Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Assembly of the translocase motor onto the preprotein-conducting channel.

ABSTRACT: Bacterial protein secretion is catalysed by the SecYEG protein-conducting channel complexed with the SecA ATPase motor. To gain insight into the SecA-SecYEG interaction we used peptide arrays, thermodynamic quantification, mutagenesis and functional assays. Our data reveal that: (i) SecA binds with low affinity on several, peripheral, exposed SecYEG sites. This largely electrostatic association is modulated by temperature and nucleotides. (ii) Binding sites cluster in five major binding 'regions': three that are exclusively cytoplasmic and two that reach the periplasm. (iii) Both the N-terminal and c-terminal regions of SecA participate in binding interactions and share some sites. (iv) Several of these sites are essential for translocase catalysis. Our data provide residue-level dissection of the SecYEG-SecA interaction. Two models of assembly of SecA on dimeric SecYEG are discussed.

SUBMITTER: Karamanou S

PROVIDER: S-EPMC2603171 | biostudies-literature | 2008 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Assembly of the translocase motor onto the preprotein-conducting channel.

Karamanou Spyridoula S Bariami Vassiliki V Papanikou Efrosyni E Kalodimos Charalampos G CG Economou Anastassios A

Molecular microbiology 20080822 2

Bacterial protein secretion is catalysed by the SecYEG protein-conducting channel complexed with the SecA ATPase motor. To gain insight into the SecA-SecYEG interaction we used peptide arrays, thermodynamic quantification, mutagenesis and functional assays. Our data reveal that: (i) SecA binds with low affinity on several, peripheral, exposed SecYEG sites. This largely electrostatic association is modulated by temperature and nucleotides. (ii) Binding sites cluster in five major binding 'regions ...[more]

PMID: 18761620

Similar Datasets

The preprotein conducting channel at the inner envelope membrane of plastids.

Project description:The preprotein translocation at the inner envelope membrane of chloroplasts so far involves five proteins: Tic110, Tic55, Tic40, Tic22 and Tic20. The molecular function of these proteins has not yet been established. Here, we demonstrate that Tic110 constitutes a central part of the preprotein translocation pore. Dependent on the presence of intact Tic110, radiolabelled preprotein specifically interacts with isolated inner envelope vesicles as well as with purified, recombinant Tic110 reconstituted into liposomes. Circular dichroism analysis reveals that Tic110 consists mainly of beta-sheets, a structure typically found in pore proteins. In planar lipid bilayers, recombinant Tic110 forms a cation-selective high-conductance channel with a calculated inner pore opening of 1.7 nm. Purified transit peptide causes strong flickering and a voltage-dependent block of the channel. Moreover, at the inner envelope membrane, a peptide-sensitive channel is described that shows properties basically identical to the channel formed by recombinant Tic110. We conclude that Tic110 has a distinct preprotein binding site and functions as a preprotein translocation pore at the inner envelope membrane.

| S-EPMC126020 | biostudies-literature

The assembly pathway of the mitochondrial carrier translocase involves four preprotein translocases.

Project description:The mitochondrial inner membrane contains preprotein translocases that mediate insertion of hydrophobic proteins. Little is known about how the individual components of these inner membrane preprotein translocases combine to form multisubunit complexes. We have analyzed the assembly pathway of the three membrane-integral subunits Tim18, Tim22, and Tim54 of the twin-pore carrier translocase. Tim54 displayed the most complex pathway involving four preprotein translocases. The precursor is translocated across the intermembrane space in a supercomplex of outer and inner membrane translocases. The TIM10 complex, which translocates the precursor of Tim22 through the intermembrane space, functions in a new posttranslocational manner: in case of Tim54, it is required for the integration of Tim54 into the carrier translocase. Tim18, the function of which has been unknown so far, stimulates integration of Tim54 into the carrier translocase. We show that the carrier translocase is built via a modular process and that each subunit follows a different assembly route. Membrane insertion and assembly into the oligomeric complex are uncoupled for each precursor protein. We propose that the mitochondrial assembly machinery has adapted to the needs of each membrane-integral subunit and that the uncoupling of translocation and oligomerization is an important principle to ensure continuous import and assembly of protein complexes in a highly active membrane.

| S-EPMC2447139 | biostudies-literature

The preprotein translocase YidC controls respiratory metabolism in Mycobacterium tuberculosis.

