Translocation Machinery Research Articles

A New Signal From B-Type Natriuretic Peptide in ST-Elevation Myocardial Infarction

A New Signal From B-Type Natriuretic Peptide in ST-Elevation Myocardial Infarction

Malaria, Plasmodium falciparum and its apicoplast

Malaria, which is caused by species of the parasite genus Plasmodium, remains a major global health problem. A vestigial plastid homologous with the chloroplasts of plants and algae was discovered in malaria and related parasites from the phylum Apicomplexa and has radically changed our view of the evolutionary origins of these disease-causing protists. We now recognize that this large group of parasites had a photosynthetic ancestry and were converted into parasitism early in the evolution of animals. Apicomplexans have probably been parasitizing the animal kingdom for more than 500 million years. The relic plastid persists in most apicomplexans and is an essential component. Perturbation of apicoplast function or inheritance results in parasite death, making the organelle a promising target for chemotherapy. Plastids, including those of malaria parasites, are essentially reduced endosymbiotic bacteria living inside a eukaryotic host. This means that plastids have bacterial-type metabolic pathways and housekeeping processes, all of which are vulnerable to antibacterial compounds. Indeed, many antibacterials kill malaria parasites by blocking essential processes in the plastid. Furthermore, a range of herbicides that target plastid metabolism of undesired plants are also parasiticidal, making them potential new leads for antimalarial drugs. In the present review, we examine the evolutionary origins of the malaria parasite's plastid by endosymbiosis and outline the recent findings on how the organelle imports nuclear-encoded proteins through a set of translocation machineries in the membranes that bound the organelle.

Peroxisome biogenesis and positioning

Plant peroxisomes are extremely dynamic, moving and undergoing changes of shape in response to metabolic and environmental signals. Matrix proteins are imported via one of two import pathways, depending on the targeting signal within the protein. Each pathway has a specific receptor but utilizes common membrane-bound translocation machinery. Current models invoke receptor recycling, which may involve cycles of ubiquitination. Some components of the import machinery may also play a role in proteolytic turnover of matrix proteins, prompting parallels with the endoplasmic-reticulum-associated degradation pathway. Peroxisome membrane proteins, some of which are imported post-translationally, others of which may traffic to peroxisomes via the endoplasmic reticulum, use distinct proteinaceous machinery. The isolation of mutants defective in peroxisome biogenesis has served to emphasize the important role of peroxisomes at all stages of the plant life cycle.

The action of cardiolipin on the bacterial translocon

Cardiolipin is an ever-present component of the energy-conserving inner membranes of bacteria and mitochondria. Its modulation of the structure and dynamism of the bilayer impacts on the activity of their resident proteins, as a number of studies have shown. Here we analyze the consequences cardiolipin has on the conformation, activity, and localization of the protein translocation machinery. Cardiolipin tightly associates with the SecYEG protein channel complex, whereupon it stabilizes the dimer, creates a high-affinity binding surface for the SecA ATPase, and stimulates ATP hydrolysis. In addition to the effects on the structure and function, the subcellular distribution of the complex is modified by the cardiolipin content of the membrane. Together, the results provide rare and comprehensive insights into the action of a phospholipid on an essential transport complex, which appears to be relevant to a broad range of energy-dependent reactions occurring at membranes.

Mutations affecting the extreme C terminus of Escherichia coli haemolysin A reduce haemolytic activity by altering the folding of the toxin

Escherichia coli haemolysin A (HlyA), an RTX toxin, is secreted probably as an unfolded intermediate, by the type I (ABC transporter-dependent) pathway, utilizing a C-terminal secretion signal. However, the mechanism of translocation and post-translocation folding is not understood. We identified a mutation (hlyA99) at the extreme C terminus, which is dominant in competition experiments, blocking secretion of the wild-type toxin co-expressed in the same cell. This suggests that unlike recessive mutations which affect recognition of the translocation machinery, the hlyA99 mutation interferes with some later step in secretion. Indeed, the mutation reduced haemolytic activity of the toxin and the activity of beta-lactamase when the latter was fused to a C-terminal 23 kDa fragment of HlyA carrying the hlyA99 mutation. A second mutant (hlyAdel6), lacking the six C-terminal residues of HlyA, also showed reduced haemolytic activity and neither mutant protein regained normal haemolytic activity in in vitro unfolding/refolding experiments. Tryptophan fluorescence spectroscopy indicated differences in structure between the secreted forms of wild-type HlyA and the HlyA Del6 mutant. These results suggested that the mutations affected the correct folding of both HlyA and the beta-lactamase fusion. Thus, we propose a dual function for the HlyA C terminus involving an important role in post-translocation folding as well as targeting HlyA for secretion.

