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1.
How bacterial protein toxins enter cells: the role of partial unfolding in membrane translocation 总被引:3,自引:0,他引:3
Erwin London 《Molecular microbiology》1992,6(22):3277-3282
Bacterial protein toxins translocate across membranes by processes that are still mysterious. Studies on diphtheria toxin have shown that partial unfolding processes play a major role in toxin membrane insertion and translocation. Similar unfolding behaviour is seen with other bacterial toxins. The lessons gained from this behaviour allow us to propose novel mechanisms for toxin translocation. 相似文献
2.
Vibrio cholerae MARTX toxin heterologous translocation of beta‐lactamase and roles of individual effector domains on cytoskeleton dynamics
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Jazel S. Dolores Shivani Agarwal Martina Egerer Karla J. F. Satchell 《Molecular microbiology》2015,95(4):590-604
The Vibrio cholerae MARTXVc toxin delivers three effector domains to eukaryotic cells. To study toxin delivery and function of individual domains, the rtxA gene was modified to encode toxin with an in‐frame beta‐lactamase (Bla) fusion. The hybrid RtxA::Bla toxin was Type I secreted from bacteria; and then Bla was translocated into eukaryotic cells and delivered by autoprocessing, demonstrating that the MARTXVc toxin is capable of heterologous protein transfer. Strains that produce hybrid RtxA::Bla toxins that carry one effector domain in addition to Bla were found to more efficiently translocate Bla. In cell biological assays, the actin cross‐linking domain (ACD) and Rho‐inactivation domain (RID) are found to cross‐link actin and inactivate RhoA, respectively, when other effector domains are absent, with toxin autoprocessing required for high efficiency. The previously unstudied alpha‐beta hydrolase domain (ABH) is shown here to activate CDC42, although the effect is ameliorated when RID is also present. Despite all effector domains acting on cytoskeleton assembly, the ACD was sufficient to rapidly inhibit macrophage phagocytosis. Both the ACD and RID independently disrupted polarized epithelial tight junction integrity. The sufficiency of ACD but strong selection for retention of RID and ABH suggests these two domains may primarily function by modulating cell signaling. 相似文献
3.
Azevedo JE Costa-Rodrigues J Guimarães CP Oliveira ME Sã-Miranda C 《Cell biochemistry and biophysics》2004,41(3):451-468
Peroxisomal matrix proteins are synthesized on free cytosolic ribosomes and posttranslationally imported into the organelle.
Translocation of these newly synthesized proteins across the peroxisomal membrane requires the concerted action of many different
proteins, the majority of which were already identified. However, not much is known regarding the mechanism, of protein translocation
across this membrane system. Here, we discuss recent mechanistic and structural data. These results point to a model in which
proteins en route to the peroxisomal matrix are translocated across the organelle membrane by their own receptor in a process
that occurs, through a large membrane protein assembly. 相似文献
4.
5.
Michael C. Brothers Brett Geissler Grant S. Hisao Brenda A. Wilson Karla J. F. Satchell Chad M. Rienstra 《Biomolecular NMR assignments》2014,8(2):225-228
1H, 13C, and 15N chemical shift assignments are presented for the isolated four-helical bundle membrane localization domain from the domain of unknown function 5 (DUF5) effector (MLDVvDUF5) of the MARTX toxin from Vibrio vulnificus in its solution state. We have assigned 97 % of all backbone and side-chain carbon atoms, including 96 % of all backbone residues. Secondary chemical shift analysis using TALOS+ demonstrates four helices that align with those predicted by structure homology modeling using the MLDs of Pasteurella multocida toxin (PMT) and the clostridial TcdB and TcsL toxins as templates. Future studies will be towards solving the structure and determining the dynamics in the solution state. 相似文献
6.