Project description:The YidC-Oxa1-Alb3 preprotein translocases play a vital role in membrane insertion of proteins in eukaryotes and bacteria. In a recent study we observed that Rv3921c, which encodes putative YidC translocase in Mycobacterium tuberculosis (Mtb), is essential for in vitro growth of bacteria. However, the exact function of this particular protein remains to identify in mycobacterial pathogens. By performing a systematic study here we show that YidC of Mtb is an envelope protein, which is required for production of ATP and maintenance of cellular redox balance. Drastic effects of depletion of Rv3921c on the expression of hypoxic genes, ATP synthases, and many proteins of central metabolic and respiratory pathways shed a significant light on the function of YidC towards controlling respiratory metabolism in Mtb. Association of YidC with proteins such as succinate dehydrogenases and ubiquinol-cytochrome C reductase further confirms its role in respiration. Finally we demonstrate that YidC is required for the intracellular survival of Mtb in human macrophages.

| S-EPMC4863248 | biostudies-literature

Tim50, a novel component of the TIM23 preprotein translocase of mitochondria.

Project description:The preprotein translocase of the inner membrane of mitochondria (TIM23 complex) is the main entry gate for proteins of the matrix and the inner membrane. We isolated the TIM23 complex of Neurospora crassa. Besides Tim23 and Tim17, it contained a novel component, referred to as Tim50. Tim50 spans the inner membrane with a single transmembrane segment and exposes a large hydrophilic domain in the intermembrane space. Tim50 is essential for viability of yeast. Mitochondria from cells depleted of Tim50 displayed strongly reduced import kinetics of preproteins using the TIM23 complex. Tim50 could be cross-linked to preproteins that were halted at the level of the translocase of the outer membrane (TOM complex) or spanning both TOM and TIM23 complexes. We suggest that Tim50 plays a crucial role in the transfer of preproteins from the TOM complex to the TIM23 complex through the intermembrane space.

| S-EPMC145450 | biostudies-literature

Preprotein mature domains contain translocase targeting signals that are essential for secretion.

Project description:Secretory proteins are only temporary cytoplasmic residents. They are typically synthesized as preproteins, carrying signal peptides N-terminally fused to their mature domains. In bacteria secretion largely occurs posttranslationally through the membrane-embedded SecA-SecYEG translocase. Upon crossing the plasma membrane, signal peptides are cleaved off and mature domains reach their destinations and fold. Targeting to the translocase is mediated by signal peptides. The role of mature domains in targeting and secretion is unclear. We now reveal that mature domains harbor their own independent targeting signals (mature domain targeting signals [MTSs]). These are multiple, degenerate, interchangeable, linear or 3D hydrophobic stretches that become available because of the unstructured states of targeting-competent preproteins. Their receptor site on the cytoplasmic face of the SecYEG-bound SecA is also of hydrophobic nature and is located adjacent to the signal peptide cleft. Both the preprotein MTSs and their receptor site on SecA are essential for protein secretion. Evidently, mature domains have their own previously unsuspected distinct roles in preprotein targeting and secretion.

| S-EPMC5412566 | biostudies-literature

Interactions of mitochondrial presequence peptides with the mitochondrial outer membrane preprotein translocase TOM.

Project description:TOM protein-conducting channels serve as the main entry sites into mitochondria for virtually all mitochondrial proteins. When incorporated into lipid bilayers, they form large, relatively nonspecific ion channels that are blocked by peptides derived from mitochondrial precursor proteins. Using single-channel electrical recordings, we analyzed the interactions of mitochondrial presequence peptides with single TOM pores. The largest conductance state of the translocon represents the likely protein-conducting conformation of the channel. The frequency (but not the duration) of the polypeptide-induced blockage is strongly modulated by the substrate concentration. Structural differences between substrates are reflected in characteristic blockage frequencies and duration of blockage. To our knowledge, this study provides first quantitative data regarding the kinetics of polypeptide interaction with the mitochondrial TOM machinery.

| S-EPMC2913200 | biostudies-literature

Preprotein-controlled catalysis in the helicase motor of SecA.

Project description:The cornerstone of the functionality of almost all motor proteins is the regulation of their activity by binding interactions with their respective substrates. In most cases, the underlying mechanism of this regulation remains unknown. Here, we reveal a novel mechanism used by secretory preproteins to control the catalytic cycle of the helicase 'DEAD' motor of SecA, the preprotein translocase ATPase. The central feature of this mechanism is a highly conserved salt-bridge, Gate1, that controls the opening/closure of the nucleotide cleft. Gate1 regulates the propagation of binding signal generated at the Preprotein Binding Domain to the nucleotide cleft, thus allowing the physical coupling of preprotein binding and release to the ATPase cycle. This relay mechanism is at play only after SecA has been previously 'primed' by binding to SecYEG, the transmembrane protein-conducting channel. The Gate1-controlled relay mechanism is essential for protein translocase catalysis and may be common in helicase motors.

| S-EPMC1894763 | biostudies-literature

Dual interaction of scaffold protein Tim44 of mitochondrial import motor with channel-forming translocase subunit Tim23.

