246 results on '"secY"'
Search Results
52. Characterization of the supporting role of SecE in protein translocation.
- Author
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Lycklama a Nijeholt, Jelger A., de Keyzer, Jeanine, Prabudiansyah, Irfan, and Driessen, Arnold J.M.
- Subjects
- *
CHROMOSOMAL translocation , *MOLECULAR chaperones , *PROTEIN stability , *BINDING sites , *PROTEIN-protein interactions , *CHROMOSOMES - Abstract
Highlights: [•] Sites of interaction between SecY and SecE remain immobile during channel opening. [•] The hinge region of SecE is essential for translocation. [•] SecE functions to guide and stabilize the open pore state of SecY. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
53. Detection and Multigene Typing of ' Candidatus Phytoplasma solani'-Related Strains Infecting Tomato and Potato Plants in Different Regions of Turkey.
- Author
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Çağlar BK and Şimşek E
- Abstract
' Candidatus Phytoplasma solani' (' Ca . P. solani') is a crop pathogen that is a member of the 16SrXII-A ribosomal subgroup. It is also known as stolbur phytoplasma and causes yield losses in several important crops, especially in Solanaceous crops. Different strains of the pathogen are regularly reported all over the world, particularly in the Mediterranean region. In this study, the determination of genetic diversity for the pathogen infecting tomatoes and potatoes was carried out by using multilocus sequence typing analysis for the Tuf , Sec Y, and Vmp 1 genes to gain insight into the epidemiology of ' Ca . P. solani' in Turkey. Genetic diversity of the phytoplasmas was investigated by sequence-based phylogenetic analyses and in silico RFLP analysis of related genes. It was determined that all ' Ca . P. solani'-related strains infecting tomatoes and potatoes were tuf-b, which is linked to field bindweed ( Convolvulus arvensis L.). Tomato or potato-infecting ' Ca . P. solani'-related strains showed similarities with each other; however, the isolates collected from different plants showed genetic differences in terms of the Sec Y gene. This study indicates that the highest genetic variability of collected samples was found in the Vmp 1 gene. Rsa I-RFLP analysis of TYPH10F/R amplicons showed that potato-infecting ' Ca . P. solani'-related strains were found to be similar to some existing V types. However, the V-type of tomato-infecting isolates is not similar to any previously reported V-type. The results indicate that there could be an important genetic diversity of ' Ca . P. solani'-related phytoplasmas in Turkey. This could indicate various ways in which the pathogen has adapted to the two host plants as a consequence of the various Vmp 1 gene rearrangements seen in these two plant hosts. Obtained results also indicate that the epidemiology of ' Ca . P. solani'-related phytoplasmas in the tomato and potato agroecosystem may be better understood with the use of molecular data on the complex of vmp-types.
- Published
- 2022
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54. Protein translocation by the SecA ATPase occurs by a power‐stroke mechanism
- Author
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Catipovic, Marco A, Bauer, Benedikt W, Loparo, Joseph J, and Rapoport, Tom A
- Published
- 2019
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55. The bacterial Sec-translocase: structure and mechanism.
- Author
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Lycklama a Nijeholt, Jelger A. and Driessen, Arnold J. M.
- Subjects
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CELL membranes , *ADENOSINE triphosphate , *GRAM-negative bacteria , *MEMBRANE fusion , *ESCHERICHIA coli - Abstract
Most bacterial secretory proteins pass across the cytoplasmic membrane via the translocase, which consists of a protein-conducting channel SecYEG and an ATP-dependent motor protein SecA. The ancillary SecDF membrane protein complex promotes the final stages of translocation. Recent years have seen a major advance in our understanding of the structural and biochemical basis of protein translocation, and this has led to a detailed model of the translocation mechanism. [ABSTRACT FROM AUTHOR]
- Published
- 2012
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- View/download PDF
56. Ancestral and Derived Protein Import Pathways in the Mitochondrion of Reclinomonas americana.
- Author
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Tong, Janette, Dolezal, Pavel, Selkrig, Joel, Crawford, Simon, Simpson, Alastair G.B., Noinaj, Nicholas, Buchanan, Susan K., Gabriel, Kipros, and Lithgow, Trevor
- Abstract
The evolution of mitochondria from ancestral bacteria required that new protein transport machinery be established. Recent controversy over the evolution of these new molecular machines hinges on the degree to which ancestral bacterial transporters contributed during the establishment of the new protein import pathway. Reclinomonas americana is a unicellular eukaryote with the most gene-rich mitochondrial genome known, and the large collection of membrane proteins encoded on the mitochondrial genome of R. americana includes a bacterial-type SecY protein transporter. Analysis of expressed sequence tags shows R. americana also has components of a mitochondrial protein translocase or “translocase in the inner mitochondrial membrane complex.” Along with several other membrane proteins encoded on the mitochondrial genome Cox11, an assembly factor for cytochrome c oxidase retains sequence features suggesting that it is assembled by the SecY complex in R. americana. Despite this, protein import studies show that the RaCox11 protein is suited for import into mitochondria and functional complementation if the gene is transferred into the nucleus of yeast. Reclinomonas americana provides direct evidence that bacterial protein transport pathways were retained, alongside the evolving mitochondrial protein import machinery, shedding new light on the process of mitochondrial evolution. [ABSTRACT FROM PUBLISHER]
- Published
- 2011
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57. Structural determinants of protein translocation in bacteria: conformational flexibility of SecA IRA1 loop region.
- Author
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Palladino, Pasquale, Saviano, Gabriella, Tancredi, Teodorico, Benedetti, Ettore, Rossi, Filomena, and Ragone, Raffaele
- Subjects
PEPTIDES ,TYROSINE ,ESCHERICHIA coli ,PROTEIN conformation ,ENZYMES - Abstract
The article discusses a study which suggests a predominant flexible state for the synthetic wild-type peptide SecA and its analog SecA[788-804]Y794A except for peptides corresponding to SecA helices. It notes that the high flexibility of the SecA domain is independent of the presence of the functional loop tyrosine but can be fundamental SecA loop docking in SecY cavity and subsequent complex activation. The peptides E. coli were synthesized in groups. It suggests that the conformational flexibility of the peptides is not affected by Y794A substitution.
- Published
- 2011
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58. Free-energy cost for translocon-assisted insertion of membrane proteins.
- Author
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Gumbart, James, Chipot, Christophe, and Schulten, Klaus
- Subjects
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MEMBRANE proteins , *AMINO acids , *MOLECULAR dynamics , *ARGININE , *PERTURBATION theory , *LEUCINE - Abstract
Nascent membrane proteins typically insert in a sequential fashion into the membrane via a protein-conducting channel, the Sec translocon. How this process occurs is still unclear, although a thermodynamic partitioning between the channel and the membrane environment has been proposed. Experiment- and simulation-based scales for the insertion free energy of various amino acids are, however, at variance, the former appearing to lie in a narrower range than the latter. Membrane insertion of arginine, for instance, requires 14-17 kcal/mol according to molecular dynamics simulations, but only 2-3 kcal/mol according to experiment. We suggest that this disagreement is resolved by assuming a two-stage insertion process wherein the first step, the insertion into the translocon, is energized by protein synthesis and, therefore, has an effectively zero free-energy cost; the second step, the insertion into the membrane, invokes the translocon as an intermediary between the fully hydrated and the fully inserted locations. Using free-energy perturbation calculations, the effective transfer free energies from the translocon to the membrane have been determined for both arginine and leucine amino acids carried by a background polyleucine helix. Indeed, the insertion penalty for arginine as well as the insertion gain for leucine from the translocon to the membrane is found to be significantly reduced compared to direct insertion from water, resulting in the same compression as observed in the experiment-based scale. [ABSTRACT FROM AUTHOR]
- Published
- 2011
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59. In vitro synthesis and oligomerization of the mechanosensitive channel of large conductance, MscL, into a functional ion channel
- Author
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Price, Claire E., Kocer, Armagan, Kol, Stefan, van der Berg, Jan Pieter, and Driessen, Arnold J.M.
- Subjects
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ION channels , *MEMBRANE proteins , *CYTOPLASM , *GENE expression , *LIPOSOMES , *OLIGOMERS - Abstract
Abstract: Elucidation of high-resolution structures of integral membrane proteins is drastically lagging behind that of cytoplasmic proteins. In vitro synthesis and insertion of membrane proteins into synthetic membranes could circumvent bottlenecks associated with the overexpression of membrane proteins, producing sufficient membrane-inserted, correctly folded protein for structural studies. Using the mechanosensitive channel of large conductance, MscL, as a model protein we show that in vitro synthesized MscL inserts into YidC-containing proteoliposomes and oligomerizes to form a homopentamer. Using planar membrane bilayers, we show that MscL forms functional ion channels capable of ion transport. These data demonstrate that membrane insertion of MscL is YidC mediated, whereas subsequent oligomerization into a functional homopentamer is a spontaneous event. [ABSTRACT FROM AUTHOR]
- Published
- 2011
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60. Identification of vaginal Leptospira in cervical-vaginal mucus of slaughtered pigs in the amazon region.
