Bacterial Protein Translocation Research Articles

Transport of cytochrome c derivatives by the bacterial Tat protein translocation system.

An experimental system developed previously for the heterologous expression of c-type cytochromes in Escherichia coli Q1has been adapted to monitor protein transfer across the bacteria's cytoplasmic membrane. Apocytochrome, lacking the haem cofactor and probably in an unfolded state, was readily transferred across the cytoplasmic membrane when fused to a Sec-specific signal peptide. Furthermore, cytochrome fused to a signal peptide regarded as specific for the twin arginine transport (Tat) system was translocated in an unfolded state by the Sec apparatus. After maturation and folding in the cytoplasm, Tat-mediated transfer of holocytochrome to the periplasm occurred. We conclude that, in addition to the nature of the specific signal peptide, the folding state of a particular protein also governs its acceptance by a given transport system.

Sec, drugs and rock’n’roll: antibiotic targeting of bacterial protein translocation

A large number of bacterial proteins are active in extracytoplasmic locations. Targeting and membrane translocation of the vast majority of these secretory and membrane polypeptides is mediated by the Sec pathway. Protein secretion requires the co-ordinated and sequential action of targeting factors on the cis-side of the membrane, a complex membrane-embedded protein translocase and maturation enzymes on the trans-side. Recently, significant advances in the molecular genetics and biochemistry of the Sec pathway have revealed that several of the Sec pathway components are essential for bacterial viability and/or pathogenicity. Moreover, several biochemical assays and structural insights have become available. Importantly, some of the Sec components are unique to bacteria. These developments raise the possibility that the bacterial protein translocase and other Sec pathway components could become formidable targets for antibacterial drug discovery.

Specificity of signal peptide recognition in tat-dependent bacterial protein translocation.

The bacterial twin arginine translocation (Tat) pathway translocates across the cytoplasmic membrane folded proteins which, in most cases, contain a tightly bound cofactor. Specific amino-terminal signal peptides that exhibit a conserved amino acid consensus motif, S/T-R-R-X-F-L-K, direct these proteins to the Tat translocon. The glucose-fructose oxidoreductase (GFOR) of Zymomonas mobilis is a periplasmic enzyme with tightly bound NADP as a cofactor. It is synthesized as a cytoplasmic precursor with an amino-terminal signal peptide that shows all of the characteristics of a typical twin arginine signal peptide. However, GFOR is not exported to the periplasm when expressed in the heterologous host Escherichia coli, and enzymatically active pre-GFOR is found in the cytoplasm. A precise replacement of the pre-GFOR signal peptide by an authentic E. coli Tat signal peptide, which is derived from pre-trimethylamine N-oxide (TMAO) reductase (TorA), allowed export of GFOR, together with its bound cofactor, to the E. coli periplasm. This export was inhibited by carbonyl cyanide m-chlorophenylhydrazone, but not by sodium azide, and was blocked in E. coli tatC and tatAE mutant strains, showing that membrane translocation of the TorA-GFOR fusion protein occurred via the Tat pathway and not via the Sec pathway. Furthermore, tight cofactor binding (and therefore correct folding) was found to be a prerequisite for proper translocation of the fusion protein. These results strongly suggest that Tat signal peptides are not universally recognized by different Tat translocases, implying that the signal peptides of Tat-dependent precursor proteins are optimally adapted only to their cognate export apparatus. Such a situation is in marked contrast to the situation that is known to exist for Sec-dependent protein translocation.

Archaeal protein translocation crossing membranes in the third domain of life.

Proper cell function relies on correct protein localization. As a first step in the delivery of extracytoplasmic proteins to their ultimate destinations, the hydrophobic barrier presented by lipid-based membranes must be overcome. In contrast to the well-defined bacterial and eukaryotic protein translocation systems, little is known about how proteins cross the membranes of archaea, the third and most recently described domain of life. In bacteria and eukaryotes, protein translocation occurs at proteinaceous sites comprised of evolutionarily conserved core components acting in concert with other, domain-specific elements. Examination of available archaeal genomes as well as cloning of individual genes from other archaeal strains reveals the presence of homologues to selected elements of the bacterial or eukaryotic translocation machines. Archaeal genomic searches, however, also reveal an apparent absence of other, important components of these two systems. Archaeal translocation may therefore represent a hybrid of the bacterial and eukaryotic models yet may also rely on components or themes particular to this domain of life. Indeed, considering the unique chemical composition of the archaeal membrane as well as the extreme conditions in which archaea thrive, the involvement of archaeal-specific translocation elements could be expected. Thus, understanding archaeal protein translocation could reveal the universal nature of certain features of protein translocation which, in some cases, may not be readily obvious from current comparisons of bacterial and eukaryotic systems. Alternatively, elucidation of archaeal translocation could uncover facets of the translocation process either not yet identified in bacteria or eukaryotes, or which are unique to archaea. In the following, the current status of our understanding of protein translocation in archaea is reviewed.

