In vivo cross-linking of the SecA and SecY subunits of the Escherichia coli preprotein translocase

Precursor protein translocation across the Escherichia coli inner membrane is mediated by the translocase, which is composed of a heterotrimeric integral membrane protein complex with SecY, SecE, and SecG as subunits and peripherally bound SecA. Cross-linking experiments were conducted to study which proteins are associated with SecA in vivo. Formaldehyde treatment of intact cells results in the specific cross-linking of SecA to SecY. Concurrently with the increased membrane association of SecA, an elevated amount of cross-linked product was obtained in cells harboring overproduced SecYEG complex. Cross-linked SecA copurified with hexahistidine-tagged SecY and not with SecE. The data indicate that SecA and SecY coexist as a stable complex in the cytoplasmic membrane in vivo.     

The SecDFyajC domain of preprotein translocase controls preprotein movement by regulating SecA membrane cycling

Escherichia coli preprotein translocase comprises a membrane-embedded hexameric complex of SecY, SecE, SecG, SecD, SecF and YajC (SecYEGDFyajC) and the peripheral ATPase SecA. The energy of ATP binding and hydrolysis promotes cycles of membrane insertion and deinsertion of SecA and catalyzes the movement of the preprotein across the membrane. The proton motive force (PMF), though not essential, greatly accelerates late stages of translocation. We now report that the SecDFyajC domain of translocase slows the movement of preprotein in transit against both reverse and forward translocation and exerts this control through stabilization of the inserted form of SecA. This mechanism allows the accumulation of specific translocation intermediates which can then complete translocation under the driving force of the PMF. These findings establish a functional relationship between SecA membrane insertion and preprotein translocation and show that SecDFyajC controls SecA membrane cycling to regulate the movement of the translocating preprotein.     

The SecA subunit of Escherichia coli preprotein translocase is exposed to the periplasm

SecA undergoes conformational changes during translocation, inserting domains into and across the membrane or enhancing the protease resistance of these domains. We now show that some SecA bound at SecYEG is accessible from the periplasm to a membrane-impermeant probe in cells with a permeabilized outer membrane but an intact plasma membrane.     

Syd, a SecY-interacting protein, excludes SecA from the SecYE complex with an altered SecY24 subunit

Syd is an Escherichia coli cytosolic protein that interacts with SecY. Overproduction of this protein causes a number of protein translocation-related phenotypes, including the strong toxicity against the secY24 mutant cells. Previously, this mutation was shown to impair the interaction between SecY and SecE, the two fundamental subunits of the membrane-embedded part of protein translocase. We have now studied in vitro the mechanisms of the Syd-directed inhibition of protein translocation. Pro-OmpA translocation into inverted membrane vesicles (IMVs) prepared from the secY24 mutant cells as well as the accompanied translocation ATPase activity of SecA were rapidly inhibited by purified Syd protein. In the course of protein translocation, high affinity binding of preprotein-bearing SecA to the translocase on the IMV is followed by ATP-driven insertion of the 30-kDa SecA segment into the membrane. Our experiments using 125I-labeled SecA and the secY24 mutant IMV showed that Syd abolished both the high affinity SecA binding and the SecA insertion. Syd was even able to release the inserted form of SecA that had been stabilized by a nonhydrolyzable ATP analog. Syd affected markedly the proteolytic digestion pattern of the IMV-integrated SecY24 protein, suggesting that Syd exerts its inhibitory effect by interacting directly with the SecY24 protein. In accordance with this notion, a SecY24 variant with a second site mutation (secY249) resisted the Syd action both in vivo and in vitro. Thus, Syd acts against the SecY24 form of translocase, in which SecY-SecE interaction has been compromised, to exclude the SecA motor protein from the SecYE channel complex.     