- Author
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Gomes, Yuri Almeida, Medeiros, L.S., Di Azevedo, Maria Isabel Nogueira, Loureiro, Ana Paula, Loria de Melo, Juliana dos Santos, Carvalho-Costa, Filipe Anibal, and Lilenbaum, Walter
- Subjects
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LEPTOSPIRA , *GENITALIA , *LEPTOSPIRA interrogans , *ABORTION , *SWINE - Abstract
Swine genital leptospirosis is an infectious disease that leads to economic losses due to abortions, stillbirths, and reproductive failures. Considering the scarcity of studies regarding this condition, the objective of the present study was to identify and analyse leptospires infecting the reproductive tract of female pigs slaughtered in the Amazon region. Cervical-vaginal mucus (CVM) from 150 non-pregnant females were collected and submitted to molecular analysis. Initially, polymerase chain reaction (PCR) based on the lipL32 gene was performed. A total of 26.7% (40/150) samples were positive, indicating the presence of Leptospira sp. DNA. Subsequently, positive lipL32- PCR samples were evaluated using secY nested-PCR and sequencing procedures. Eleven amplicons could be sequenced and were identified as Leptospira interrogans (100% identity). Results from phylogenetic analyses led to identification of a putative strain of L. interrogans serogroup Australis, which is indicative of this being a serogroup. In the present study, there was detection of female pigs with leptospires in CVM indicating the possibility of venereal transmission. The large number of genital positive cases could indicate that genital leptospirosis syndrome could also be relevant on to swine production. • Identification of leptospires in Cervico-vaginal mucus in swine. • Detection of leptospires in cervico-vaginal mucus indicates possible venereal transmission. • Genital leptospirosis syndrome could also be relevant in swine. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
61. Protein transport across the endoplasmic reticulum membrane.
- Author
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Rapoport, Tom A.
- Subjects
- *
PROTEINS , *PROKARYOTES , *ENDOPLASMIC reticulum , *CHROMOSOMAL translocation , *CELL membranes - Abstract
A decisive step in the biosynthesis of many eukaryotic proteins is their partial or complete translocation across the endoplasmic reticulum membrane. A similar process occurs in prokaryotes, except that proteins are transported across or are integrated into the plasma membrane. In both cases, translocation occurs through a protein-conducting channel that is formed from a conserved, heterotrimeric membrane protein complex, the Sec61 or SecY complex. Structural and biochemical data suggest mechanisms that enable the channel to function with different partners, to open across the membrane and to release laterally hydrophobic segments of membrane proteins into lipid. [ABSTRACT FROM AUTHOR]
- Published
- 2008
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62. Sec- and Tat-mediated protein secretion across the bacterial cytoplasmic membrane—Distinct translocases and mechanisms
- Author
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Natale, Paolo, Brüser, Thomas, and Driessen, Arnold J.M.
- Subjects
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BIOLOGICAL transport , *CELL membranes , *FUNGUS-bacterium relationships , *SURFACE chemistry - Abstract
Abstract: In bacteria, two major pathways exist to secrete proteins across the cytoplasmic membrane. The general Secretion route, termed Sec-pathway, catalyzes the transmembrane translocation of proteins in their unfolded conformation, whereupon they fold into their native structure at the trans-side of the membrane. The Twin-arginine translocation pathway, termed Tat-pathway, catalyses the translocation of secretory proteins in their folded state. Although the targeting signals that direct secretory proteins to these pathways show a high degree of similarity, the translocation mechanisms and translocases involved are vastly different. [Copyright &y& Elsevier]
- Published
- 2008
- Full Text
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63. Protein Translocation Across the Bacterial Cytoplasmic Membrane.
- Author
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Driessen, Arnold J. M. and Nouwen, Nico
- Subjects
- *
PROTEINS , *CELL membranes , *BACTERIA , *MOLECULAR chaperones , *PROTONS - Abstract
About 25% to 30% of the bacterial proteins function in the cell envelope or outside of the cell. These proteins are synthesized in the cytosol, and the vast majority is recognized as a ribosome-bound nascent chain by the signal recognition particle (SRP) or by the secretion-dedicated chaperone SecB. Subsequently, they are targeted to the Sec translocase in the cytoplasmic membrane, a multimeric membrane protein complex composed of a highly conserved protein-conducting channel, SecYEG, and a peripherally bound ribosome or ATP-dependent motor protein SecA. The Sec translocase mediates the translocation of proteins across the membrane and the insertion of membrane proteins into the cytoplasmic membrane. Translocation requires the energy sources of ATP and the proton motive force (PMF) while the membrane protein insertion is coupled to polypeptide chain elongation at the ribosome. This review summarizes the present knowledge of the mechanism and structure of the Sec translocase, with a special emphasis on unresolved questions and topics of current research. [ABSTRACT FROM AUTHOR]
- Published
- 2008
- Full Text
- View/download PDF
64. Characterization of putative membrane protein genes of the ‘Candidatus Phytoplasma asteris’, chrysanthemum yellows isolate.
- Author
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Galetto, Luciana, Fletcher, Jacqueline, Bosco, Domenico, Turina, Massimo, Wayadande, Astri, and Marzachì, Cristina
- Subjects
- *
PHYTOPLASMAS , *MEMBRANE proteins , *GENETIC regulation , *ASTERS , *GENETIC vectors , *CHRYSANTHEMUMS , *EPITOPES , *SCIENTIFIC method , *MICROBIOLOGY education , *PHYSIOLOGY - Abstract
To characterize potentially important surface-exposed proteins of the phytoplasma causing chrysanthemum yellows (CY), new primers were designed based on the conserved regions of 3 membrane protein genes of the completely sequenced onion yellows and aster yellows witches’ broom phytoplasmas and were used to amplify CY DNA. The CY genes secY, amp, and artI, encoding the protein translocase subunit SecY, the antigenic membrane protein Amp and the arginine transporter ArtI, respectively, were cloned and completely sequenced. Alignment of CY-specific secY sequences with the corresponding genes of other phytoplasmas confirmed the 16S rDNA-based classification, while amp sequences were highly variable within the ‘Candidatus Phytoplasma asteris’. Five CY partial sequences were cloned into the pRSetC expression vector, and 3 of the encoded protein fragments (Amp 64/651, Amp 64/224, ArtI 131/512) were expressed as fusion antigens for the production of CY-specific polyclonal antibodies (A416 against Amp 64/224; A407 against ArtI 131/512). A416 recognized, in Western blots, the full-length Amp from CY-infected plants (periwinkle, daisy) and insect vectors (Euscelidius variegatus, Macrosteles quadripunctulatus). A416 also reacted to European aster yellows, to primula yellows phytoplasmas, to northern Italian strains of ‘Ca. Phytoplasma asteris’ from lettuce and gladiolus, but it did not react to American aster yellows phytoplasma. [ABSTRACT FROM AUTHOR]
- Published
- 2008
- Full Text
- View/download PDF
65. Kinetics and Energetics of the Translocation of Maltose Binding Protein Folding Mutants
- Author
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Tomkiewicz, Danuta, Nouwen, Nico, and Driessen, Arnold J.M.
- Subjects
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CARRIER proteins , *PROTEIN folding , *ESCHERICHIA , *PROTEIN conformation - Abstract
Abstract: Protein translocation in Escherichia coli is mediated by the translocase that, in its minimal form, comprises a protein-conducting pore (SecYEG) and a motor protein (SecA). The SecYEG complex forms a narrow channel in the membrane that allows passage of secretory proteins (preproteins) in an unfolded state only. It has been suggested that the SecA requirement for translocation depends on the folding stability of the mature preprotein domain. Here we studied the effects of the signal sequence and SecB on the folding and translocation of folding stabilizing and destabilizing mutants of the mature maltose binding protein (MBP). Although the mutations affect the folding of the precursor form of MBP, these are drastically overruled by the combined unfolding stabilization of the signal sequence and SecB. Consequently, the translocation kinetics, the energetics and the SecA and SecB dependence of the folding mutants are indistinguishable from those of wild-type preMBP. These data indicate that unfolding of the mature domain of preMBP is likely not a rate-determining step in translocation when the protein is targeted to the translocase via SecB. [Copyright &y& Elsevier]
- Published
- 2008
- Full Text
- View/download PDF
66. Bacterial Sec-translocase Unfolds and Translocates a Class of Folded Protein Domains
- Author
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Nouwen, Nico, Berrelkamp, Greetje, and Driessen, Arnold J.M.
- Subjects
- *
CHROMOSOMAL translocation , *BACTERIAL immunoglobulin-binding proteins , *MICROBIAL peptides , *BACTERIAL proteins - Abstract
Abstract: It is generally assumed that preprotein substrates must be presented in an unfolded state to the bacterial Sec-translocase in order to be translocated. Here, we have examined the ability of the Sec-translocase to translocate folded preproteins. Tightly folded human cardiac Ig-like domain I27 fused to the C terminus of proOmpA is translocated efficiently by the Sec-translocase and the translocation kinetics are determined by the extent of folding of the titin I27 domain. Accumulation of specific translocation intermediates around the fusion point that undergo translocation progress upon ATP binding suggests that the motor protein SecA plays an important and decisive role in promoting unfolding of the titin I27 domain. It is concluded that the bacterial Sec-translocase is capable of actively unfolding preproteins. [Copyright &y& Elsevier]
- Published
- 2007
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67. Arginine 357 of SecY is needed for SecA-dependent initiation of preprotein translocation
- Author
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de Keyzer, Jeanine, Regeling, Anouk, and Driessen, Arnold J.M.
- Subjects
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ARGININE , *CHROMOSOMAL translocation , *ADENOSINE triphosphatase , *MEMBRANE proteins - Abstract
Abstract: The Escherichia coli SecYEG complex forms a transmembrane channel for both protein export and membrane protein insertion. Secretory proteins and large periplasmic domains of membrane proteins require for translocation in addition the SecA ATPase. The conserved arginine 357 of SecY is essential for a yet unidentified step in the SecA catalytic cycle. To further dissect its role, we have analysed the requirement for R357 in membrane protein insertion. Although R357 substitutions abolish post-translational translocation, they allow the translocation of periplasmic domains targeted co-translationally by an N-terminal transmembrane segment. We propose that R357 is essential for the initiation of SecA-dependent translocation only. [Copyright &y& Elsevier]
- Published
- 2007
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68. Corrigendum: Dynamic nature of SecA and Its associated proteins in Escherichia coli.