Translocation of Helicobacter pylori CagA into gastric epithelial cells by type IV secretion.

The Gram-negative bacterium Helicobacter pylori is a causative agent of gastritis and peptic ulcer disease in humans. Strains producing the CagA antigen (cagA(+)) induce strong gastric inflammation and are strongly associated with gastric adenocarcinoma and MALT lymphoma. We show here that such strains translocate the bacterial protein CagA into gastric epithelial cells by a type IV secretion system, encoded by the cag pathogenicity island. CagA is tyrosine-phosphorylated and induces changes in the tyrosine phosphorylation state of distinct cellular proteins. Modulation of host cells by bacterial protein translocation adds a new dimension to the chronic Helicobacter infection with yet unknown consequences.

Improving protein secretion by engineering components of the bacterial translocation machinery.

The increased insight into the mechanism of bacterial protein translocation has resulted in new concepts for the production of heterologous proteins. The periplasm of gram-negative bacteria is revealed to have a role as a 'protein construction compartment', which can be used to fold complex proteins. Passage across the outer membrane, however, remains a challenge due to the high selectivity of the outer membrane translocase. In gram-positive bacteria, slow folding at the membrane-cell-wall interface can make heterologous proteins vulnerable to degradation by wall-associated proteases. The recent identification of thiol-disulfide oxidoreductases in Bacillus subtilis might open the possibility of secreting proteins containing multiple disulfide bonds from this host.

A Component of the Chloroplast Protein Import Apparatus Functions in Bacteria

Toc36 is a family of 44-kDa envelope polypeptides previously identified as components of the chloroplast protein import apparatus by virtue of their close physical proximity to translocating proteins. An indication of their function thus remains at large. A heterologous in vivo approach for studying the function of Toc36 was developed in this study by introducing a member of Toc36 into E. coli to assess its effect on bacterial protein translocation. The presence of Toc36 enhances the translocation of two bacterial periplasmic proteins in a manner resembling the chloroplast system. Translocation of the two bacterial periplasmic proteins was less sensitive to sodium azide, resembling more the azide-insensitive nature of the chloroplast protein import process. Mutated Toc36 proteins were not capable of causing the same effect as that observed for unaltered Toc36. Toc36 was also capable of complementing bacterial strains with temperature-sensitive secA mutations that affected protein translocation. The combined results provide evidence that Toc36 plays a central role in the chloroplast protein translocation process.

SecA homolog in protein transport within chloroplasts: evidence for endosymbiont-derived sorting.

The SecA protein is an essential, azide-sensitive component of the bacterial protein translocation machinery. A SecA protein homolog (CPSecA) now identified in pea chloroplasts was purified to homogeneity. CPSecA supported protein transport into thylakoids, the chloroplast internal membrane network, in an azide-sensitive fashion. Only one of three pathways for protein transport into thylakoids uses the CPSecA mechanism. The use of a bacteria-homologous mechanism in intrachloroplast protein transport provides evidence for conservative sorting of proteins within chloroplasts.

A point mutation converts Escherichia coli FtsZ septation GTPase to an ATPase.

The cell division protein FtsZ, essential to initiate septum formation in Escherichia coli, is a GTPase. The thermosensitive ftsZ84 mutation, which impairs the ability of FtsZ to bind and hydrolyze GTP in vitro, maps to a short glycine-rich FtsZ segment. This region is conserved in eubacterial FtsZ homologs and is strikingly similar to the proposed GTP binding motif in the eukaryotic cytoskeletal protein tubulin. Here we show that in contrast to FtsZ, FtsZ84 protein has a Mg(2+)-dependent ATPase activity in vitro. This activity, unlike the wild-type GTPase, is specifically inhibited by sodium azide, a known antagonist of F-type ATPases and the bacterial SecA protein translocation ATPase (Oliver, D., Cabelli, R. J., Dolan, K. M., and Jarosik, G. P. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 8227-8231). Conversely, aluminum fluoride abolishes FtsZ GTPase activity but only partially affects FtsZ84 ATPase. Affinity-purified anti-FtsZ antibody blocks FtsZ84 ATPase activity, indicating that this enzymatic function is intrinsic to the mutant protein. This is, to our knowledge, the first example of a missense mutation that converts a GTPase to an ATPase.

Bacterial protein translocation: kinetic and thermodynamic role of ATP and the protonmotive force.

The energetic mechanism of preprotein export in Escherichia coli has been a source of controversy for many years. In vitro studies of translocation reactions that use purified soluble and membrane components have not clarified the main features of this mechanism. Translocation occurs through consecutive steps which each have distinct energy requirements. Initiation of translocation requires ATP and the SecA protein. Most of the further steps can be driven by the protonmotive force (delta p).

