The archaeal Sec-dependent protein translocation pathway

Over the past three decades, transport of proteins across cellular membranes has been studied extensively in various model systems. One of the major transport routes, the so-called Sec pathway, is conserved in all domains of life. Very little is known about this pathway in the third domain of life, archaea. The core components of the archaeal, bacterial and eucaryal Sec machinery are similar, although the archaeal components appear more closely related to their eucaryal counterparts. Interestingly, the accessory factors of the translocation machinery are similar to bacterial components, which indicates a unique hybrid nature of the archaeal translocase complex. The mechanism of protein translocation in archaea is completely unknown. Based on genomic sequencing data, the most likely system for archaeal protein translocation is similar to the eucaryal co-translational translocation pathway for protein import into the endoplasmic reticulum, in which a protein is pushed across the translocation channel by the ribosome. However, other models can also be envisaged, such as a bacterial-like system in which a protein is translocated post-translationally with the aid of a motor protein analogous to the bacterial ATPase SecA. This review discusses the different models. Furthermore, an overview is given of some of the other components that may be involved in the protein translocation process, such as those required for protein targeting, folding and post-translational modification.     

Inactivation of the elongation factor Tu by mosquitocidal toxin-catalyzed mono-ADP-ribosylation

The mosquitocidal toxin (MTX) produced by Bacillus sphaericus strain SSII-1 is an approximately 97-kDa single-chain toxin which contains a 27-kDa enzyme domain harboring ADP-ribosyltransferase activity and a 70-kDa putative binding domain. Due to cytotoxicity toward bacterial cells, the 27-kDa enzyme fragment cannot be produced in Escherichia coli expression systems. However, a nontoxic 32-kDa N-terminal truncation of MTX can be expressed in E. coli and subsequently cleaved to an active 27-kDa enzyme fragment. In vitro the 27-kDa enzyme fragment of MTX ADP-ribosylated numerous proteins in E. coli lysates, with dominant labeling of an approximately 45-kDa protein. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry combined with peptide mapping identified this protein as the E. coli elongation factor Tu (EF-Tu). ADP ribosylation of purified EF-Tu prevented the formation of the stable ternary EF-Tuaminoacyl-tRNAGTP complex, whereas the binding of GTP to EF-Tu was not altered. The inactivation of EF-Tu by MTX-mediated ADP-ribosylation and the resulting inhibition of bacterial protein synthesis are likely to play important roles in the cytotoxicity of the 27-kDa enzyme fragment of MTX toward E. coli.     

Inactivation of the Elongation Factor Tu by Mosquitocidal Toxin-Catalyzed Mono-ADP-Ribosylation

The mosquitocidal toxin (MTX) produced by Bacillus sphaericus strain SSII-1 is an ~97-kDa single-chain toxin which contains a 27-kDa enzyme domain harboring ADP-ribosyltransferase activity and a 70-kDa putative binding domain. Due to cytotoxicity toward bacterial cells, the 27-kDa enzyme fragment cannot be produced in Escherichia coli expression systems. However, a nontoxic 32-kDa N-terminal truncation of MTX can be expressed in E. coli and subsequently cleaved to an active 27-kDa enzyme fragment. In vitro the 27-kDa enzyme fragment of MTX ADP-ribosylated numerous proteins in E. coli lysates, with dominant labeling of an ~45-kDa protein. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry combined with peptide mapping identified this protein as the E. coli elongation factor Tu (EF-Tu). ADP ribosylation of purified EF-Tu prevented the formation of the stable ternary EF-Tuaminoacyl-tRNAGTP complex, whereas the binding of GTP to EF-Tu was not altered. The inactivation of EF-Tu by MTX-mediated ADP-ribosylation and the resulting inhibition of bacterial protein synthesis are likely to play important roles in the cytotoxicity of the 27-kDa enzyme fragment of MTX toward E. coli.     

Evolution of the prokaryotic protein translocation complex: a comparison of archaeal and bacterial versions of SecDF

Protein translocation across the prokaryotic plasma membrane occurs at the translocon, an evolutionarily conserved membrane-embedded proteinaceous complex. Together with the core components SecYE, prokaryotic translocons also contain auxilliary proteins, such as SecDF. Alignment of bacterial and archaeal SecDF protein sequences reveals the presence of a similar number of homologous regions within each protein. Moreover, the conserved sequence domains in the archaeal proteins are located in similar positions as their bacterial counterparts. When these domains are, however, compared along Bacteria-Archaea lines, a much lower degree of similarity is detected. In Bacteria, SecDF are thought to modulate the membrane association of SecA, the ATPase that provides the driving force for bacterial protein secretion. As no archaeal version of SecA has been detected, the sequence differences reported here may reflect functional differences between bacterial and archaeal SecDF proteins, and by extension, between the bacterial and archaeal protein translocation processes. Moreover, the apparent absence of SecDF in several completed archaeal genomes suggests that differences may exist in the process of protein translocation within the archaeal domain itself.     