- Author
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Adachi, Shun, Murakawa, Yasuhiro, and Hiraga, Sota
- Subjects
ESCHERICHIA coli ,DNA topoisomerase I ,PROTEINS - Abstract
Keywords: chromosome partition; SecA; SecY; AcpP; MukB; DNA topoisomerase EN chromosome partition SecA SecY AcpP MukB DNA topoisomerase N.PAG N.PAG 2 11/30/20 20201125 NES 201125 In the original article, there was a mistake in Figure 2 as published. (A) SecA-GFP uv4 in wild-type cells (MQ318) growing at 30°C. (E) SecA-GFP uv4 in acpP D38V(Ts) mutant cells (MQ456) growing at the non-permissive temperature of 42°C. [Extracted from the article]
- Published
- 2020
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69. Identification of Two Interaction Sites in SecY that Are Important for the Functional Interaction with SecA
- Author
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van der Sluis, Eli O., Nouwen, Nico, Koch, Joachim, de Keyzer, Jeanine, van der Does, Chris, Tampé, Robert, and Driessen, Arnold J.M.
- Subjects
- *
GLUTATHIONE , *SERUM albumin , *BLOOD proteins , *CELL membranes - Abstract
Abstract: The motor protein SecA drives the translocation of (pre-)proteins across the SecYEG channel in the bacterial cytoplasmic membrane by nucleotide-dependent cycles of conformational changes often referred to as membrane insertion/de-insertion. Despite structural data on SecA and an archaeal homolog of SecYEG, the identity of the sites of interaction between SecA and SecYEG are unknown. Here, we show that SecA can be cross-linked to several residues in cytoplasmic loop 5 (C5) of SecY, and that SecA directly interacts with a part of transmembrane segment 4 (TMS4) of SecY that is buried in the membrane region of SecYEG. Mutagenesis of either the conserved Arg357 in C5 or Glu176 in TMS4 interferes with the catalytic activity of SecA but not with binding of SecA to SecYEG. Our data explain how conformational changes in SecA could be directly coupled to the previously proposed opening mechanism of the SecYEG channel. [Copyright &y& Elsevier]
- Published
- 2006
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70. Intracellular trafficking of fluorescently tagged proteins associated with pathogenesis in Candida albicans.
- Author
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Lee, Samuel A., Khalique, Zachary, Gale, Cheryl A., and Wong, Brian
- Subjects
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CANDIDA albicans , *PROTEINS , *SECRETION , *MICROBIAL virulence , *PATHOGENIC microorganisms - Abstract
Proteins that enter the secretory pathway play important roles in virulence and pathogenesis in Candida albicans , but our understanding of the trafficking of these proteins is in its early stages. In Saccharomyces cerevisiae , dominant negative alleles of YPT1 and SEC4 interrupt secretory traffic at pre- and post-Golgi steps, respectively. We therefore used a dominant negative genetic approach to examine the intracellular trafficking of several proteins associated with virulence or azole resistance. When the dominant negative ypt1 (N121I) allele of C. albicans was overexpressed, yellow-fluorescent protein (YFP) tagged forms of two plasma membrane transporters (Cdr1p and Ftr1p) and the vacuolar membrane ABC transporter Mlt1p accumulated in intracellular structures that appeared related to the ER, but localization of Cdc10p and Int1p was unaffected. When the dominant negative sec4 (S28N) allele of C. albicans was overexpressed, Cdr1p and Ftr1p accumulated intracellularly, and localization of Mlt1p, Cdc10p and Int1p was unaffected. These results imply that (i) Cdr1p and Ftr1p are transported to the plasma membrane by the general secretory pathway, (ii) Mlt1p enters the secretory pathway but is diverted to the vacuole at an early post-Golgi step, and (iii) like Cdc10p, Int1p does not enter the general secretory pathway. [ABSTRACT FROM AUTHOR]
- Published
- 2005
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71. Disruption of rpmJ Encoding Ribosomal Protein L36 Decreases the Expression of secY Upstream of the spc Operon and Inhibits Protein Translocation in Escherichia coli.
- Author
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Ikegami, Ayao, Nishiyama, Ken-ichi, Matsuyama, Shin-ichi, and Tokuda, Hajime
- Subjects
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GENES , *MEMBRANE proteins , *CHLORAMPHENICOL , *ACETYLTRANSFERASES , *MESSENGER RNA , *PROTEIN synthesis , *CHROMOSOMAL translocation - Abstract
Examines the development of rpmJ gene null strains, AY101 and AY201, by replacing rpmJ with tetA, which encodes a membrane protein, and cat, which encodes a cytoplasmic chloramphenicol acetyltransferase, respectively. Decrease in the level of SecY mRNA in both disruptants; Correction of the phenotypes caused by the rpmJ null mutation by a plasmid carrying SecY; Roles of RpmJ in protein synthesis and translocation.
- Published
- 2005
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72. Targeting and insertion of heterologous membrane proteins in E. coli
- Author
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Raine, Amanda, Ullers, Ronald, Pavlov, Michael, Luirink, Joen, Wikberg, Jarl E.S., and Ehrenberg, Måns
- Subjects
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PROTEIN synthesis , *ESCHERICHIA coli , *MEMBRANE proteins - Abstract
Membrane targeting and insertion of the archaeal Halobacter halobium proton pump bacterioopsin (Bop) and the human melanocortin 4 receptor (MC4R) were studied in vitro, using E. coli components for protein synthesis, membrane targeting and insertion. These heterologous proteins are targeted to E. coli membranes in a signal recognition particle (SRP) dependent manner and inserted into the membrane co-translationally. Furthermore, we demonstrate that nascent chains of Bop and MC4R first interact with SecY and then with YidC as they move through the translocon. Our results suggest that the initial stages of membrane targeting and insertion of heterologous proteins in E. coli proceed by the pathway used for native E. coli membrane proteins. No significant pausing of protein elongation was observed in the presence of E. coli SRP, in line with the suggestion that translational arrest requires an Alu domain, which is absent in SRP from E. coli. [Copyright &y& Elsevier]
- Published
- 2003
- Full Text
- View/download PDF
73. Isolation of fusion proteins containing SecY and SecE, components of the protein translocation complex from the halophilic archaeon Haloferax volcanii.
- Author
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Irihimovitch, Vered, Ring, Gabriela, Elkayam, Tsiona, Konrad, Zvia, and Eichler, Jerry
- Subjects
PROTEINS ,CHROMOSOMAL translocation ,CELLULOSE ,CELLS ,BIOMOLECULES ,IMMUNOGLOBULINS - Abstract
By exploiting the salt-insensitive interaction of the cellulose-binding domain (CBD) of the Clostridium thermocellum cellulosome with cellulose, purification of CBD-fused versions of SecY and SecE, components of the translocation apparatus of the halophilic archaeon Haloferax volcanii, was undertaken. Following transformation of Haloferax volcanii cells with CBD-SecY- or -SecE-encoding plasmids, cellulose-based purification led to the capture of stably expressed, membrane-bound 68 and 25 kDa proteins, respectively. Both fusion proteins were recognized by antibodies raised against the CBD. Thus, CBD–cellulose interactions can be employed as a salt-insensitive affinity purification system for the capture of complexes containing the Haloferax volcanii translocation apparatus components SecY and SecE. [ABSTRACT FROM AUTHOR]
- Published
- 2003
- Full Text
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74. SecY–SecY and SecY–SecG contacts revealed by site-specific crosslinking
- Author
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van der Sluis, Eli O., Nouwen, Nico, and Driessen, Arnold J.M.
- Subjects
- *
CHROMOSOMAL translocation , *ESCHERICHIA coli - Abstract
Protein translocation across the cytoplasmic membrane of Escherichia coli is mediated by the integral membrane complex SecYEG and the peripherally bound ATPase SecA. To probe the environment of the cytoplasmic domains of SecY within the SecYEG complex, we introduced single cysteine residues in each of the six cytoplasmic domains. Neighbouring SecY molecules with a single cysteine residue in cytoplasmic domains C1, C2 or C6 formed a disulfide bond upon oxidation. The presence of the disulfide bond between two C2 domains reversibly inhibited protein translocation. Chemical crosslinking showed that the C2 and C3 domains are in close proximity of SecG and chemical modification of the cysteine residue in the C5 domain with N-ethyl-maleimide or fluorescein-5-maleimide inactivates the SecYEG complex. Taken together, our data give novel insights in the interactions between subunits of the SecYEG complex and emphasise the importance of cytoplasmic domain C5 for SecY functioning. [Copyright &y& Elsevier]
- Published
- 2002
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75. The F286Y mutation of PrlA4 tempers the signal sequence suppressor phenotype by reducing the SecA binding affinity.
- Author
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de Keyzer, Jeanine, van der Does, Chris, Swaving, Jelto, and Driessen, Arnold J.M.
