Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Stoichiometry of SecYEG in the active translocase of Escherichia coli varies with precursor species.

ABSTRACT: We have established a reconstitution system for the translocon SecYEG in proteoliposomes in which 55% of the accessible translocons are active. This level corresponds to the fraction of translocons that are active in vitro when assessed in their native environment of cytoplasmic membrane vesicles. Assays using these robust reconstituted proteoliposomes and cytoplasmic membrane vesicles have revealed that the number of SecYEG units involved in an active translocase depends on the precursor undergoing transfer. The active translocase for the precursor of periplasmic galactose-binding protein contains twice the number of heterotrimeric units of SecYEG as does that for the precursor of outer membrane protein A.

SUBMITTER: Mao C

PROVIDER: S-EPMC3718118 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Stoichiometry of SecYEG in the active translocase of Escherichia coli varies with precursor species.

Mao Chunfeng C Cheadle Carl E CE Hardy Simon J S SJ Lilly Angela A AA Suo Yuying Y Sanganna Gari Raghavendar Reddy RR King Gavin M GM Randall Linda L LL

Proceedings of the National Academy of Sciences of the United States of America 20130701 29

We have established a reconstitution system for the translocon SecYEG in proteoliposomes in which 55% of the accessible translocons are active. This level corresponds to the fraction of translocons that are active in vitro when assessed in their native environment of cytoplasmic membrane vesicles. Assays using these robust reconstituted proteoliposomes and cytoplasmic membrane vesicles have revealed that the number of SecYEG units involved in an active translocase depends on the precursor underg ...[more]

PMID: 23818593

Similar Datasets

Stoichiometry and architecture of active DNA replication machinery in Escherichia coli.

Project description:The multiprotein replisome complex that replicates DNA has been extensively characterized in vitro, but its composition and architecture in vivo is unknown. Using millisecond single-molecule fluorescence microscopy in living cells expressing fluorescent derivatives of replisome components, we have examined replisome stoichiometry and architecture. Active Escherichia coli replisomes contain three molecules of the replicative polymerase, rather than the historically accepted two. These are associated with three molecules of tau, a clamp loader component that trimerizes polymerase. Only two of the three sliding clamps are always associated with the core replisome. Single-strand binding protein has a broader spatial distribution than the core components, with 5 to 11 tetramers per replisome. This in vivo technique could provide single-molecule insight into other molecular machines.

| S-EPMC2859602 | biostudies-literature

The active repertoire of Escherichia coli peptidoglycan amidases varies with physiochemical environment.

Project description:Nearly all bacteria are encased in peptidoglycan, an extracytoplasmic matrix of polysaccharide strands crosslinked through short peptide stems. In the Gram-negative model organism Escherichia coli, more than 40 synthases and autolysins coordinate the growth and division of the peptidoglycan sacculus in the periplasm. The precise contribution of many of these enzymes to peptidoglycan metabolism remains unclear due to significant apparent redundancy, particularly among the autolysins. E. coli produces three major LytC-type-N-acetylmuramoyl-L-alanine amidases, which share a role in separating the newly formed daughter cells during cytokinesis. Here, we reveal two of the three amidases that exhibit growth medium-dependent changes in activity. Specifically, we report acidic growth conditions stimulate AmiB-and to a lesser extent, AmiC-amidase activity. Combining genetic, biochemical, and computational analyses, we demonstrate that low pH-dependent stimulation of AmiB is mediated through the periplasmic amidase activators NlpD, EnvC, and ActS (formerly known as YgeR). Although NlpD and EnvC promote amidase activity across pH environments, ActS preferentially stimulates AmiB activity in acidic conditions. Altogether, our findings support partially overlapping roles for E. coli amidases and their regulators in cell separation and illuminate the physiochemical environment as an important mediator of cell wall enzyme activity.

| S-EPMC8295211 | biostudies-literature

Comparison of Single and Multiple Turnovers of SecYEG in Escherichia coli.