The preprotein translocase of the mitochondrial inner membrane: function and evolution

Growing mitochondria acquire most of their proteins by the uptake of mitochondrial preproteins from the cytosol. To mediate this protein import, both mitochondrial membranes contain independent protein transport systems: the Tom machinery in the outer membrane and the Tim machinery in the inner membrane. Transport of proteins across the inner membrane and sorting to the different inner mitochondrial compartments is mediated by several protein complexes which have been identified in the past years. A complex containing the integral membrane proteins Tim17 and Tim23 constitutes the import channel for preproteins containing amino-terminal hydrophilic presequences. This complex is associated with Tim44 which serves as an adaptor protein for the binding of mtHsp70 to the membrane. mtHsp70, a 70 kDa heat shock protein of the mitochondrial matrix, drives the ATP-dependent import reaction of the processed preprotein after cleavage of the presequence. Preproteins containing internal targeting information are imported by a separate import machinery, which consists of the intermembrane-space proteins Tim9, Tim10, and Tim12, and the inner membrane proteins Tim22 and Tim54. The proteins Tim17, Tim22, and Tim23 have in common a similar topology in the membrane and a homologous amino acid sequence. Moreover, they show a sequence similarity to OEP16, a channel-forming amino acid transporter in the outer envelope of chloroplasts, and to LivH, a component of a prokaryotic amino acid permease, defining a new PRAT-family of preprotein and amino acid transporters.     

The catalytic cycle of the escherichia coli SecA ATPase comprises two distinct preprotein translocation events

Poly(ADP-ribosyl)transferase (pADPRT) is a nuclear protein which catalyzes the polymerization of ADP-ribose using NAD+ as substrate, as well as the transfer of ADP-ribose polymers to itself and other protein acceptors. The catalytic activity of pADPRT strictly depends on the presence of DNA single-strand breaks. In this report, protein-protein interaction of pADPRT was found to depend on both the extent of automodification with poly(ADP-ribose) and the presence of DNA. Specific binding of radiolabeled pADPRT to transblotted proteins was first tested in blot overlay experiments. For radiolabeling, use was made of the ability of the enzyme to incorporate [32P]ADP-ribose from [32P]NAD+. Varying the concentration of NAD+, two different forms of automodified pADPRT were obtained: oligo(ADP-ribosyl)ated pADPRT with less than 20 ADP-ribose units per chain, and poly(ADP-ribosyl)ated pADPRT with polymer lengths of up to 200 ADP-ribose residues. Interaction of these probes with transblotted HeLa nuclear extracts, purified histones, and distinct regions of recombinant pADPRT was investigated. While the oligo(ADP-ribosyl)ated enzyme associated preferentially with transblotted purified histones, or pADPRT present in HeLa nuclear extracts, poly(ADP-ribosyl)ated pADPRT bound to a variety of transblotted proteins in the nuclear extracts. In the presence of DNA, both the oligo- and the poly(ADP-ribosyl)ated enzymes bound to the transblotted recombinant zinc finger domain of pADPRT even at high salt concentrations. In the absence of DNA, the transblotted automodification domain of pADPRT appeared to be the region involved in self-association. In another set of experiments, unmodified or poly(ADP-ribosyl)ated pADPRT was immobilized on Sepharose. Affinity precipitation of recombinant pADPRT domains confirmed the specific interaction of pADPRT with its zinc finger region and the automodification domain, whereas no interaction was observed with the NAD+ binding domain. Affinity precipitation of HeLa nuclear extracts with poly(ADP-ribosyl)ated pADPRT-Sepharose led to the enrichment of a number of proteins, whereas nuclear proteins bound to the unmodified pADPRT-Sepharose in a smaller extent. The results suggest that protein-protein interaction of the human pADPRT is governed by its functional state.     