- Subjects
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CHROMOSOMAL translocation , *PROTEIN-protein interactions , *SIGNAL peptides , *BILAYER lipid membranes , *BINDING sites , *SUPPRESSOR mutation , *HUMAN phenotype - Abstract
SecYEG forms the protein-conducting channel of the Escherichia coli translocase. It binds the peripheral ATPase SecA that drives the preprotein translocation reaction. PrlA4 is a double mutant of SecY that enables the translocation of preproteins with a defective or even missing signal sequence. The effect of the individual mutations, F286Y and I408N, was studied with SecYEG proteoliposomes. SecY(I408N) is responsible for the increased translocation of preproteins with a defective and normal signal sequence, and exhibits a stronger prl phenotype than PrlA4. This activity correlates with an elevated SecA-translocation ATPase and SecA binding affinity. SecY(F286Y) supports only a low SecA binding affinity, preprotein translocation and SecA translocation ATPase activity. These results suggest that the second site F286Y mutation reduces the strength of the I408N mutation of PrlA4 by lowering the SecA binding affinity. [ABSTRACT FROM AUTHOR]
- Published
- 2002
- Full Text
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76. Evolutionarily conserved binding of ribosomes to the translocation channel via the large ribosomal RNA.
- Author
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Prinz, Anke, Behrens, Christina, Rapoport, Tom A., Hartmann, Enno, and Kalies, Kai-Uwe
- Subjects
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RIBOSOMES , *ENDOPLASMIC reticulum , *PROTEINS , *BIOLOGICAL membranes , *RNA , *MAMMALS - Abstract
During early stages of cotranslational protein translocation across the endoplasmic reticulum (ER) membrane the ribosome is targeted to the heterotrimeric Sec61p complex, the major component of the protein-conducting channel. We demonstrate that this interaction is mediated by the 28S rRNA of the eukaryotic large ribosomal subunit. Bacterial ribosomes also bind via their 23S rRNA to the bacterial homolog of the Sec61p complex, the SecYEG complex. Eukaryotic ribosomes bind to the SecYEG complex, and prokaryotic ribosomes to the Sec61p complex. These data indicate that rRNA-mediated interaction of ribosomes with the translocation channel occurred early in evolution and has been conserved. [ABSTRACT FROM AUTHOR]
- Published
- 2000
- Full Text
- View/download PDF
77. SecYEG assembles into a tetramer to form the active protein translocation channel.
- Author
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Manting, Erik H., van der Does, Chris, Remigy, Hervé, Engel, Andreas, and Driessen, Arnold J. M.
- Subjects
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ESCHERICHIA coli , *CELL membranes , *BACTERIAL cell walls , *TETRAMERS (Oligomers) - Abstract
Translocase mediates preprotein translocation across the Escherichia coli inner membrane. It consists of the SecYEG integral membrane protein complex and the peripheral ATPase SecA. Here we show by functional assays, negative-stain electron microscopy and mass measurements with the scanning transmission microscope that SecA recruits SecYEG complexes to form the active translocation channel. The active assembly of SecYEG has a side length of 10.5 nm and exhibits an ∼5 nm central cavity. The mass and structure of this SecYEG as well as the subunit stoichiometry of SecA and SecY in a soluble translocaseprecursor complex reveal that translocase consists of the SecA homodimer and four SecYEG complexes. [ABSTRACT FROM AUTHOR]
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- 2000
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78. Characterization of a Mutant Form of SecA That Alleviates a SecY Defect at Low Temperature and Shows a Synthetic Defect with SecY Alteration at High Temperature1.
- Author
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Nakatogawa, Hitoshi, Mori, Hiroyuki, Matsumoto, Gen, and Ito, Koreaki
- Subjects
MUTANT proteins ,PROTEIN binding ,TRYPSIN inhibitors ,TEMPERATURE ,HYDROLYSIS - Abstract
The secY205 mutant is cold-sensitive for protein export, with an in vitro defect in supporting ATP- and preprotein-dependent insertion of SecA into the membrane. We characterized SecA81 with a Gly516 to Asp substitution near the minor ATP-binding region, which suppresses the secY205 defect at low temperature and exhibits an allele-specific synthetic defect with the same SecY alteration at 42°C. The overproduced SecA81 aggregated in vivo at temperatures above 37°C. Purified SecA81 exhibited markedly enhanced intrinsic and membrane ATPase activities at 30°C, while it was totally inactive at 42°C. The trypsin digestion patterns indicated that SecA81 has some disorder in the central region of SecA, which encompasses residues 421–575. This conformational abnormality may result in unregulated ATPase at low temperature as well as the thermosensitivity of the mutant protein. In the presence of both proOmpA and the wild-type membrane vesicles, however, the thermosensitivity was alleviated, and SecA81 was able to catalyze significant levels of proOmpA-stimulated ATP hydrolysis as well as proOmpA translocation at 42°C. While SecA81 was able to overcome the SecY205 defect at low temperature, the SecY205 membrane vesicles could not significantly support the translocation ATPase or the proOmpA translocation activity of SecA81 at 42°C. The inactivated SecA81 molecules seemed to jam the translocate since it interfered with translocase functions at 42°C. Based on these results, we propose that under preprotein-translocating conditions, the SecYEG channel can stabilize and activate SecA, and that this aspect is defective for the SecA81-SecY205 combination. The data also suggest that the conformation of the central region of SecA is important for the regulation of ATP hydrolysis and for the productive interaction of SecA with SecY. [ABSTRACT FROM AUTHOR]
- Published
- 2000
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79. Yet another job for the bacterial ribosome
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Origi, Andrea, Natriashivili, Ana, Knüpffer, Lara, Fehrenbach, Clara, Denks, Kärt, Asti, Rosella, and Koch, Hans-Georg
- Subjects
Models, Molecular ,Ribosomal Proteins ,SecA ,Molecular Biology and Physiology ,SecYEG ,uL23 ,Ribosome ,Binding, Competitive ,environment and public health ,Microbiology ,ribosomes ,03 medical and health sciences ,0302 clinical medicine ,Ribosomal protein ,Virology ,protein targeting ,Escherichia coli ,signal recognition particle ,030304 developmental biology ,0303 health sciences ,Signal recognition particle ,SecYEG Translocon ,Binding Sites ,SecA Proteins ,Chemistry ,Escherichia coli Proteins ,Cell Membrane ,Molecular Mimicry ,SecY ,Translation (biology) ,Microreview ,QR1-502 ,Transport protein ,Secretory protein ,ribosome ,Protein Biosynthesis ,Mutation ,Biophysics ,bacteria ,protein transport ,Signal recognition particle binding ,030217 neurology & neurosurgery ,Protein Binding ,Research Article - Abstract
Bacterial protein transport via the conserved SecYEG translocon is generally classified as either cotranslational, i.e., when transport is coupled to translation, or posttranslational, when translation and transport are separated. We show here that the ATPase SecA, which is considered to bind its substrates posttranslationally, already scans the ribosomal tunnel for potential substrates. In the presence of a nascent chain, SecA retracts from the tunnel but maintains contact with the ribosomal surface. This is remarkably similar to the ribosome-binding mode of the signal recognition particle, which mediates cotranslational transport. Our data reveal a striking plasticity of protein transport pathways, which likely enable bacteria to efficiently recognize and transport a large number of highly different substrates within their short generation time., Bacteria execute a variety of protein transport systems for maintaining the proper composition of their different cellular compartments. The SecYEG translocon serves as primary transport channel and is engaged in transporting two different substrate types. Inner membrane proteins are cotranslationally inserted into the membrane after their targeting by the signal recognition particle (SRP). In contrast, secretory proteins are posttranslationally translocated by the ATPase SecA. Recent data indicate that SecA can also bind to ribosomes close to the tunnel exit. We have mapped the interaction of SecA with translating and nontranslating ribosomes and demonstrate that the N terminus and the helical linker domain of SecA bind to an acidic patch on the surface of the ribosomal protein uL23. Intriguingly, both also insert deeply into the ribosomal tunnel to contact the intratunnel loop of uL23, which serves as a nascent chain sensor. This binding pattern is remarkably similar to that of SRP and indicates an identical interaction mode of the two targeting factors with ribosomes. In the presence of a nascent chain, SecA retracts from the tunnel but maintains contact with the surface of uL23. Our data further demonstrate that ribosome and membrane binding of SecA are mutually exclusive, as both events depend on the N terminus of SecA. Our study highlights the enormous plasticity of bacterial protein transport systems and reveals that the discrimination between SRP and SecA substrates is already initiated at the ribosome.
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- 2019
80. Multilokusna sekvenčna tipizacija fitoplazme 'Candidatus Phytoplasma solani' v slovenskih vinorodnih deželah
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Mermal, Sara and Dermastia, Marina
- Subjects
Candidatus Phytoplasma solani ,udc:601.4:577.21:632.3(043.2) ,PCR ,stamp ,epidemiologija ,secY ,phytoplasma ,tuf ,epidemiology ,fitoplazme - Abstract
'Candidatus Phytoplasma solani' je bakterija brez celične stene, ki je obligatni parazit rastlin. Je povzročiteljica bolezni na številnih rastlinah na vinski trti na primer povzroča navadno trsno rumenico. S sekvenciranjem produktov ugnezdene verižne reakcije s polimerazo (nPCR) genov tuf, secY in stamp smo določili molekulsko raznolikost sevov 'Ca. P. solani' izoliranih iz vinske trte. Z analizo njihovega časovnega in geografskega pojavljanja v Sloveniji ter primerjavo rezultatov z rezultati podobnih analiz v Evropi, še posebej v okoliških državah, smo določili možno pot širjenja navadne trsne rumenice med Slovenijo in okoliškimi državami. 'Candidatus Phytoplasma solani' is a bacteria without a cell wall, which is an obligatory parasite of plants. It causes diseases in many plants, for example: Bois Noir on grapevine. By sequencing nested polymerase chain reaction products of tuf, secY and stamp genes, we determined the molecular diversity of 'Ca. P. solani' strains isolated from grapevines. By analyzing their occurrence in Slovenia on a geographical and time scale and then comparing the results with similar analysis in Europe, especially in surrounding countries, we determined possible pathways by which spreading of Bois Noir grapevine disease, between Slovenia and its neighbouring countries, can occur.