Protein targeting and secretion

Part 1 General principles: subcellular principles targeting sequences and patches cytoplasmic synthesis of proteins the pathways that proteins take through cells ways of identifying targeting sequences evolutionary relationships in targeting the role of targeting sequences in protein folding the role of chaperone proteins. Part 2 Bacterial protein translocation: experimental approaches for determining protein localization the structure of prokaryotic signal peptides the membrane trigger hypothesis and M13 coat protein components of the export machinery translocation from the inner to the outer membrane the novel secretion of haemolysin from Escherichia coli. Part 3 Protein translocation at the rough endoplasmic reticulum: historical background the use of in vitro systems for studying protein translocation structure and function of signal peptides proteins involved in eukaryotic translocation endoplasmic reticulum of yeast. Part 4 Assembly of membrane proteins: mode of attachment of proteins to membranes topography of integral membrane proteins mechanisms of assembly hydrophobic modifications of proteins. Part 5 Protein sorting and motivation: regulated and bulk flow export form the endoplasmic reticulum glycosylation budding and fusion of transport vesicles sorting at the trans-Golgi network endocytosis sorting in polarized cells alternative pathways of secretion. Part 6 Import of proteins from the cytosol: mitochondria chloroplasts peroxisomes the nucleus.

Eukaryotic and prokaryotic signal peptides direct secretion of a bacterial endoglucanase by mammalian cells.

It is well established that hydrophobic signal sequences direct proteins into or across the endoplasmic reticulum membrane in eukaryotes and cell membranes in prokaryotes. Although it is recognized that eukaryote proteins are efficiently secreted by bacterial systems, the export of bacterial proteins by eukaryotes has received little attention. To investigate membrane translocation of bacterial proteins by mammalian cells, the secretion of a bacterial endoglucanase (endoglucanase E) from stably transfected Chinese hamster ovary cells has been examined. We report that a functional endoglucanase is secreted when fused to prokaryote or eukaryote signal peptides. Furthermore, the endoglucanase was post-translationally modified before secretion. Data presented in this paper suggest that secretion of bacterial proteins by eukaryote cells may be a general phenomenon and infer that there are no specific requirements with respect to the origin of the signal sequences.

In vitro translocation of bacterial secretory proteins and energy requirements

The recent establishment of in vitro assay systems has made biochemical studies on the process of membrane translocation of secretory proteins possible. This review summarizes what we have learned, using these in vitro systems, concerning the biochemical process of protein translocation, with special reference to energy requirements. Both ATP and the protonmotive force participate in the translocation reaction. The requirement of ATP is obligatory, whereas that of the protonmotive force differs, in terms of its level, with the secretory protein species. The possible roles of ATP and the protonmotive force in protein translocation are discussed with special reference to the function of SecA, an essential component of the secretory machinery. The effect of positive charges, which precede or follow the hydrophobic domain of signal peptides, on translocation is also discussed.

ProOmpA spontaneously folds in a membrane assembly competent state which trigger factor stabilizes.

The precursor protein proOmpA can translocate across purified Escherichia coli inner membrane vesicles in the absence of any other soluble proteins. ProOmpA, purified 2000-fold in the presence of 8 M urea, is competent for translocation following rapid renaturation via dilution. ATP, the transmembrane electrochemical potential, and functional secY protein are essential for the translocation of proOmpA renatured by dilution. The kinetics of its translocation and the level of translocation at each concentration of ATP are indistinguishable from that of proOmpA renatured by dialysis with trigger factor. After dilution, the proOmpA rapidly loses its competence for membrane assembly. However, this competence is stabilized by trigger factor. Assembly-competent proOmpA is in a protease-sensitive conformation, whereas proOmpA which has lost this competence is more resistant to degradation. This suggests that the primary role for trigger factor in in vitro protein translocation is to maintain precursor proteins in a translocation-competent conformation. We propose that a properly folded precursor protein and ATP are the only soluble components which are essential for bacterial protein translocation.

A bacterial secretory protein requires signal recognition particle for translocation across mammalian endoplasmic reticulum.

In vitro transcription of DNA from plasmid pBR322 was coupled to cell-free translation in a wheat germ system. The major translation product was pre-beta-lactamase. Upon addition of dog pancreas microsomes, the precursor was processed to authentic beta-lactamase as shown by partial NH2-terminal sequence analysis. Processing was linked to translocation into the microsomal vesicles. Salt-extracted microsomes did not process pre-beta lactamase but could be reactivated by purified signal recognition particle, which is the functional component of the salt wash (Walter, P., and Blobel, G. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 7112-7116). Signal recognition particle alone caused a drastic translation arrest that could be released by salt-depleted membranes. These data are consistent with those obtained for eukaryotic proteins and suggest that co-translational translocation of both bacterial and eukaryotic secretory proteins across the endoplasmic reticulum require identical components.