Project description:Precursor proteins are translocated across the cytoplasmic membrane in Escherichia coli by the general secretory, or Sec, pathway. The main components of the pathway are the integral membrane heterotrimeric SecYEG complex and the peripheral membrane ATPase, SecA. In this study, we have applied an in vitro assay using inverted cytoplasmic membrane vesicles to investigate the complex cycle that leads to translocation. We compared the apparent rate constants for nine precursors under two experimental conditions, single turnover and multiple turnovers. For each precursor, the rate constant for a single turnover was higher than for multiple turnovers, indicating that a different step limits the rate under the two conditions. We conclude that the rate-limiting step for a single turnover is an early step in the initial phase of transit through the channel, whereas the rate of multiple turnovers is limited by the resetting of the translocon. The presence of the chaperone SecB during multiple turnovers increased the maximal amplitude translocated for the three precursor species tested, pGBP, pPhoA, and proOmpA, and also increased the apparent rate constants for both pGBP and pPhoA. The rate constant for proOmpA was decreased by the presence of SecB.IMPORTANCE Vastly different experimental techniques and conditions have been used to study export in E. coli We demonstrated that altering experimental conditions can change the step that is observed during study. Investigators should consider specific experimental conditions when comparing data from different laboratories, as well as when comparing data from different experiments within a laboratory. We have shown that each precursor species has inherent properties that determine the translocation rate; thus generalizations from studies of a single species must be made with caution. A summary of advantages and disadvantages in use of nine precursors is presented.

| S-EPMC7685557 | biostudies-literature

Escherichia coli ClpB is a non-processive polypeptide translocase.

Project description:Escherichia coli caseinolytic protease (Clp)B is a hexameric AAA+ [expanded superfamily of AAA (ATPase associated with various cellular activities)] enzyme that has the unique ability to catalyse protein disaggregation. Such enzymes are essential for proteome maintenance. Based on structural comparisons to homologous enzymes involved in ATP-dependent proteolysis and clever protein engineering strategies, it has been reported that ClpB translocates polypeptide through its axial channel. Using single-turnover fluorescence and anisotropy experiments we show that ClpB is a non-processive polypeptide translocase that catalyses disaggregation by taking one or two translocation steps followed by rapid dissociation. Using single-turnover FRET experiments we show that ClpB containing the IGL loop from ClpA does not translocate substrate through its axial channel and into ClpP for proteolytic degradation. Rather, ClpB containing the IGL loop dysregulates ClpP leading to non-specific proteolysis reminiscent of ADEP (acyldepsipeptide) dysregulation. Our results support a molecular mechanism where ClpB catalyses protein disaggregation by tugging and releasing exposed tails or loops.

| S-EPMC4692069 | biostudies-literature

Noncompetitive binding of PpiD and YidC to the SecYEG translocon expands the global view on the SecYEG interactome in <i>Escherichia coli</i>.

Project description:The SecYEG translocon constitutes the major protein transport channel in bacteria and transfers an enormous variety of different secretory and inner-membrane proteins. The minimal core of the SecYEG translocon consists of three inner-membrane proteins, SecY, SecE, and SecG, which, together with appropriate targeting factors, are sufficient for protein transport in vitro However, in vivo the SecYEG translocon has been shown to associate with multiple partner proteins, likely allowing the SecYEG translocon to process its diverse substrates. To obtain a global view on SecYEG plasticity in Escherichia coli, here we performed a quantitative interaction proteomic analysis, which identified several known SecYEG-interacting proteins, verified the interaction of SecYEG with quality-control proteins, and revealed several previously unknown putative SecYEG-interacting proteins. Surprisingly, we found that the chaperone complex PpiD/YfgM is the most prominent interaction partner of SecYEG. Detailed analyses of the PpiD-SecY interaction by site-directed cross-linking revealed that PpiD and the established SecY partner protein YidC use almost completely-overlapping binding sites on SecY. Both PpiD and YidC contacted the lateral gate, the plug domain, and the periplasmic cavity of SecY. However, quantitative MS and cross-linking analyses revealed that despite having almost identical binding sites, their binding to SecY is noncompetitive. This observation suggests that the SecYEG translocon forms different substrate-independent subassemblies in which SecYEG either associates with YidC or with the PpiD/YfgM complex. In summary, the results of this study indicate that the PpiD/YfgM chaperone complex is a primary interaction partner of the SecYEG translocon.

| S-EPMC6916481 | biostudies-literature

Molecular communication of the membrane insertase YidC with translocase SecYEG affects client proteins.