The sorting route of cytochrome b2 branches from the general mitochondrial import pathway at the preprotein translocase of the inner membrane

Cytochrome b2 is synthesized in the cytosol with a bipartite presequence. The first part of the presequence targets the protein to mitochondria and mediates translocation into the mitochondrial matrix compartment; the second part contains the sorting signal that is required for delivery of the protein to the intermembrane space. The localization of the structures that recognize the sorting signal is unclear. Here we show that upon import in vivo, the sorting signal of cytochrome b2 causes an early divergence from the general matrix import pathway and thereby prevents translocation of a folded C-terminal domain into mitochondria. By co-immunoprecipitations we find that translocation intermediates of cytochrome b2 are associated with Tim23, a component of the inner membrane protein import machinery. Cytochrome b2 constructs with an alteration in the sorting signal are mistargeted to the matrix of wild-type mitochondria. In mitochondria containing a mutant form of Tim23, however, the translocation of the altered sorting signal across the inner membrane is inhibited, and cytochrome b2 is correctly sorted to the intermembrane space. We suggest that the sorting signal of cytochrome b2 is recognized within the inner membrane in close vicinity to Tim23.     

The largest subunits of RNA polymerase from gastric helicobacters are tethered

The rpoB and rpoC genes of eubacteria and archaea, coding respectively for the beta- and beta'-like subunits of DNA-dependent RNA polymerase, are organized in an operon with rpoB always preceding rpoC. The genome sequence of the gastric pathogen Helicobacter pylori (strain 26695) revealed homologs of two genes in one continuous open reading frame that potentially could encode one 2890-amino acid-long beta-beta' fusion protein. Here, we show that this open reading frame does in fact encode a fused beta-beta' polypeptide. In addition, we establish by DNA sequencing that rpoB and rpoC are also fused in each of four other unrelated strains of H. pylori, as well as in Helicobacter felis, another member of the same genus. In contrast, the rpoB and rpoC genes are separate in two members of the related genus Campylobacter (Campylobacter jejuni and Campylobacter fetus) and encode separate RNA polymerase subunits. The Campylobacter genes are also unusual in overlapping one another rather than being separated by a spacer as in other Gram-negative bacteria. We propose that the unique organization of rpoB and rpoC in H. pylori may contribute to its ability to colonize the human gastric mucosa.     

Characterization of the secA gene of Streptomyces lividans encoding a protein translocase which complements and Escherichia coli mutant defective in the ATPase activity of SecA

The secA gene of Streptomyces lividans was cloned using as probe a 57-mer oligonucleotide based on conserved sequences of the Escherichia coli secA and the Bacillus subtilis div genes. It encodes a protein of 946 amino acids (aa) with a deduced M(r) of 106,079, with high similarity to all known SecA proteins. All the previously described conserved motifs of SecA proteins were conserved in the S. lividans protein. The secA gene of S. lividans restored sensitivity to sodium azide in E. coli SecA4 (AzR) a mutant with an azide-resistant (ATPase defective) SecA protein. However, it did not complement the temperature-sensitive mutation in E. coli MM52 (SecAts) (a conditional lethal mutant defective in protein translocation) allowing only poor growth at the nonpermissive temperature. secA homologous sequences were present in 11 different species of Streptomyces and Nocardia.     

PKC1, encoding a protein kinase C, and FAT1, encoding a fatty acid transporter protein, are neighbors in Cochliobolus heterostrophus

This study aimed at analysing spatial and spatio-temporal aspects of foot-pad dermatitis in Swedish broilers. The information on disease prevalence and severity was based on a two-year foot-health surveillance programme where information on producer, breed, feed manufacturer, region, abattoir, date of slaughter and several other variables was recorded. The level of clustering in space was analysed on 2-digit zipcode level using Moran's I test which measures similarity of location. The level of clustering in space was also analysed using the Ipop test, which takes the population at risk into consideration. The examination of time-space interaction was carried out using the Barton method and the Knox method. We found a significant (p < 0.001) clustering of regions with respect to foot-pad dermatitis score using Moran's I test, and a significant (p < 0.0001) clustering in space also when related to the number of flocks delivered from each region. The flocks with very high prevalence of foot-pad dermatitis were significantly (p < 0.05) clustered in both time and space, i.e. the flocks with high prevalence of lesions came from the same geographic area during the same time periods. This information will permit us to focus the control efforts within the foot-health surveillance programme on specific regions in specific time periods, thus making the programme more effective.     