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- 2019
81. Geographical and Temporal Diversity of ' Candidatus Phytoplasma solani' in Wine-Growing Regions in Slovenia and Austria.
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Mehle N, Kavčič S, Mermal S, Vidmar S, Pompe Novak M, Riedle-Bauer M, Brader G, Kladnik A, and Dermastia M
- Abstract
As the causal agent of the grapevine yellows disease Bois noir, ' Candidatus Phytoplasma solani' has a major economic impact on grapevines. To improve the control of Bois noir, it is critical to understand the very complex epidemiological cycles that involve the multiple " Ca . P. solani" host plants and insect vectors, of which Hyalesthes obsoletus is the most important. In the present study, multiple genotyping of the tuf , secY, stamp , and vmp1 genes was performed. This involved archived grapevine samples that were collected during an official survey of grapevine yellows throughout the wine-growing regions of Slovenia (from 2003 to 2016), plus samples from Austrian grapevines, stinging nettle, field bindweed, and insect samples (collected from 2012 to 2019). The data show that the tuf-b2 type of the tuf gene has been present in eastern Slovenia since at least 2003. The hypotheses that the occurrence of the haplotypes varies due to the geographical position of Slovenia on the Italian-Slovenian Karst divide and that the haplotypes are similar between Slovenian and Austrian Styria were confirmed. The data also show haplotype changes for host plants and H . obsoletus associated with ' Ca . P. solani,' which might be linked to new epidemiological cycles of this phytoplasma that involve not just new plant sources and new insect vectors, but also climate and land-use changes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mehle, Kavčič, Mermal, Vidmar, Pompe Novak, Riedle-Bauer, Brader, Kladnik and Dermastia.)
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- 2022
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82. PrlA4 prevents the rejection of signal sequence defective preproteins by stabilizing the SecA-SecY interaction during the initiation of translocation.
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van der Wolk, Jeroen P. W., Fekkes, Peter, Boorsma, Andre, Huie, Janet L., Silhavy, Thomas J., and Driessen, Arnold J. M.
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- *
ESCHERICHIA coli , *CELL membranes , *CHROMOSOMAL translocation , *ENZYMES , *GENETIC mutation , *PROTEIN precursors - Abstract
In Escherichia coli, precursor proteins are translocated across the cytoplasmic membrane by translocase. This multisubunit enzyme consists of a preprotein-binding and ATPase domain, SecA, and the SecYEG complex as the integral membrane domain. PrlA4 is a mutant of SecY that enables the translocation of preproteins with a defective, or missing, signal sequence. Inner membranes of the prlA4 strain efficiently translocate · 8proOmpA, a proOmpA derivative with a non-functional signal sequence. Owing to the signal sequence mutation, · 8proOmpA binds to the translocase with a lowered affinity and the recognition is not restored by the prlA4 SecY. At the ATP-dependent initiation of translocation, the binding affinity of SecA for SecYEG is lowered causing the premature loss of bound preproteins from the translocase. The prlA4 membranes, however, bind SecA with a much higher affinity than the wild-type, and during initiation, the SecA and preprotein remain bound at the translocation site allowing an improved efficiency of translocation. It is concluded that the prlA4 strain prevents the rejection of defective preproteins from the export pathway by stabilizing SecA at the SecYEG complex. [ABSTRACT FROM AUTHOR]
- Published
- 1998
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83. The Escherichia coli SRP and SecB targeting pathways converge at the translocon.
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Valent, Quido A., Scotti, Pier A., High, Stephen, De Gier, Jan-Willem L., Von Heijne, Gunnar, Lentzen, Georg, Wintermeyer, Wolfgang, Oudega, Bauke, and Luirink, Joen
- Subjects
- *
ESCHERICHIA coli , *PROTEINS , *CELL membranes , *CHROMOSOMAL translocation - Abstract
Two distinct protein targeting pathways can direct proteins to the Escherichia coli inner membrane. The Sec pathway involves the cytosolic chaperone SecB that binds to the mature region of pre-proteins. SecB targets the pre-protein to SecA that mediates preprotein translocation through the SecYEG translocon. The SRP pathway is probably used primarily for the targeting and assembly of inner membrane proteins. It involves the signal recognition particle (SRP) that interacts with the hydrophobic targeting signal of nascent proteins. By using a protein cross-linking approach, we demonstrate here that the SRP pathway delivers nascent inner membrane proteins at the membrane. The SRP receptor FtsY, GTP and inner membranes are required for release of the nascent proteins from the SRP. Upon release of the SRP at the membrane, the targeted nascent proteins insert into a translocon that contains at least SecA, SecY and SecG. Hence, as appears to be the case for several other translocation systems, multiple targeting mechanisms deliver a variety of precursor proteins to a common membrane translocation complex of the E.coli inner membrane. [ABSTRACT FROM AUTHOR]
- Published
- 1998
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84. SecY and SecA interact to allow SecA insertion and protein translocation across the Escherichia coli plasma membrane.
- Author
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Matsumoto, Gen, Yoshihisa, Tohru, and Ito, Koreaki
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- *
GENETIC mutation , *CHROMOSOMAL translocation , *ESCHERICHIA coli , *CELL membranes , *AMINO acids , *PROTEINS - Abstract
SecA, the preprotein-driving ATPase in Escherichia coli, was shown previously to insert deeply into the plasma membrane in the presence of ATP and a preprotein; this movement of SecA was proposed to be mechanistically coupled with preprotein translocation. We now address the role played by SecY, the central subunit of the membrane-embedded heterotrimeric complex, in the SecA insertion reaction. We identified a secY mutation (secY205), affecting the most carboxyterminal cytoplasmic domain, that did not allow ATP and preprotein-dependent productive SecA insertion, while allowing idling insertion without the preprotein. Thus, the secY205 mutation might affect the SecYEG ‘channel’ structure in accepting the preprotein—SecA complex or its opening by the complex. We isolated secA mutations that allele-specifically suppressed the secY205 translocation defect in vivo. One mutant protein, SecA36, with an amino acid alteration near the high-affinity ATP-binding site, was purified and suppressed the in vitro translocation defect of the inverted membrane vesicles carrying the SecY205 protein. The SecA36 protein could also insert into the mutant membrane vesicles in vitro. These results provide genetic evidence that SecA and SecY specifically interact, and show that SecY plays an essential role in insertion of SecA in response to a preprotein and ATP and suggest that SecA drives protein translocation by inserting into the membrane in vivo. [ABSTRACT FROM AUTHOR]
- Published
- 1997
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85. Roles of the Conserved Cytoplasmic Region and Non-Conserved Carboxy-Terminal Region of SecE in Escherichia coli Protein Translocase1.
- Author
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Kontinen, Vesa P., Yamanaka, Miyuki, Nishiyama, Ken-ichi, and Tokuda, Hajime
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ESCHERICHIA coli ,ENTEROBACTERIACEAE ,BACILLUS subtilis ,PROTEINS ,LOW temperatures - Abstract
SecE, an essential membrane component of the Escherichia coli protein translocase, consists of 127 ami no acid residues. Only a part of the second putative cytoplasmic region comprising some 13 residues is essential for the SecE function as long as the proper topological arrangement is retained. The Trp84 and Pro85 residues of this region are conserved in all eubacterial SecE homologues. The conservation of positively charged residues corresponding to ArgSO and Lys81 is also substantial. We deleted or replaced these residues to assess their roles in the SecE function. Deletion of the Arg80-Lys81 dipeptide did not abolish the SecE function whereas that of Trp84 or Pro85 caused a loss of the function. Strikingly, however, replacement of Pro85 with either Gly, Ser, or Ala, and that of Trp84 with Lys did not abolish the SecE function. These results indicate that the strong conservation of these residues does not reflect their obligatory requirement for the SecE function. A chimeric SecE possessing the cytoplasmic region of the E. coli SecE and the following region of the Bacillus subtilis SecE was able to form the translocation machinery together with SecA, SecY, and SecG. Although a Leu to Arg mutation at position 108 has been thought to cause a loss of signal recognition fidelity and thereby suppress a signal sequence defect, the same mutation at position 111 caused a complete loss of the function. The levels of SecY and SecG in the secEcsE501 mutant, which expresses SecE at a decreased level and is sensitive to low temperature, increased upon the expression of functional SecE derivatives, irrespective of the site of mutation, suggesting that the levels of SecY and SecG are co-operatively determined by the level of functional, but not non-functional, SecE. Based on these results, the SecE function in the translocase is discussed. [ABSTRACT FROM AUTHOR]
- Published
- 1996
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86. Phylogeny and expression of the secA gene from a chromophytic alga – implications for the evolution of plastids and sec-dependent protein translocation.