MeTaDoR: a comprehensive resource for membrane targeting domains and their host proteins 总被引:1,自引:0,他引:1
MOTIVATION: Protein-lipid interactions play a central role in cellular signaling and membrane trafficking and at the core of these interactions are domains specialized in lipid binding and membrane targeting. Considering the importance of these domains, we have created MeTaDoR, a comprehensive resource dedicated to membrane targeting domains (MTDs). RESULT: MeTaDoR begins with a brief introduction about all the important MTDs including their subcellular localization and structural features. Sequences of all known MTDs are then provided in two formats: standard Prosite format and a parsed tab-delimited format that provides a manually curated classification into binding or non-binding. Structures of all MTDs and host proteins known so far are provided with links to PDB and Pfam databases. Membrane-binding orientation of these proteins, whether experimentally determined or proposed, is also provided with links to the appropriate literature. To facilitate molecular dynamics studies of these proteins, the force-field parameters for many non-standard lipids that commonly interact with these proteins are also provided. Finally, an online server for predicting membrane-binding proteins and a search function with various search fields are included. The resource is publicly available and will be updated on a regular basis. 相似文献
7.
Zachary C. Ruhe Josephine Y. Nguyen Christina M. Beck David A. Low Christopher S. Hayes 《Molecular microbiology》2014,94(2):466-481
Contact‐dependent growth inhibition (CDI) is a mode of bacterial competition orchestrated by the CdiB/CdiA family of two‐partner secretion proteins. The CdiA effector extends from the surface of CDI+ inhibitor cells, binds to receptors on neighbouring bacteria and delivers a toxin domain derived from its C‐terminal region (CdiA‐CT). Here, we show that CdiA‐CT toxin translocation requires the proton‐motive force (pmf) within target bacteria. The pmf is also critical for the translocation of colicin toxins, which exploit the energized Ton and Tol systems to cross the outer membrane. However, CdiA‐CT translocation is clearly distinct from known colicin‐import pathways because ΔtolA ΔtonB target cells are fully sensitive to CDI. Moreover, we provide evidence that CdiA‐CT toxins can be transferred into the periplasm of de‐energized target bacteria, indicating that transport across the outer membrane is independent of the pmf. Remarkably, CDI toxins transferred under de‐energized conditions remain competent to enter the target‐cell cytoplasm once the pmf is restored. Collectively, these results indicate that outer‐ and inner‐membrane translocation steps can be uncoupled, and that the pmf is required for CDI toxin transport from the periplasm to the target‐cell cytoplasm. 相似文献
8.
Solute translocation across the mammalian lysosome membrane 总被引:2,自引:0,他引:2
9.
Yersinia pseudotuberculosis YopD mutants that genetically separate effector protein translocation from host membrane disruption
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Walter Adams Jessica Morgan Laura Kwuan Victoria Auerbuch 《Molecular microbiology》2015,96(4):764-778
The Yersinia type III secretion system (T3SS) translocates Yop effector proteins into host cells to manipulate immune defenses such as phagocytosis and reactive oxygen species (ROS) production. The T3SS translocator proteins YopB and YopD form pores in host membranes, facilitating Yop translocation. While the YopD amino and carboxy termini participate in pore formation, the role of the YopD central region between amino acids 150–227 remains unknown. We assessed the contribution of this region by generating Y. pseudotuberculosis yopDΔ150–170 and yopDΔ207–227 mutants and analyzing their T3SS functions. These strains exhibited wild‐type levels of Yop secretion in vitro and enabled robust pore formation in macrophages. However, the yopDΔ150–170 and yopDΔ207–227 mutants were defective in Yop translocation into CHO cells and splenocyte‐derived neutrophils and macrophages. These data suggest that YopD‐mediated host membrane disruption and effector Yop translocation are genetically separable activities requiring distinct protein domains. Importantly, the yopDΔ150–170 and yopDΔ207–227 mutants were defective in Yop‐mediated inhibition of macrophage cell death and ROS production in neutrophil‐like cells, and were attenuated in disseminated Yersinia infection. Therefore, the ability of the YopD central region to facilitate optimal effector protein delivery into phagocytes, and therefore robust effector Yop function, is important for Yersinia virulence. 相似文献
10.
Deletion mutants in the transit sequence of preferredoxin were used in label transfer cross-linking assays to map the interactions of the transit sequence with the import machinery. The deletion mutants gave distinct cross-linking patterns to the Toc and Tic components of the import machinery, consistent with the binding and import properties obtained in in vitro import assays. The cross-linking results revealed two separate properties of the transit peptide: first the presentation of specific binding domains for the initial interaction with outer membrane components, and second the presence of different domains for interaction with the outer and inner membrane components of the transport machinery for full envelope translocation. The N-terminal Delta6-14 deletion blocked import of the precursor at the Toc components, whereas the more internal deletion Delta15-25 blocked import at the Tic components. The information for association with the outer and inner membrane components therefore resides in two separate but partly overlapping domains in the first 25 amino acids of the transit sequence. 相似文献
11.