Project description:Proteins destined for the mitochondrial matrix are targeted to the inner membrane Tim17/23 translocon by their presequences. Inward movement is driven by the matrix-localized, Hsp70-based motor. The scaffold Tim44, interacting with the matrix face of the translocon, recruits other motor subunits and binds incoming presequence. The basis of these interactions and their functional relationships remains unclear. Using site-specific in vivo crosslinking and genetic approaches in Saccharomyces cerevisiae, we found that both domains of Tim44 interact with the major matrix-exposed loop of Tim23, with the C-terminal domain (CTD) binding Tim17 as well. Results of in vitro experiments showed that the N-terminal domain (NTD) is intrinsically disordered and binds presequence near a region important for interaction with Hsp70 and Tim23. Our data suggest a model in which the CTD serves primarily to anchor Tim44 to the translocon, whereas the NTD is a dynamic arm, interacting with multiple components to drive efficient translocation.

| S-EPMC5422074 | biostudies-literature

Mitsugumin 23 forms a massive bowl-shaped assembly and cation-conducting channel.

Project description:Mitsugumin 23 (MG23) is a 23 kDa transmembrane protein localized to the sarcoplasmic/endoplasmic reticulum and nuclear membranes in a wide variety of cells. Although the characteristics imply the participation in a fundamental function in intracellular membrane systems, the physiological role of MG23 is unknown. Here we report the biochemical and biophysical characterization of MG23. Hydropathicity profile and limited proteolytic analysis proposed three transmembrane segments in the MG23 primary structure. Chemical cross-linking analysis suggested a homo-oligomeric assembly of MG23. Ultrastructural observations detected a large symmetrical particle as the predominant component and a small asymmetric assembly as the second major component in highly purified MG23 preparations. Single-particle three-dimensional reconstruction revealed that MG23 forms a large bowl-shaped complex equipped with a putative central pore, which is considered an assembly of the small asymmetric subunit. After reconstitution into planar phospholipid bilayers, purified MG23 behaved as a voltage-dependent, cation-conducting channel, permeable to both K(+) and Ca(2+). A feature of MG23 gating was that multiple channels always appeared to be gating together in the bilayer. Our observations suggest that the bowl-shaped MG23 can transiently assemble and disassemble. These building transitions may underlie the unusual channel gating behavior of MG23 and allow rapid cationic flux across intracellular membrane systems.

| S-EPMC3065873 | biostudies-other

Dynamics of the preprotein translocation channel of the outer membrane of mitochondria.

Project description:The protein translocase of the outer mitochondrial membrane (TOM) serves as the main entry site for virtually all mitochondrial proteins. Like many other protein translocases it also has an ion channel activity that can be used to study the dynamical properties of this supramolecular complex. We have purified TOM core complex and Tom40, the main pore forming subunit, from mitochondria of the filamentous fungus Neurospora crassa and incorporated them into planar lipid bilayers. We then examined their single channel properties to provide a detailed description of the conformational dynamics of this channel in the absence of its protein substrate. For isolated TOM core complex we have found at least six conductance states. Transitions between these states were voltage-dependent with a bell-shaped open probability distribution and distinct kinetics depending on the polarity of the applied voltage. The states with the largest conductance followed an Ohmic I/V characteristic consistent with a large cylindrical pore with very little interaction with the permeating ions. For the lower conductance states, however, we have observed inverted S-shaped nonlinear current-voltage curves reminiscent to those of much narrower pores where the permeating ions have to surmount an electrostatic energy barrier. At low voltages (<+/-70 mV), purified Tom40 protein did not show any transitions between its conductance states. Prolonged exposure to higher voltages induced similar gating behavior to what we observed for TOM core complex. This effect was time-dependent and reversible, indicating that Tom40 forms not only the pore but also contains the "gating machinery" of the complex. However, for proper functioning, additional proteins (Tom22, Tom7, Tom6, and Tom5) are required that act as a modulator of the pore dynamics by significantly reducing the energy barrier between different conformational states.

| S-EPMC2479589 | 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