- Author
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Valentin, Klaus
- Abstract
In bacteria many periplasmatic proteins are exported via the sec-dependent pathway. A homologous apparatus was found to be involved in the transport of proteins across the thylakoid membrane in plastids. In the present study additional data on the phylogeny and expression of one of the genes essential in this process, secA, is presented. For the first time, transcriptional activity of secA in the plastid was detected. When secA is used as a phylogenetic marker for plastid evolution it demonstrates a large phylogenetic distance between chlorophytic and non-chlorophytic (i.e. rhodophytic) primary plastids. This distance could be explained by assuming polyphyly for major plastid lineages. Moreover, it was found that two types of secA genes may exist in plastids. Whether or not these are involved in different protein translocation processes is presently unknown. In an attempt to identify further candidates, i.e. non-photosynthesis-related proteins, for sec-dependent protein transport, an SbpA protein was detected in chromophytic plastids by the use of a peptide antibody. [ABSTRACT FROM AUTHOR]
- Published
- 1997
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87. A model for the evolution of the plastid sec apparatus inferred from secY gene phylogeny.
- Author
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Vogel, Heiko, Fischer, Sabine, and Valentin, Klaus
- Abstract
Plastids possess a bacteria-like sec apparatus that is involved in protein import into the thylakoid lumen. We have analyzed one of the genes essential for this process, secY. A secY gene from the unicellular red alga Cyanidium caldarium was found to be transcriptionally active, demonstrating for the first time that secY is functional in a plastid. Unlike the situation seen in bacteria the C. caldarium gene is transcribed monocistronically, despite the fact that it is part of a large ribosomal gene cluster that resembles bacterial spc operons. A molecular phylogeny is presented for 8 plastid-encoded secY genes, four of which have not been published yet. In this analysis plastid secY genes fall into two classes. One of these, comprising of genes from multicellular red algae and Cryptophyta, clusters in a neighbour-joining tree with a cyanobacterial counterpart. Separated from the aforesaid are secY genes from Chromophyta, Glaucocystophyta and a unicellular red alga. All plastid and cyanobacterial sequences are located on the same branch, separated from bacterial homologues. We postulate that the two classes of secY genes are paralogous, i.e. their gene products are involved in different protein translocation processes. Based on this assumption a model for the evolution of the plastid sec apparatus is presented. [ABSTRACT FROM AUTHOR]
- Published
- 1996
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88. The Journal of Membrane Biology / Driving Forces of Translocation Through Bacterial Translocon SecYEG
- Author
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Knyazev, Denis G., Kuttner, Roland, Zimmermann, Mirjam, Sobakinskaya, Ekaterina, and Pohl, Peter
- Subjects
Proton motive force ,SecY ,Translocation - Abstract
This review focusses on the energetics of protein translocation via the Sec translocation machinery. First we complement structural data about SecYEG’s conformational rearrangements by insight obtained from functional assays. These include measurements of SecYEG permeability that allow assessment of channel gating by ligand binding and membrane voltage. Second we will discuss the power stroke and Brownian ratcheting models of substrate translocation and the role that the two models assign to the putative driving forces: (i) ATP (SecA) and GTP (ribosome) hydrolysis, (ii) interaction with accessory proteins, (iii) membrane partitioning and folding, (iv) proton motive force (PMF), and (v) entropic contributions. Our analysis underlines how important energized membranes are for unravelling the translocation mechanism in future experiments. Version of record
- Published
- 2018
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89. Extra-renal bovine leptospirosis: Molecular characterization of the Leptospira interrogans Sejroe serogroup on the uterus of non-pregnant cows.
- Author
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Di Azevedo, Maria Isabel Nogueira, Pires, Bruno C., Libonati, Hugo, Pinto, Priscila S., Cardoso Barbosa, Lucas Figueiredo, Carvalho-Costa, Filipe Anibal, and Lilenbaum, Walter
- Subjects
- *
LEPTOSPIRA interrogans , *LEPTOSPIROSIS , *UTERUS , *ESTRUS , *GENITALIA , *COWS , *FEMALE reproductive organs , *SYMPTOMS - Abstract
• Uterus is an important extra-renal site of bovine genital leptospirosis. • Leptospires were detected on uteri by PCR and sequencing. • Leptospiral uterine infection are associated to members of the Sejroe serogroup. Bovine genital leptospirosis is a chronic disease that causes reproductive disorders such as abortions, stillbirths, and estrus repetition, as well as economic losses. Despite clinical signs related to reproductive failure, the majority of studies have focused on the detection of Leptospira spp. in the urine, while few have considered the reproductive tract. Consequently, the aim of the present study was to investigate the uterus as an important extra-renal site of leptospiral infection in cows. A total of 42 non-pregnant cows were studied at a slaughterhouse. Blood samples and uterine fragments were collected for serology and molecular analysis, respectively. Concerning serologic results, 20.5 % presented as reactive, all of them against the Sejroe serogroup. Regarding lipL32 PCR, 26.2 % (11/42) of samples were positive for pathogenic Leptospira sp. Sequencing the secY gene short region enabled nine strains to be characterized, all of which were L. interrogans, with high identity (98.8 %–99.8 %) with serovar Hardjo. The use of molecular tools substantially improved the sensitivity of Leptospira sp. detection at species level and demonstrated that the uterus is an important site of bovine leptospiral infection. The findings of the present study reinforce our understanding that leptospiral uterine infection are associated to members of the Sejroe serogroup. [ABSTRACT FROM AUTHOR]
- Published
- 2020
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90. The First Proteomic Study of Nostoc sp. PCC 7120 Exposed to Cyanotoxin BMAA under Nitrogen Starvation.
- Author
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Koksharova, Olga A., Butenko, Ivan O., Pobeguts, Olga V., Safronova, Nina A., and Govorun, Vadim M.
- Subjects
- *
NOSTOC , *AMINO acid metabolism , *PROTEOMICS , *DNA ligases , *GLUTAMINE synthetase , *METABOLIC regulation , *CYANOBACTERIAL toxins - Abstract
The oldest prokaryotic photoautotrophic organisms, cyanobacteria, produce many different metabolites. Among them is the water-soluble neurotoxic non-protein amino acid beta-N-methylamino-L-alanine (BMAA), whose biological functions in cyanobacterial metabolism are of fundamental scientific and practical interest. An early BMAA inhibitory effect on nitrogen fixation and heterocyst differentiation was shown in strains of diazotrophic cyanobacteria Nostoc sp. PCC 7120, Nostocpunctiforme PCC 73102 (ATCC 29133), and Nostoc sp. strain 8963 under conditions of nitrogen starvation. Herein, we present a comprehensive proteomic study of Nostoc (also called Anabaena) sp. PCC 7120 in the heterocyst formation stage affecting by BMAA treatment under nitrogen starvation conditions. BMAA disturbs proteins involved in nitrogen and carbon metabolic pathways, which are tightly co-regulated in cyanobacteria cells. The presented evidence shows that exogenous BMAA affects a key nitrogen regulatory protein, PII (GlnB), and some of its protein partners, as well as glutamyl-tRNA synthetase gltX and other proteins that are involved in protein synthesis, heterocyst differentiation, and nitrogen metabolism. By taking into account the important regulatory role of PII, it becomes clear that BMAA has a severe negative impact on the carbon and nitrogen metabolism of starving Nostoc sp. PCC 7120 cells. BMAA disturbs carbon fixation and the carbon dioxide concentrating mechanism, photosynthesis, and amino acid metabolism. Stress response proteins and DNA repair enzymes are upregulated in the presence of BMAA, clearly indicating severe intracellular stress. This is the first proteomic study of the effects of BMAA on diazotrophic starving cyanobacteria cells, allowing a deeper insight into the regulation of the intracellular metabolism of cyanobacteria by this non-protein amino acid. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
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91. Proposal of A New Bois Noir Epidemiological Pattern Related to 'Candidatus Phytoplasma Solani' Strains Characterized by A Possible Moderate Virulence in Tuscany.
- Author
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Pierro, Roberto, Panattoni, Alessandra, Passera, Alessandro, Materazzi, Alberto, Luvisi, Andrea, Loni, Augusto, Ginanni, Marco, Lucchi, Andrea, Bianco, Piero Attilio, and Quaglino, Fabio
- Subjects
RHIZOCTONIA solani ,CANDIDATUS ,HOST plants ,GRAPE diseases & pests ,GRAPES ,VITIS vinifera ,WEEDS - Abstract
Bois noir (BN), associated with 'Candidatus Phytoplasma solani' (CaPsol), is the most widespread disease of the grapevine yellows complex worldwide. In this work, BN epidemiology was investigated in a case study vineyard where an unusual CaPsol strain, previously detected only in other host plants, was found to be prevalent in grapevine. Experimental activities included: symptom observation; sampling of symptomatic vines, Auchenorrhyncha specimens, and weeds; molecular detection and typing of CaPsol strains; statistical analyses for determining possible relationships between CaPsol relative concentration, strain type, and symptom severity. Among insects, Reptalus quinquecostatus was the most abundant and was found to be highly infected by CaPsol, while Hyalesthes obsoletus, the main CaPsol vector, was not caught. Moreover, R. quinquecostatus harbored CaPsol strains carrying uniquely the stamp sequence variant St10, also identified as prevalent in vines and in the majority of weeds, and all the secY variants identified in the vineyard. Statistical analyses revealed that CaPsol strains carrying the St10 variant are not associated with severe symptoms, suggesting their possible moderate virulence. Based on such evidence, a new BN epidemiological pattern related to these CaPsol strains and involving grapevine, R. quinquecostatus, and/or weeds is proposed. Furthermore, the possible presence of other players (vectors and weeds) involved in CaPsol transmission to grapevines was highlighted. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
92. Membrane protein insertion and assembly by the bacterial holo-translocon SecYEG-SecDF-YajC-YidC
- Author
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Ian Collinson, Imre Berger, Timothy R. Dafforn, Sara Alvira, Sarah C. Lee, Gabriele Deckers-Hebestreit, Remy Martin, Joanna Komar, Jelger A. Lycklama a Nijeholt, Christiane Schaffitzel, and Ryan J. Schulze
- Subjects
0301 basic medicine ,Sec61 ,BrisSynBio ,Biology ,Biochemistry ,Ribosome ,insertion ,holo-translocon ,03 medical and health sciences ,Bacterial Proteins ,Inner membrane ,membrane protein ,Molecular Biology ,Research Articles ,Signal recognition particle ,030102 biochemistry & molecular biology ,YidC ,Escherichia coli Proteins ,Bristol BioDesign Institute ,SecY ,Membrane Proteins ,Cell Biology ,Translocon ,Transmembrane domain ,Spectrometry, Fluorescence ,030104 developmental biology ,Secretory protein ,Membrane protein ,Biophysics ,synthetic biology ,Research Article - Abstract
Protein secretion and membrane insertion occur through the ubiquitous Sec machinery. In this system, insertion involves the targeting of translating ribosomes via the signal recognition particle and its cognate receptor to the SecY (bacteria and archaea)/Sec61 (eukaryotes) translocon. A common mechanism then guides nascent transmembrane helices (TMHs) through the Sec complex, mediated by associated membrane insertion factors. In bacteria, the membrane protein ‘insertase’ YidC ushers TMHs through a lateral gate of SecY to the bilayer. YidC is also thought to incorporate proteins into the membrane independently of SecYEG. Here, we show the bacterial holo-translocon (HTL) — a supercomplex of SecYEG–SecDF–YajC–YidC — is a bona fide resident of the Escherichia coli inner membrane. Moreover, when compared with SecYEG and YidC alone, the HTL is more effective at the insertion and assembly of a wide range of membrane protein substrates, including those hitherto thought to require only YidC.