The Drosophila trithorax group proteins BRM, ASH1 and ASH2 are subunits of distinct protein complexes

The trithorax group gene brahma (brm) encodes an activator of Drosophila homeotic genes that functions as the ATPase subunit of a large protein complex. To determine if BRM physically interacts with other trithorax group proteins, we purified the BRM complex from Drosophila embryos and analyzed its subunit composition. The BRM complex contains at least seven major polypeptides. Surprisingly, the majority of the subunits of the BRM complex are not encoded by trithorax group genes. Furthermore, a screen for enhancers of a dominant-negative brm mutation identified only one trithorax group gene, moira (mor), that appears to be essential for brm function in vivo. Four of the subunits of the BRM complex are related to subunits of the yeast chromatin remodeling complexes SWI/SNF and RSC. The BRM complex is even more highly related to the human BRG1 and hBRM complexes, but lacks the subunit heterogeneity characteristic of these complexes. We present biochemical evidence for the existence of two additional complexes containing trithorax group proteins: a 2 MDa ASH1 complex and a 500 kDa ASH2 complex. These findings suggest that BRM plays a role in chromatin remodeling that is distinct from the function of most other trithorax group proteins.     

Tim9, a new component of the TIM22.54 translocase in mitochondria

We have identified Tim9, a new component of the TIM22.54 import machinery, which mediates transport of proteins into the inner membrane of mitochondria. Tim9, an essential protein of Saccharomyces cerevisiae, shares sequence similarity with Tim10 and Tim12. Tim9 is located in the mitochondrial intermembrane space and is organized into two distinct hetero-oligomeric assemblies with Tim10 and Tim12. One complex contains Tim9 and Tim10. The other complex contains Tim9, Tim10 and Tim12 and is tightly associated with Tim22 in the inner membrane. The TIM9.10 complex is more abundant than the TIM9.10.12 complex and mediates partial translocation of mitochondrial carriers proteins across the outer membrane. The TIM9.10.12 complex assists further translocation into the inner membrane in association with TIM22.54.     

Isolation and characterization of subunits of DB58, a lectin from the stems and leaves of Dolichos biflorus

The DB58 lectin of the stems and leaves of Dolichos biflorus is a heterodimer composed of two closely related subunits, alpha and beta. These subunits were dissociated from one another in urea and isolated by high-performance anion-exchange chromatography. Steric exclusion chromatography of the isolated subunits in 6 M guanidine hydrochloride showed molecular weights of 30,900 and 29,800 for the alpha and beta subunits, respectively. The subunits have very similar amino acid compositions and are glycosylated at each of their two N-glycosylation consensus sites. Each of the subunits had weak carbohydrate binding activity. Reverse-phase chromatography of tryptic digests of the subunits showed identical peptide maps with the exception of peaks identified as COOH-terminal peptides. Analyses of these peptides, COOH-terminal amino acid analyses, and the small differences in amino acid composition between the 2 subunits establish that the beta subunit differs from the alpha subunit by the absence of 11 or 12 amino acids from its COOH terminus. This structural difference, combined with information from previous biosynthetic studies, establishes that the beta subunit is derived from the alpha subunit by posttranslational proteolytic modification at the COOH terminus. The heterogeneity in the extent of truncation suggests that this conversion occurs by sequential removal of amino acids rather than by endoproteolytic cleavage. The possible physiological significance of this modification is discussed.     

钢包精炼炉的谐波危害及治理

文章介绍了谐波危害的多方面表现,分析了包钢炼钢厂LC单调谐滤波器谐波产生的原因,研究认为钢包精炼炉发生的多次事故都是由谐波引起,提出谐波治理的方案,并评价了谐波治理的效果.     