TAT-Pathway-Dependent Lipoproteins as a Niche-Based Adaptation in Prokaryotes

Bacterial lipoproteins, characterized by the N-terminal N-acyl S-diacylglyceryl Cysteine, are key membrane proteins in bacterial homeostasis. It is generally thought that during the modification lipoprotein precursors are translocated via the Sec-machinery in an unfolded state. The recent discovery of twin-arginine translocation (TAT) machinery, meant for exporting folded-proteins, and the presence of TAT-type signal sequences in co-factor-containing (hence already folded) lipoproteins, prompted us to investigate its role and significance in lipoprotein biosynthesis. We systematically analyzed 696 prokaryotic genomes using an algorithm based on DOLOP and TatP rules to predict TAT-pathway-dependent lipoprotein substrates. Occurrence of the deduced TAT-pathway-dependent lipoprotein substrates in relation to genome size, presence or absence of TAT machinery, and extent of its usage for lipoprotein export and habitat types revealed that unlike the host-obligates, the free-living prokaryotes in complex hostile environments (e.g., soil) depend more on TAT-exported lipoproteins. Functional classification of the predicted TAT-dependent lipoproteins revealed enrichment in hydrolases and oxido-reductases, which are fast-folding and co-factor-containing proteins. The role of the TAT pathway in the export of folded-lipoproteins and in niche-specific adaptation for survival has important implications not only in lipoprotein biosynthesis, but also for protein and metabolic engineering applications.

CpSecA, a thylakoid protein translocase subunit, is essential for photosynthetic development in Arabidopsis

The endosymbiont-derived Sec-dependent protein sorting pathway is essential for protein import into the thylakoid lumen and is important for the proper functioning of the chloroplast. Two loss-of-function mutants of cpSecA, the ATPase subunit of the chloroplast Sec translocation machinery, were analysed in Arabidopsis. The homozygous mutants were albino and seedling lethal under autotrophic conditions and remained dwarf and infertile with an exogenous carbon supply. They were subject to oxidative stress and accumulated superoxide under normal lighting conditions. Electron microscopy revealed that the chloroplast of the mutants had underdeveloped thylakoid structures. Histochemical GUS assay of the AtcpSecA::GUS transgenic plants confirmed that AtcpSecA was expressed in green organs in a light-inducible way. Real-time RT-PCR and microarray analysis revealed repressed transcription of nucleus- and chloroplast- encoded subunits of photosynthetic complexes, and induced transcription of chloroplast protein translocation machinery and mitochondrion-encoded respiratory complexes in the mutants. It is inferred that AtcpSecA plays an essential role in chloroplast biogenesis, the absence of which triggered a retrograde signal, eventually leading to a reprogramming of chloroplast and mitochondrial gene expression.

Protein translocation through the Sec61/SecY channel

Special codes are embedded in the primary sequence of newly synthesized proteins to determine their final destination. Protein translocation across biological membranes requires co-operation between the targeting and translocation machineries. A conserved membrane channel, the Sec61/SecY complex, mediates protein translocation across or integration into the endoplasmic reticulum membrane in eukaryotes and the plasma membrane in prokaryotes. A combination of recent biochemical and structural data provides novel insights into the mechanism of how the channel allows polypeptide movement into the exoplasmic space and the lipid bilayer.

Primary metabolism of chickpea is the initial target of wound inducing early sensed Fusarium oxysporum f. sp. ciceri race I.

BackgroundBiotrophic interaction between host and pathogen induces generation of reactive oxygen species that leads to programmed cell death of the host tissue specifically encompassing the site of infection conferring resistance to the host. However, in the present study, biotrophic relationship between Fusarium oxysporum and chickpea provided some novel insights into the classical concepts of defense signaling and disease perception where ROS (reactive oxygen species) generation followed by hypersensitive responses determined the magnitude of susceptibility or resistant potentiality of the host.Methodology/Principal FindingsMicroscopic observations detected wound mediated in planta pathogenic establishment and its gradual progression within the host vascular tissue. cDNA-AFLP showed differential expression of many defense responsive elements. Real time expression profiling also validated the early recognition of the wound inducing pathogen by the host. The interplay between fungus and host activated changes in primary metabolism, which generated defense signals in the form of sugar molecules for combating pathogenic encounter.Conclusions/SignificanceThe present study showed the limitations of hypersensitive response mediated resistance, especially when foreign encounters involved the food production as well as the translocation machinery of the host. It was also predicted from the obtained results that hypersensitivity and active species generation failed to impart host defense in compatible interaction between chickpea and Fusarium. On the contrary, the defense related gene(s) played a critical role in conferring natural resistance to the resistant host. Thus, this study suggests that natural selection is the decisive factor for selecting and segregating out the suitable type of defense mechanism to be undertaken by the host without disturbing its normal metabolism, which could deviate from the known classical defense mechanisms.