- Published
- 2016
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93. Molecular analysis of leptospires from serogroup Sejroe obtained from asymptomatic cattle in Rio de Janeiro — Brazil reveals genetic proximity to serovar Guaricura
- Author
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Walter Lilenbaum, A. P. Loureiro, Sylvie Brémont, Priscila S. Pinto, Camila Hamond, Pascale Bourhy, Universidade Federal Fluminense [Rio de Janeiro] (UFF), Centre Collaborateur FAO/OMS pour l'épidémiologie de la leptospirose, Institut Pasteur [Paris], This study was supported by FAPERJ — Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro. PP is a CAPES fellow. CH, WL are CNPq fellows. APL and WL are FAPERJ fellows., The authors thank the abbatoir owner (Frigorifico Esteves), who allowed collection of samples. We are also thankful to MSc. L. Narduche, H. Libonati (UFF) for their help in collecting samples, to Dr. Silvio Vasconcellos (USP) for providing the anti-Guaricura antiserum, Leonardo Almeida, Fabio Navarro (Yale University) for their help in English writing and Jonh Kastelic (University of Calgary) for the critical English revision, and Institut Pasteur [Paris] (IP)
- Subjects
Male ,0301 basic medicine ,Serotype ,MESH: Amino Acid Sequence ,Cattle Diseases ,MESH: Base Sequence ,Guaricura ,Genotype ,MESH: Cattle Diseases ,MESH: Animals ,MESH: Genetic Variation ,MESH: Phylogeny ,Phylogeny ,Leptospira ,biology ,Leptospirosis ,rrs ,MESH: Cattle ,[SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology ,secY ,Female ,MESH: Leptospirosis / microbiology ,Brazil ,030106 microbiology ,MESH: Sequence Alignment ,MESH: Leptospira / metabolism ,Microbiology ,Serogrouping ,03 medical and health sciences ,Genetic variation ,medicine ,Animals ,Amino Acid Sequence ,Genetic diversity ,Base Sequence ,General Veterinary ,Sejroe ,MESH: Leptospira / genetics ,Leptospira santarosai ,Genetic Variation ,biology.organism_classification ,medicine.disease ,Virology ,MESH: Male ,Cattle ,MESH: Brazil ,Sequence Alignment ,MESH: Female - Abstract
International audience; Bovine leptospirosis causes substantial reproductive failure in cattle, mainly due to infections with serovar (sv) Hardjo infection. Notwithstanding, other serovars from the serogroup (sg) Sejroe could also have important roles in bovine leptospirosis. The objective was to investigate genetic diversity of serogroup Sejroe isolates obtained from asymptomatic cattle in the state of Rio de Janeiro, Brazil. Urine and vaginal fluid (VF) were collected from clinically healthy cattle immediately after slaughter. Five isolates were recovered and characterized (serogrouping) as belonging to sg Sejroe. Sequencing of rrs and secY genes further identified them as Leptospira santarosai. Analysis of secY sequences indicated a high level of sequence homology to sv Guaricura strains. Based on culture and sequence data, we inferred that other members of sg Sejroe may be important in bovine leptospiral infection, particularly genotypes of L. santarosai serovar Guaricura.
- Published
- 2016
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94. PLANT PATHOGEN FORENSICS: A DETECTIVE STORY OF TRAC(K)ING PHYTOPLASMA EMERGENCE AND SPREAD BY MLST
- Author
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Plavec, Jelena, Križanac, Ivana, Budinšćak, Željko, Škorić, Dijana, Šeruga Musić, Martina, Katalinić, Maja, and Kovarik, Zrinka
- Subjects
phytoplasma ,genotyping ,infection ,plants ,insects ,secY ,imp ,phylogenetic analysis - Abstract
Uncultivable and insect-transmitted bacteria from the genus ‘Candidatus Phytoplasma’ are associated with diseases affecting various plant species worldwide, including many economically important crops. Classification and detection of phytoplasmas are based mainly on the 16S rRNA gene analysis. However, for the elucidation of a complex pathosystem or a distinction between strains, genotyping of additional housekeeping and specific variable genes is applied. Multi-locus sequence typing (MLST) is widely used in population, evolutionary and epidemiological studies of diverse bacterial pathogens. Yet unassigned to a species taxon within the ‘Ca. Phytoplasma’ genus, Flavescence dorée (FD) phytoplasma is one of the main agents associated with grapevine yellows diseases in Europe. It is transmitted by insect vector Scaphoideus titanus Ball. and possesses a high epidemic potential. First report of FD phytoplasma in Croatia was given in 2009 for restricted continental areas of the country. The main objective of this study was to demonstrate tracking and tracing of FD phytoplasmas emergence and spread by MLST approach. In the 4-year period, over 500 samples of grapevine, S. titanus and wild plants from different regions of Croatia were analyzed. Triplex real- time PCR was performed for detection together with PCR/RFLP of 16S rDNA. Subsequently, secY, map and uvrB-degV genes were amplified and sequenced and the phylogenetic analysis was performed. Continuous emergence of new locations affected by FD over the years and a clear trend of the disease spread with Istrian peninsula heavily affected since 2014 was revealed. FD-related phytoplasmas were found for the first time in alder and invasive tree species Ailanthus altissima Mill. Phylogenetic analyses of map gene revealed the existence of 2 distinct FD clusters: map-FD2 and map-FD3, with the latter being prevalent. Different genotypes within both FD clusters could be distinguished based on additional SNPs. Furthermore, MLST revealed a strong relatedness between strains detected in grapevines and insects, showing their affiliation to the same pathosystem. Finding of distinct FD clusters and different distribution of genotypes based on MLST suggests separate routes of the disease introduction in Croatia and spread of the disease.
- Published
- 2016
95. CSI Vineyard: Trac(k)ing of Grapevine Yellows Phytoplasmas Emergence and Spread by MLST
- Author
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Plavec, Jelena, Križanac, Ivana, Budinšćak, Željko, Škorić, Dijana, Šeruga Musić, Martina, and Antolović, Roberto
- Subjects
Bois noir ,Flavescence dorée ,Hyalesthes obsoletus ,Scaphoideus titanus ,weeds ,map ,uvrB-degV ,secY ,tuf ,vmp1 ,16S rRNA gene - Abstract
Insect-transmitted plant pathogens from the genus ‘Candidatus Phytoplasma’ are associated with numerous diseases affecting plant species worldwide. Since their axenic cultivation in a pure culture is still challenging, phytoplasma detection, diagnosis and classification are still based primarily on the 16S rRNA gene analysis. However, when a distinction between closely related strains or the elucidation of a complex pathosystem is needed, information obtained solely on the basis of highly conserved 16S rDNA is not adequate. Thus, the genotyping of additional housekeeping genes together with specific variable genes is performed. Multi-locus sequence typing (MLST) is commonly used tool in population, evolutionary and epidemiological studies of various bacterial pathogens and is recognized as a reproducible approach. In Euro-Mediterranean region, economically important grapevine yellows (GY) diseases are mainly associated with phytoplasmas belonging to the ribosomal groups 16SrV-C and -D (flavescence dorée ; FD) and 16SrXII-A (stolbur ; bois noir ; BN, ‘Ca. Phytoplasma solani’) causing similar symptoms but differing in epidemiology. The main objective of this work was to demonstrate tracking and tracing of both BN and FD phytoplasmas emergence and spread by MLST approach. In the period 2012-2016, over 500 samples of grapevine, insect vectors and wild plants were taken from different viticultural regions of Croatia. Total nucleic acids were extracted and triplex real-time PCR assay was performed, together with PCR/RFLP. Subsequently, distinctive sets of gene fragments differing in conservation and evolution were amplified and sequenced and the phylogenetic analysis was performed. Real-time PCR revealed the continuous emergence of new locations and areas affected by FD over the years and a clear trend of the disease spread with Istria as a new hotspot heavily affected since 2014. For FD phytoplasma, MLST demonstrated the existence of 2 map, 4 uvrB-degV and 5 secY genotypes with a strong relatedness between strains, showing their affiliation to the same pathosystem. MLST analysis of BN isolates revealed the presence of at least 17 different comprehensive genotypes encompassing 3 tuf, 5 secY, 7 vmp1 and 10 stamp genotypes and the prevalent ones were identified. Finding of distinct BN and FD clusters and different distribution of genotypes based on MLST suggests separate routes of the disease introduction in Croatia and spread of the disease.