Project description:The membrane insertase YidC inserts newly synthesized proteins by its hydrophobic slide consisting of the two transmembrane (TM) segments TM3 and TM5. Mutations in this part of the protein affect the insertion of the client proteins. We show here that a quintuple mutation, termed YidC-5S, inhibits the insertion of the subunit a of the FoF1 ATP synthase but has no effect on the insertion of the Sec-independent M13 procoat protein and the C-tail protein SciP. Further investigations show that the interaction of YidC-5S with SecY is inhibited. The purified and fluorescently labeled YidC-5S did not approach SecYEG when both were co-reconstituted in proteoliposomes in contrast to the co-reconstituted YidC wild type. These results suggest that TM3 and TM5 are involved in the formation of a common YidC-SecYEG complex that is required for the insertion of Sec/YidC-dependent client proteins.

| S-EPMC7886851 | biostudies-literature

Mfd translocase is necessary and sufficient for transcription-coupled repair in Escherichia coli.

Project description:Nucleotide excision repair in Escherichia coli is stimulated by transcription, specifically in the transcribed strand. Previously, it was shown that this transcription-coupled repair (TCR) is mediated by the Mfd translocase. Recently, it was proposed that in fact the majority of TCR in E. coli is catalyzed by a second pathway ("backtracking-mediated TCR") that is dependent on the UvrD helicase and the guanosine pentaphosphate (ppGpp) alarmone/stringent response regulator. Recently, we reported that as measured by the excision repair-sequencing (XR-seq), UvrD plays no role in TCR genome-wide. Here, we tested the role of ppGpp and UvrD in TCR genome-wide and in the lacZ operon using the XR-seq method, which directly measures repair. We found that the mfd mutation abolishes TCR genome-wide and in the lacZ operon. In contrast, the relA-spoT- mutant deficient in ppGpp synthesis carries out normal TCR. We conclude that UvrD and ppGpp play no role in TCR in E. coli.

| S-EPMC5682952 | biostudies-literature

Escherichia coli biofilm: mono-species culture vs. mixed-species culture

Project description:Transcription profile of Escherichia coli cells in mono-species pure biofilms was compared to that of E. coli cells in E. coli-Stenotrophomonas maltophilia dual-species biofilms. E. coli cells were separated from dual-species biofilms before total RNA extraction to eliminate possible cross hybridization from S. maltophilia transcripts. The separation method was developed by combining the use of reagent RNAlater and immuno-magnetic separation. Pure E. coli biofilms were processed with the same separation protocol before RNA extraction.

2013-12-30 | GSE24912 | GEO

Direct visualization of the <i>E. coli</i> Sec translocase engaging precursor proteins in lipid bilayers.

Project description:Escherichia coli exports proteins via a translocase comprising SecA and the translocon, SecYEG. Structural changes of active translocases underlie general secretory system function, yet directly visualizing dynamics has been challenging. We imaged active translocases in lipid bilayers as a function of precursor protein species, nucleotide species, and stage of translocation using atomic force microscopy (AFM). Starting from nearly identical initial states, SecA more readily dissociated from SecYEG when engaged with the precursor of outer membrane protein A as compared to the precursor of galactose-binding protein. For the SecA that remained bound to the translocon, the quaternary structure varied with nucleotide, populating SecA2 primarily with adenosine diphosphate (ADP) and adenosine triphosphate, and the SecA monomer with the transition state analog ADP-AlF3. Conformations of translocases exhibited precursor-dependent differences on the AFM imaging time scale. The data, acquired under near-native conditions, suggest that the translocation process varies with precursor species.

| S-EPMC6561738 | biostudies-literature

Promiscuous targeting of polytopic membrane proteins to SecYEG or YidC by the Escherichia coli signal recognition particle.

Project description:Protein insertion into the bacterial inner membrane is facilitated by SecYEG or YidC. Although SecYEG most likely constitutes the major integration site, small membrane proteins have been shown to integrate via YidC. We show that YidC can also integrate multispanning membrane proteins such as mannitol permease or TatC, which had been considered to be exclusively integrated by SecYEG. Only SecA-dependent multispanning membrane proteins strictly require SecYEG for integration, which suggests that SecA can only interact with the SecYEG translocon, but not with the YidC insertase. Targeting of multispanning membrane proteins to YidC is mediated by signal recognition particle (SRP), and we show by site-directed cross-linking that the C-terminus of YidC is in contact with SRP, the SRP receptor, and ribosomal proteins. These findings indicate that SRP recognizes membrane proteins independent of the downstream integration site and that many membrane proteins can probably use either SecYEG or YidC for integration. Because protein synthesis is much slower than protein transport, the use of YidC as an additional integration site for multispanning membrane proteins may prevent a situation in which the majority of SecYEG complexes are occupied by translating ribosomes during cotranslational insertion, impeding the translocation of secretory proteins.

| S-EPMC3268725 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data