Reducible Poly(amido ethylenimine)-Based Gene Delivery System for Improved Nucleus Trafficking of Plasmid DNA

In a nonviral gene delivery system, localization of a plasmid DNA in the nucleus is a prerequisite for expression of a desired therapeutic protein encoded in the plasmid DNA. In this study, a reducible polymer-based gene delivery system for improved intracellular trafficking and nuclear translocation of plasmid DNA is introduced. The system is consisted of two components, a plasmid DNA having repeated binding sequence for a karyophilic protein, NFkappaB, and a reducible polymer. A reducible poly(amido ethylenimine), poly(TETA-CBA), was synthesized by a Michael-type addition polymerization between cystamine bisacrylamide and triethyl tetramine. The polymer forming tight complexes with plasmid DNA could be degraded in the reductive cytosol to release the plasmid DNA. The triggered release mechanism in the cytosol could facilitate the interaction between cytosolic NFkappaB and the plasmid DNA having repeated NFkappaB biding motif. Upon activation of NFkappaB by interleukin-1beta (IL-1beta), most of the plasmid distributed in the cytoplasm was localized within the nucleus, resulting in significantly higher gene transfection efficiency than controls with nondegradable PEI. The current study suggests an alternative way of improving transfection efficiency by taking advantage of endogenous transport machinery for intracellular trafficking and nuclear translocation of a plasmid DNA.

Twin arginine translocase pathway and fast-folding lipoprotein biosynthesis in E. coli: interesting implications and applications

Bacterial lipoproteins, an important class of membrane proteins, are generally thought to be translocated in an unfolded state by the well-studied Sec machinery, whereas the role of TAT, meant for folded proteins, is hardly investigated. Using appropriately engineered fast-folding Enhanced Green Fluorescence Protein (EGFP), as a model, here we show that TAT is essential for not only translocating fast-folding lipoprotein but also its lipid modification. EGFP was lipid-modified and targeted to the outer membrane's outer surface with a prototypical TAT signal sequence containing lipobox but not with the lipoprotein or TAT signal sequence. Justifiably signal sequences of many substrate-binding and co-factor-containing lipoproteins contained both TAT-box and lipobox (Shruthi et al., submitted). Cytoplasmic accumulation of unmodified precursors of engineered EGFP in a tatC mutant implicated this TAT-box-recognizing component in lipid-modification. Similar observations reported earlier with Sec components and murein lipoprotein led us to propose that the translocation-competent and translocase-associated (Sec or TAT) precursor form is prerequisite to initiation of lipid-modification in vivo. The above missing links between translocation and lipid modification machineries in vivo is important to our understanding of bacterial lipoprotein biosynthesis and its utility as a protein engineering tool for potent applications in synthetic biology and nanobiotechnology like display, arrays on bacterial surfaces, vaccines and biosensors.

Facilitated Polypeptide Translocation through a Protein Pore

Facilitated translocation of proteins through a transmembrane protein pore is a ubiquitous and fundamental process in biology. Protein translocation machineries possess various binding sites within the pore interior, but a clear mechanistic understanding of how the interaction of the polypeptides with the binding site alters the underlying kinetics is still missing. Here, we employed standard protein engineering and single-channel electrical recordings to obtain detailed kinetic information of polypeptide translocation through the staphylococcal α-hemolysin (αHL) transmembrane pore, a robust, tractable, and versatile β-barrel protein. Binding sites comprised of rings of negatively-charged aspartic acid residues, engineered at different positions within the β barrel, produced significant alterations in the functional features pf the protein pore, facilitating the transport of cationic polypeptides from one side of the membrane to the other. The translocation of polypeptides through the engineered protein pore was dependent on the position of the binding site, the length of the polypeptide as well as its hydrophobic index. Acknowledgements. This research was supported by grants from the National Science Foundation (DMR-0706517 and HRD-0703452) and the National Institutes of Health (R01 GM088403) as well as by the Syracuse Biomaterials Institute (SBI).