Modes of action of tunicamycin, liposidomycin B, and mureidomycin A: inhibition of phospho-N-acetylmuramyl-pentapeptide translocase from Escherichia coli

Using a continuous fluorescence-based enzyme assay, we have characterized the antibacterial agents tumicamycin and liposidomycin B as inhibitors of solubilized Escherichia coli phospho-N-acetylmuramyl-pentapeptide translocase. Tunicamycin exhibited reversible inhibition (Ki = 0.55 +/- 0.1 microM) which was noncompetitive with respect to the lipid acceptor substrate and competitive with respect to the fluorescent substrate analog, dansyl-UDPMurNAc-pentapeptide. Liposidomycin B exhibited slow-binding inhibition (Ki = 80 +/- 15 nM) which was competitive with respect to the lipid acceptor substrate and noncompetitive with respect to dansyl-UDPMurNAc-pentapeptide. These results provide insight into the molecular mechanisms of action of these two classes of nucleoside antibiotics.     

AMPA receptor subunits are differentially expressed in parvalbumin- and calretinin-positive neurons of the rat hippocampus

Recent studies suggest a functional diversity of native alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate-type glutamate receptor channels (AMPARs). In several types of interneurons, AMPARs are characterized by higher Ca2+ permeability and faster kinetics than AMPARs in principal cells. We studied the expression profile of AMPAR subunits in the hippocampal parvalbumin (PV)- and calretinin (CR)-positive cells, which represent different populations of non-principal cells. To this end, non-radioactive in situ hybridization with AMPAR subunit specific cRNAs was combined with immunocytochemistry for PV or CR. Double-immunolabelling using antibodies against AMPAR subunits and PV or CR was also performed. PV-containing neurons represent a fairly homogeneous population of cells expressing high levels of GluR-A and GluR-D mRNAs, moderate levels of GluR-C and low levels of GluR-B mRNAs in all the examined regions of hippocampus. The vast majority of CR-containing cells have a much lower expression of GluR-A, -C and -D mRNA than PV-positive neurons, although similarly featuring low levels of GluR-B mRNA. Only a subpopulation of CR-containing cells, the spiny neurons of the dentate gyrus and CA3 region of the hippocampus were characterized by a strong expression of GluR-A and -D subunit mRNAs. The differential pattern found for the AMPAR subunit mRNA expression was confirmed by immunocytochemistry at protein level. Despite the common feature of low GluR-B subunit expression, PV- and CR-containing interneurons differ with respect to the density and combination of their expressed AMPAR subunits. The different combination of subunits might subserve different properties of the AMPA channels featured by these cell types, with implications for the functioning of the hippocampal network.     

The genes encoding the delta subunits of dinitrogenases 2 and 3 are required for mo-independent diazotrophic growth by Azotobacter vinelandii

vnfG and anfG encode the delta subunits of alternative nitrogenases 2 and 3 in Azotobacter vinelandii, respectively. As a first step towards elucidating the role of these subunits, diazotrophic growth and acetylene reduction studies were conducted on mutants containing alterations in the genes encoding these subunits. Mutants containing a stop codon (C36stop) or an in-frame deletion in anfG were unable to grow in N-free, Mo-deficient medium (Anf-). Mutants in which cysteine 36 of AnfG (a residue conserved between VnfG and AnfG) was changed to Ala or Ser were Anf+. Thus, this conserved cysteine is not essential for the function of AnfG in dinitrogenase 3. A mutant with a stop codon in vnfG (C17stop) grew after a lag of 25 h in N-free, Mo-deficient medium containing V2O5. However, a Nif- Anf- strain with this mutation was unable to grow under these conditions. This shows that the vnfG gene product is required for nitrogenase 2-dependent growth. Strains with mutations in vnfG and anfG reduced acetylene to different degrees. This indicates that the delta subunits are not required for acetylene reduction by nitrogenases 2 and 3.