomonas reinhardtii ccs mutants, which are pleiotropically deficient in both the membrane-anchored cytochrome f and the soluble cytochrome c6, revealed a minimum of six loci, plastid ccsA and nuclear CCS1 through CCS5, that are required for the conversion of the chloroplast apocytochromes to their respective holo forms. Sequence analysis of the cloned ccsA and Ccs1 genes indicates that the predicted protein products are integral membrane proteins with homologues in cyanobacteria, some gram-positive bacteria (Bacillus subtilis, Mycobacterium spp.), beta-proteobacteria (Neisseria spp.) and an epsilon-proteobacterium (Helicobacter pylori). CcsA and Ccs1 require each other for accumulation in vivo and are therefore proposed to function in a complex, possibly with the products of some of the other CCS loci. A tryptophan-rich motif, which has been proposed to represent a heme binding site in bacterial cytochrome biogenesis proteins (CcmC and CcmF), is functionally important in plastid CcsA. As is the case for CcmC and CcmF, the tryptophan-rich sequence is predicted to occur in a loop on the p-side of the membrane, where the heme attachment reaction occurs. Conserved histidine residues in the CcsA and Ccs1 may serve as ligands to the heme iron. A multiple alignment of the tryptophan-rich regions of the CcsA-, CcmC- and CcmF-like sequences in the genome databases indicates that they represent three different families.     

Filamin translocation is an early endothelial cell inflammatory response to bradykinin: regulation by calcium, protein kinases, and protein phosphatases

Endothelial cell (EC) cytoskeletal proteins are one of the earliest primary targets of second messenger cascades generated in response to inflammatory agonists. Actin binding proteins, by modulating actin gelation-solation state and membrane-cytoskeleton interactions, in part regulate cell motility and cell-cell apposition. This in turn can also modulate interendothelial junctional diameter and permeability. Nonmuscle filamin (ABP-280), a dimeric actin-crosslinking protein, promotes orthogonal branching of F-actin and links microfilaments to membrane glycoproteins. In the present study, immunoblot analysis demonstrates that filamin protein levels are low in sparse EC cultures, increase once cell-cell contact is initiated and then decrease slightly at post-confluency. Both bradykinin and ionomycin cause filamin redistribution from the peripheral cell border to the cytosol of confluent EC. Forskolin, an activator of adenylate cyclase, blocks filamin translocation. Bradykinin activation of EC is not accompanied by significant proteolytic cleavage of filamin. Instead, intact filamin is recycled back to the membrane within 5-10 min of bradykinin stimulation. Inhibitors of calcium/calmodulin dependent protein kinase (KT-5926 and KN-62) attenuate bradykinin-induced filamin translocation. H-89, an inhibitor of cAMP-dependent protein kinase, causes translocation of filamin in unstimulated cells. Calyculin A, an inhibitor of protein phosphatases, also causes translocation of filamin in the absence of an inflammatory agent. ML-7, an inhibitor of myosin light chain kinase and phorbol myristate acetate, an activator of protein kinase C, do not cause filamin movement into the cytosol, indicating that these pathways do not modulate the translocation. Pharmacological data suggest that filamin translocation is initiated by the calcium/calmodulin-dependent protein kinase whereas the cAMP-dependent protein kinase pathway prevents translocation. Inflammatory agents therefore may increase vascular junctional permeability by increasing cytoplasmic calcium, which disassembles the microfilament dense peripheral band by releasing filamin from F-actin.     

A plant mitochondrial gene encodes a protein involved in cytochrome c biogenesis

Analysis of a transcribed region in the mitochondrial genome of Oenothera revealed an open reading frame (ORF) of 577 codons (orf577) that is also conserved in carrot, here encoding a protein of 579 amino acids (orf579). RNA editing alters the mRNA sequence of orf577 in Oenothera with 46 C to U transitions, many of which improve sequence similarity with the homologous Marchantia gene orf509. The deduced polypeptides show significant similarity with the ccl1-encoded protein involved in cytochrome c biogenesis in the photosynthetic bacterium Rhodobacter capsulatus. A highly conserved domain is also found in plastid ORFs, suggesting that these bacterial, chloroplast and mitochondrial genes encode polypeptides with analogous functions in assembly and maturation of cytochromes c.     

Dual topology of the large envelope protein of duck hepatitis B virus: determinants preventing pre-S translocation and glycosylation

The biosynthesis and topology of the large envelope protein (L protein) of hepadnaviruses was investigated using the duck hepatitis B virus (DHBV) model, which also allows the study of hepadnavirus morphogenesis in experimentally infected hepatocytes. Results from proteolysis of virus particles and from the analysis of topology and posttranslational modification of L chains synthesized in vivo or in a cell-free system both support the presence of a mixed population of L-protein molecules with their N-terminal pre-S domain located either inside or outside the virus particle. During L biosynthesis and DHBV morphogenesis, pre-S, together with the neighboring transmembrane domain (TM-I), initially remained cytoplasmically disposed and was translocated only posttranslationally. Delayed pre-S translocation into a post-endoplasmic reticulum compartment is also indicated by the absence of glycosylation at a modification-competent pre-S glycosylation site. Major features of L-protein biosynthesis and of the resulting dual topology appear to be conserved between avian and mammalian hepadnaviruses, supporting the model that pre-S domains function in part either as an internal matrix for capsid envelopment or externally as a ligand for cellular receptor binding. However, differences in the mechanisms controlling pre-S translocation were revealed by the results of mutational analyses identifying and characterizing cis-acting determinants in pre-S that delay its cotranslational translocation. Our data from DHBV demonstrate the negative influence of a cluster of positively charged amino acid residues next to TM-I, a motif that is conserved among the avian but absent from mammalian hepadnaviruses. Additional control elements, which are apparently shared between both virus groups and which may serve in chaperone binding, were mapped by deletion analysis in the central part of pre-S.