Structure and Assembly of Escherichia coli Capsules

The capsule is a cell surface structure composed of long-chain polysaccharides that envelops many isolates of Escherichia coli. It protects the cell against host defenses or physical environmental stresses, such as desiccation. The component capsular polysaccharides (CPSs) are major surface antigens in E. coli. They are named K antigens (after the German word Kapsel). Due to variations in CPS structures, more than 80 serologically unique K antigens exist in E. coli. Despite the hypervariability in CPS structures, only two capsule-assembly strategies exist in E. coli. These have led to the assignment of group 1 and group 2 capsules, and many of the key elements of the corresponding assembly pathways have been resolved. Structural features, as well as genetic and regulatory variations, give rise to additional groups 3 and 4. These employ the same biosynthesis processes described in groups 2 and 1, respectively. Each isolate possesses a distinctive set of cytosolic and inner-membrane enzymes, which generate a precise CPS structure, defining a given K serotype. Once synthesized, a multiprotein complex is needed to translocate the nascent CPS across the Gram-negative cell envelope to the outer surface of the outer membrane, where the capsule structure is assembled. While the translocation machineries for group 1 and group 2 CPSs are fundamentally different from one another, they possess no specificity for a given CPS structure. Each is conserved in all isolates producing capsules belonging to a particular group.

Tuning the Cellular Trafficking of the Lysosomal Peptide Transporter TAPL by its N-terminal Domain

The homodimeric ATP-binding cassette (ABC) transport complex TAPL (transporter associated with antigen processing-like, ABCB9) translocates a broad spectrum of peptides from the cytosol into the lumen of lysosomes. The presence of an extra N-terminal transmembrane domain (TMD0) lacking any sequence homology to known proteins distinguishes TAPL from most other ABC transporters of its subfamily. By dissecting TAPL, we could assign distinct functions to the core complex and TMD0. The core-TAPL complex, composed of six predicted transmembrane helices and a nucleotide-binding domain, is sufficient for peptide transport, showing that the core transport complex is correctly targeted to and assembled in the membrane. Strikingly, in contrast to the full-length transporter, the core translocation complex is targeted preferentially to the plasma membrane. However, TMD0 alone, comprising a putative four transmembrane helix bundle, traffics to lysosomes. Upon coexpression, TMD0 forms a stable non-covalently linked complex with the core translocation machinery and guides core-TAPL into lysosomal compartments. Therefore, TMD0 represents a unique domain, which folds independently and encodes the information for lysosomal targeting. These outcomes are discussed in respect of trafficking, folding and function of TAPL.

A Point Mutation in Sec61α1 Leads to Diabetes and Hepatosteatosis in Mice

OBJECTIVEType 2 diabetes is caused by both environmental and genetic factors. To better understand the genetic factors we used forward genetics to discover genes that have not previously been implicated in the development of hyperglycemia or diabetes.RESEARCH DESIGN AND METHODSOffspring of ethylnitrosurea-mutagenized C57BL/6 mice were bred to homozygosity, maintained on high-fat diet, and screened for hyperglycemia. The phenotype in one diabetic family of mice was mapped among hybrid F2s with single nucleotide polymorphic markers, followed by candidate gene sequencing to identify the gene harboring the causative mutation. Subsequent analysis was done on wild-type, heterozygous, and homozygous mutant mice on a pure C57BL/6 background.RESULTSDiabetes mapped to a point mutation in the Sec61a1 gene that encodes a His to Tyr substitution at amino acid 344 (Y344H). Metabolic profiling, histological examination, and electron microscopy revealed that hyperglycemia was a result of insulin insufficiency due to β-cell apoptosis brought on by endoplasmic reticulum (ER) stress. Transgenic β-cell–specific expression of Sec61a1 in mutant mice rescued diabetes, β-cell apoptosis, and ER stress. In vitro experiments showed that Sec61α1 plays a critical role in the β-cell response to glucose.CONCLUSIONSHere we phenotypically characterize diabetes in mice with a novel point mutation in a basic component of the cell's ER protein translocation machinery, Sec61α1. Translocation by the mutant protein does not appear to be affected. Rather, ER homeostasis is perturbed leading to β-cell death and diabetes.

The type III secretion system of Pseudomonas aeruginosa: infection by injection.