Although transmembrane proteins generally require membrane-embedded machinery for integration, a few can insert spontaneously into liposomes. Previously, we established that the tail-anchored (TA) protein cytochrome b(5) (b5) can posttranslationally translocate 28 residues downstream to its transmembrane domain (TMD) across protein-free bilayers (Brambillasca, S., M. Yabal, P. Soffientini, S. Stefanovic, M. Makarow, R.S. Hegde, and N. Borgese. 2005. EMBO J. 24:2533-2542). In the present study, we investigated the limits of this unassisted translocation and report that surprisingly long (85 residues) domains of different sequence and charge placed downstream of b5's TMD can posttranslationally translocate into mammalian microsomes and liposomes at nanomolar nucleotide concentrations. Furthermore, integration of these constructs occurred in vivo in translocon-defective yeast strains. Unassisted translocation was not unique to b5 but was also observed for another TA protein (protein tyrosine phosphatase 1B) whose TMD, like the one of b5, is only moderately hydrophobic. In contrast, more hydrophobic TMDs, like synaptobrevin's, were incapable of supporting unassisted integration, possibly because of their tendency to aggregate in aqueous solution. Our data resolve long-standing discrepancies on TA protein insertion and are relevant to membrane evolution, biogenesis, and physiology. 相似文献
12.
Evidence for posttranslational translocation of beta-lactamase across the bacterial inner membrane 总被引:31,自引:0,他引:31
Secretion of beta-lactamase was studied in Salmonella typhimurium infected with P22 phage carrying wild-type and mutant alleles of the structural gene. Cellular location of precursor and mature products of wild-type and temperature-sensitive and chain-terminating mutants was analyzed by cell fractionation and by trypsin accessibility in intact and lysed spheroplasts. The precursors of wild-type and all these mutants (none of which alter the signal peptide) are found sequestered within the cell, while all the mature forms have at least partially been translocated across the inner membrane. Thus most beta-lactamase molecules traverse the membrane after completion of their translation. It seems that the carboxyl terminus of beta-lactamase is not required for translocation across the inner membrane but is required for the protein to appear in the periplasm as a soluble species. 相似文献
13.
Many polypeptides have been postulated to play direct roles in secretory protein translocation based on genetic criteria, cross-linking, and antibody inhibition. Much of the excitement in the next few years will come from the resolution of current controversies. What is the nature of the ribosome receptor, and is it essential for translocation? Is BiP required for translocation in mammalian cells? Are all of the polypeptides of signal peptidase and oligosaccharyltransferase required for catalytic function, or do some of them mediate steps of protein translocation? One of the best ways to resolve these problems will be to determine the importance of each in reconstituted translocation reactions by fractionation or immunodepletion, or by analysis in a purified reaction. Another approach is to identify homologues of these molecules in S. cerevisiae and to assess their importance in in vivo translocation. Several mechanistic questions remain to be addressed as well. Does the protein translocation apparatus consist of protein, or lipid, or both? How are integral membrane proteins inserted? How is the translocon gated to admit only unfolded or partially folded secretory polypeptides and to exclude cytoplasmic molecules? The answers to these questions will illuminate a basic enigma in cell biology that has remained unanswered for many years. 相似文献
14.
15.
Energy requirements for protein translocation across the Escherichia coli inner membrane 总被引:5,自引:0,他引:5
B. L. Geller 《Molecular microbiology》1991,5(9):2093-2098
Both ATP and an electrochemical potential play roles in translocating proteins across the inner membrane of Escherichia coli. Recent discoveries have dissected the overall transmembrane movement into separate subreactions with different energy requirements, identified a translocation ATPase, and reconstituted both energy-requiring steps of the reaction from purified components. A more refined understanding of the energetics of this fundamental process is beginning to provide answers about the basic issues of how proteins move across the hydrophobic membrane barrier. 相似文献
16.