- Published
- 2016
96. The bacterial Sec-translocase: structure and mechanism
- Author
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Jelger A. Lycklama a Nijeholt and Arnold J. M. Driessen
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Models, Molecular ,SecA ,Cytoplasm ,ENDOPLASMIC-RETICULUM ,Chromosomal translocation ,membrane protein insertion ,General Biochemistry, Genetics and Molecular Biology ,Motor protein ,Structure-Activity Relationship ,ESCHERICHIA-COLI TRANSLOCASE ,Adenosine Triphosphate ,Bacterial Proteins ,BACILLUS-SUBTILIS SECA ,Escherichia coli ,LARGE CONFORMATIONAL-CHANGE ,Translocase ,PRECURSOR PROTEIN TRANSLOCATION ,PLUG DOMAIN ,Bacterial Secretion Systems ,translocon ,Adenosine Triphosphatases ,SecA Proteins ,protein translocation ,ATP-BINDING-SITE ,biology ,Escherichia coli Proteins ,Cell Membrane ,SIGNAL-SEQUENCE RECOGNITION ,Membrane Transport Proteins ,SecY ,Articles ,Cell biology ,Enzyme Activation ,Protein Transport ,Membrane ,Secretory protein ,Biochemistry ,Membrane protein complex ,biology.protein ,PREPROTEIN TRANSLOCASE ,General Agricultural and Biological Sciences ,SEC Translocation Channels ,X-RAY-STRUCTURE ,Protein Binding - Abstract
Most bacterial secretory proteins pass across the cytoplasmic membrane via the translocase, which consists of a protein-conducting channel SecYEG and an ATP-dependent motor protein SecA. The ancillary SecDF membrane protein complex promotes the final stages of translocation. Recent years have seen a major advance in our understanding of the structural and biochemical basis of protein translocation, and this has led to a detailed model of the translocation mechanism.
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- 2012
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97. Fine interaction profiling of VemP and mechanisms responsible for its translocation-coupled arrest-cancelation.
- Author
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Miyazaki R, Akiyama Y, and Mori H
- Subjects
- Escherichia coli, Protein Biosynthesis, Vibrio alginolyticus, Bacterial Proteins metabolism, SEC Translocation Channels metabolism
- Abstract
Bacterial cells utilize monitoring substrates, which undergo force-sensitive translation elongation arrest, to feedback-regulate a Sec-related gene. Vibrio alginolyticus VemP controls the expression of SecD/F that stimulates a late step of translocation by undergoing export-regulated elongation arrest. Here, we attempted at delineating the pathway of the VemP nascent-chain interaction with Sec-related factors, and identified the signal recognition particle (SRP) and PpiD (a membrane-anchored periplasmic chaperone) in addition to other translocon components and a ribosomal protein as interacting partners. Our results showed that SRP is required for the membrane-targeting of VemP, whereas PpiD acts cooperatively with SecD/F in the translocation and arrest-cancelation of VemP. We also identified the conserved Arg-85 residue of VemP as a crucial element that confers PpiD-dependence to VemP and plays an essential role in the regulated arrest-cancelation. We propose a scheme of the arrest-cancelation processes of VemP, which likely monitors late steps in the protein translocation pathway., Competing Interests: RM, YA, HM No competing interests declared, (© 2020, Miyazaki et al.)
- Published
- 2020
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98. Dynamics of Membrane Proteins Monitored by Single-Molecule Fluorescence Across Multiple Timescales.
- Author
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Fessl T, Crossley JA, Watkins D, Scholz M, Watson MA, Sabir T, Radford SE, Collinson I, and Tuma R
- Subjects
- Humans, Membrane Proteins chemistry, Membrane Proteins metabolism, Protein Conformation, Fluorescence, Fluorescence Resonance Energy Transfer methods, Membrane Proteins analysis, Single Molecule Imaging methods
- Abstract
Single-molecule techniques provide insights into the heterogeneity and dynamics of ensembles and enable the extraction of mechanistic information that is complementary to high-resolution structural techniques. Here, we describe the application of single-molecule Förster resonance energy transfer to study the dynamics of integral membrane protein complexes on timescales spanning sub-milliseconds to minutes (10
-9 -102 s).- Published
- 2020
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99. Protein Translocation Across the Bacterial Cytoplasmic Membrane
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Nico Nouwen and Arnold J. M. Driessen
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SecA ,Cytoplasm ,Translocase of the outer membrane ,Molecular Conformation ,SIGNAL-RECOGNITION PARTICLE ,Models, Biological ,Biochemistry ,translocase ,PREPROTEIN TRANSLOCATION ,Twin-arginine translocation pathway ,Bacterial Proteins ,Escherichia coli ,chaperone ,Translocase ,membrane protein ,DISTINCT ATP-BINDING ,CRYSTAL-STRUCTURE ,ESCHERICHIA-COLI SECA ,STOP-TRANSFER FUNCTION ,Integral membrane protein ,Adenosine Triphosphatases ,SecA Proteins ,Bacteria ,biology ,PRECURSOR PROTEIN ,Escherichia coli Proteins ,proton motive force ,Cell Membrane ,Peripheral membrane protein ,SecY ,Membrane Transport Proteins ,Proton-Motive Force ,Protein Structure, Tertiary ,Cell biology ,Protein Transport ,Membrane protein complex ,Translocase of the inner membrane ,biology.protein ,Protons ,Peptides ,EXPORT CHAPERONE SECB ,SEC Translocation Channels ,X-RAY-STRUCTURE ,Molecular Chaperones - Abstract
About 25% to 30% of the bacterial proteins function in the cell envelope or outside of the cell. These proteins are synthesized in the cytosol, and the vast majority is recognized as a ribosome-bound nascent chain by the signal recognition particle (SRP) or by the secretion-dedicated chaperone SecB. Subsequently, they are targeted to the Sec translocase in the cytoplasmic membrane, a multimeric membrane protein complex composed of a highly conserved protein-conducting channel, SecYEG, and a peripherally bound ribosome or ATP-dependent motor protein SecA. The Sec translocase mediates the translocation of proteins across the membrane and the insertion of membrane proteins into the cytoplasmic membrane. Translocation requires the energy sources of ATP and the proton motive force (PMF) while the membrane protein insertion is coupled to polypeptide chain elongation at the ribosome. This review summarizes the present knowledge of the mechanism and structure of the Sec translocase, with a special emphasis on unresolved questions and topics of current research.
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- 2008
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100. Kinetics and energetics of the translocation of maltose binding protein folding mutants
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Tomikiewicz, Danuta, Nouwen, Nico, Driessen, Arnold J. M., Tomkiewicz, Danuta, Groningen Biomolecular Sciences and Biotechnology, Zernike Institute for Advanced Materials, and Molecular Microbiology
- Subjects
folding ,SecA ,PRECURSOR PROTEINS ,Protein Folding ,CATALYTIC CYCLE ,Chromosomal translocation ,Protein Structure, Secondary ,Maltose-binding protein ,LEADER SEQUENCE ,Structural Biology ,Translocase ,SIGNAL SEQUENCE ,Adenosine Triphosphatases ,protein translocation ,biology ,Escherichia coli Proteins ,TRIGGER FACTOR ,Tryptophan ,TERTIARY STRUCTURE ,SecY ,Folding (chemistry) ,Protein Transport ,Biochemistry ,ESCHERICHIA-COLI ,Periplasmic Binding Proteins ,Thermodynamics ,maltose binding protein ,Endopeptidase K ,Signal peptide ,Motor protein ,Bacterial Proteins ,Escherichia coli ,Protein Precursors ,Molecular Biology ,SecA Proteins ,COLI MEMBRANE-VESICLES ,Membrane Transport Proteins ,Protein tertiary structure ,Protein Structure, Tertiary ,Kinetics ,Spectrometry, Fluorescence ,Secretory protein ,Mutagenesis ,Mutation ,PLASMA-MEMBRANE ,biology.protein ,Biophysics ,CYTOPLASMIC MEMBRANE ,Mutant Proteins ,Carrier Proteins ,SEC Translocation Channels - Abstract
Protein translocation in Escherichia coli is mediated by the translocase that, in its minimal form, comprises a protein-conducting pore (SecYEG) and a motor protein (SecA). The SecYEG complex forms a narrow channel in the membrane that allows passage of secretory proteins (preproteins) in an unfolded state only. It has been suggested that the SecA requirement for translocation depends on the folding stability of the mature preprotein domain. Here we studied the effects of the signal sequence and SecB on the folding and translocation of folding stabilizing and destabilizing mutants of the mature maltose binding protein (MBP). Although the mutations affect the folding of the precursor form of MBP, these are drastically overruled by the combined unfolding stabilization of the signal sequence and SecB. Consequently, the translocation kinetics, the energetics and the SecA and SecB dependence of the folding mutants are indistinguishable from those of wild-type preMBP. These data indicate that unfolding of the mature domain of preMBP is likely not a rate-determining step in translocation when the protein is targeted to the translocase via SecB. (c) 2008 Elsevier Ltd. All rights reserved.
- Published
- 2008
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