The Gram-negative bacterium Pseudomonas aeruginosa uses a complex type III secretion apparatus to inject effector proteins into host cells. The configuration of this secretion machinery, the activities of the proteins that are injected by it and the consequences of this process for infection are now being elucidated. This Review summarizes our current knowledge of P. aeruginosa type III secretion, including the secretion and translocation machinery, the regulation of this machinery, and the associated chaperones and effector proteins. The features of this interesting secretion system have important implications for the pathogenesis of P. aeruginosa infections and for other type III secretion systems.

Impairment of twin-arginine-dependent export by seemingly small alterations of substrate conformation

The twin-arginine translocation (Tat) machinery is able to transport fully folded proteins across bacterial and thylakoidal membranes. Previous in vivo and in vitro studies indicated that the model Tat substrate TorA–PhoA acquires Tat-competence only if its four cysteines form disulfide bonds. We now show that removal of the last 33 amino acids of PhoA, although not affecting the formation of disulfide bonds, converts TorA–PhoA into a poor Tat substrate. This finding suggests that even incomplete folding of a substrate can interfere with transport by the Tat translocase of Escherichia coli.

Effect of Crowding Agents, Signal Peptide, and Chaperone SecB on the Folding and Aggregation ofE. coliMaltose Binding Protein

SecB, a soluble cytosolic chaperone component of the Sec export pathway, binds to newly synthesized precursor proteins and prevents their premature aggregation and folding and subsequently targets them to the translocation machinery on the membrane. PreMBP, the precursor form of maltose binding protein, has a 26-residue signal sequence attached to the N-terminus of MBP and is a physiological substrate of SecB. We examine the effect of macromolecular crowding and SecB on the stability and refolding of denatured preMBP and MBP. PreMBP was less stable than MBP (DeltaT(m )= 7 +/- 0.5 K) in both crowded and uncrowded solutions. Crowding did not cause any substantial changes in the thermal stability of MBP (DeltaT(m )= 1 +/- 0.4 K) or preMBP (DeltaT(m )= 0 +/- 0.6 K), as observed in spectroscopically monitored thermal unfolding experiments. However, both MBP and preMBP were prone to aggregation while refolding under crowded conditions. In contrast to MBP aggregates, which were amorphous, preMBP aggregates form amyloid fibrils. Under uncrowded conditions, a molar excess of SecB was able to completely prevent aggregation and promote disaggregation of preformed aggregates of MBP. When a complex of the denatured protein and SecB was preformed, SecB could completely prevent aggregation and promote folding of MBP and preMBP even in crowded solution. Thus, in addition to maintaining substrates in an unfolded, export-competent conformation, SecB also suppresses the aggregation of its substrates in the crowded intracellular environment. SecB is also able to promote passive disaggregation of macroscopic aggregates of MBP in the absence of an energy source such as ATP or additional cofactors. These experiments also demonstrate that signal peptide can greatly influence protein stability and aggregation propensity.

Twin-Arginine-Dependent Translocation of SufI in the Absence of Cytosolic Helper Proteins

The twin-arginine translocation (Tat) machinery present in bacterial and thylakoidal membranes is able to transport fully folded proteins. Folding of some Tat precursor proteins requires dedicated chaperones that also sequester the signal sequence during the maturation process. Whether or not signal sequence-binding chaperones are a general prerequisite for all Tat substrate proteins is not known. Here, we have studied the propensity of Tat signal sequences of Escherichia coli to interact with general chaperones and peptidyl-prolyl-cis,trans-isomerases. Site-specific photocross-linking revealed a clear specificity for FK506-binding proteins. Nevertheless transport of the Tat substrate SufI into inverted inner membrane vesicles of E. coli was found to occur in the bona fide absence of any cytosolic chaperone. Our results suggest that in E. coli, cytosolic chaperones are not essential for the twin-arginine-dependent export of cofactor-less substrates.

Multiple roles of TorD-like chaperones in the biogenesis of molybdoenzymes

Biogenesis of prokaryote molybdoenzymes is a complex process leading to the insertion of the molybdenum cofactor in the cytoplasm into a folded apoenzyme before transport through the cell membrane. Usually, specific chaperones belonging to the TorD family are required for the maturation of the molybdoenzymes of the dimethyl sulfoxide reductase family. These chaperones play a crucial role during the biogenesis and the incorporation of the molybdenum cofactor by interacting with the core of the apoprotein. Moreover, they are also involved in the protection of the apoproteins by binding to their N-terminal extremity in an early stage of synthesis. Finally, the TorD-like proteins may possess a proofreading activity and they could target their partners to the twin arginine translocation machinery system for cross-membrane transport of prefolded proteins. The roles of these chaperones during the different steps of molybdoenzyme biogenesis are described.