The mitochondrial inner membrane has a central function for the energy metabolism of the cell. The respiratory chain generates a proton gradient across the inner mitochondrial membrane, which is used to produce ATP by the F1Fo-ATPase. To maintain the electrochemical gradient, the inner membrane represents an efficient permeability barrier for small molecules. Nevertheless, metabolites as well as polypeptide chains need to be transported across the inner membrane while the electrochemical gradient is retained. While specialized metabolite carrier proteins mediate the transport of small molecules, dedicated protein translocation machineries in the inner mitochondrial membrane (so called TIM complexes) transport precursor proteins across the inner membrane. Here we describe the organization of the TIM complexes and discuss the current models as to how they mediate the posttranslational import of proteins across and into the inner mitochondrial membrane. 相似文献
17.
Requirements for translocation of periplasmic domains in polytopic membrane proteins. 总被引:1,自引:1,他引:1
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Periplasmic domains of cytoplasmic membrane proteins require export signals for proper translocation. These signals were studied by using a MalF-alkaline phosphatase fusion in a genetic selection that allowed the isolation of mislocalization mutants. In the original construct, alkaline phosphatase is fused to the second periplasmic domain of the membrane protein, and its activity is thus confined exclusively to the periplasm. Mutants that no longer translocated alkaline phosphatase were selected by complementation of a serB mutation. A total of 11 deletions in the amino terminus were isolated, all of which spanned at least the third transmembrane segment. This domain immediately precedes the periplasmic domain to which alkaline phosphatase was fused. Our results obtained in vivo support the model that amino-terminal membrane-spanning segments are required for translocation of large periplasmic domains. In addition, we found that the inability to export the alkaline phosphatase domain could be suppressed by a mutation, prlA4, in the secretion apparatus. 相似文献
18.
We measured the density, expansivity, specific heat at constant pressure, and sound velocity of suspensions of purple membrane from Halobacterium halobium and their constituent buffers. From these quantities we calculated the apparent values for the density, expansivity, adiabatic compressibility, isothermal compressibility, specific heat at constant pressure, and specific heat at constant volume for the purple membrane. These results are discussed with respect to previously reported measurements on globular proteins and lipids. Our data suggest a simple additive model in which the protein and lipid molecules expand and compress independently of each other. However, this simple model seems to fail to describe the specific heat data. Our compressibility data suggest that bacteriorhodopsin in native purple membrane binds less water than many globular proteins in neutral aqueous solution, a finding consistent with the lipid surround of bacteriorhodopsin in purple membrane. 相似文献
19.
Posttranslational protein translocation across the membrane of the endoplasmic reticulum 总被引:1,自引:0,他引:1
Posttranslational protein translocation across the membrane of the endoplasmic reticulum is mediated by the Sec complex. This complex includes a transmembrane channel formed by multiple copies of the Sec61 protein. Translocation of a polypeptide begins when the signal sequence binds at a specific site within the channel. Binding results in the insertion of the substrate into the channel, possibly as a loop with a small segment exposed to the lumen. While bound, the signal sequence is in contact with both protein components of the channel and the lipid of the membrane. Subsequent movement of the polypeptide through the channel occurs when BiP molecules interact transiently with a luminal domain of the Sec complex, hydrolyze ATP, and bind to the substrate. Bound BiP promotes translocation by preventing the substrate from diffusing backwards through the channel, and thus acts as a molecular ratchet. 相似文献
20.
Proteins destined for secretion are translocated across or inserted into the endoplasmic reticulum membrane whereupon they fold and assemble to their native state before their subsequent transport to the Golgi apparatus. Proteins that fail to fold correctly are translocated back across the endoplasmic reticulum membrane to the cytosol where they become substrates for the cytosolic degradative machinery. Central to translocation is a protein pore in the membrane called the translocon that allows passage of proteins in and out of the endoplasmic reticulum. It is clear that the conformation of the polypeptide chain influences the translocation process and that there is a temporal relationship between modification of the chain, translocation and folding. This review will consider when and how the polypeptide chain folds, and how this might influence translocation into and out of the ER; and discuss how protein folding might affect post-translational modification of the polypeptide chain following translocation into the ER lumen. 相似文献