Effects of domain exchanges between Escherichia coli and mammalian mitochondrial EF-Tu on interactions with guanine nucleotides, aminoacyl-tRNA and ribosomes.

Escherichia coli elongation factor (EF-Tu) and the corresponding mammalian mitochondrial factor, EF-Tumt, show distinct differences in their affinities for guanine nucleotides and in their interactions with elongation factor Ts (EF-Ts) and mitochondrial tRNAs. To investigate the roles of the three domains of EF-Tu in these differences, six chimeric proteins were prepared in which the three domains were systematically switched. E. coli EF-Tu binds GDP much more tightly than EF-Tumt. This difference does not reside in domain I alone but is regulated by interactions with domains II and III. All the chimeric proteins formed ternary complexes with GTP and aminoacyl-tRNA although some had an increased or decreased activity in this assay. The activity of E. coli EF-Tu but not of EF-Tumt is stimulated by E. coli EF-Ts. The presence of any one of the domains of EF-Tumt in the prokaryotic factor reduced its interaction with E. coli EF-Ts 2-3-fold. In contrast, the presence of any of the three domains of E. coli EF-Tu in EF-Tumt allowed the mitochondrial factor to interact with bacterial EF-Ts. This observation indicates that even domain II which is not in contact with EF-Ts plays an important role in the nucleotide exchange reaction. EF-Tsmt interacts with all of the chimeras produced. However, with the exception of domain III exchanges, it inhibits the activities of the chimeras indicating that it could not be productively released to allow formation of the ternary complex. The unique ability of EF-Tumt to promote binding of mitochondrial Phe-tRNAPhe to the A-site of the ribosome resides in domains I and II. These studies indicate that the interactions of EF-Tu with its ligands is a complex process involving cross-talk between all three domains.     

A membrane-bound archaeal Lon protease displays ATP-independent proteolytic activity towards unfolded proteins and ATP-dependent activity for folded proteins

In contrast to the eucaryal 26S proteasome and the bacterial ATP-dependent proteases, little is known about the energy-dependent proteolysis in members of the third domain, Archae. We cloned a gene homologous to ATP-dependent Lon protease from a hyperthermophilic archaeon and observed the unique properties of the archaeal Lon. Lon from Thermococcus kodakaraensis KOD1 (Lon(Tk)) is a 70-kDa protein with an N-terminal ATPase domain belonging to the AAA(+) superfamily and a C-terminal protease domain including a putative catalytic triad. Interestingly, a secondary structure prediction suggested the presence of two transmembrane helices within the ATPase domain and Western blot analysis using specific antiserum against the recombinant protein clearly indicated that Lon(Tk) was actually a membrane-bound protein. The recombinant Lon(Tk) possessed thermostable ATPase activity and peptide cleavage activity toward fluorogenic peptides with optimum temperatures of 95 and 70 degrees C, respectively. Unlike the enzyme from Escherichia coli, we found that Lon(Tk) showed higher peptide cleavage activity in the absence of ATP than it did in the presence of ATP. When three kinds of proteins with different thermostabilities were examined as substrates, it was found that Lon(Tk) required ATP for degradation of folded proteins, probably due to a chaperone-like function of the ATPase domain, along with ATP hydrolysis. In contrast, Lon(Tk) degraded unfolded proteins in an ATP-independent manner, suggesting a mode of action in Lon(Tk) different from that of its bacterial counterpart.     

The archaeal molecular chaperone machine: peculiarities and paradoxes.

A major finding within the field of archaea and molecular chaperones has been the demonstration that, while some species have the stress (heat-shock) gene hsp70(dnaK), others do not. This gene encodes Hsp70(DnaK), an essential molecular chaperone in bacteria and eukaryotes. Due to the physiological importance and the high degree of conservation of this protein, its absence in archaeal organisms has raised intriguing questions pertaining to the evolution of the chaperone machine as a whole and that of its components in particular, namely, Hsp70(DnaK), Hsp40(DnaJ), and GrpE. Another archaeal paradox is that the proteins coded by these genes are very similar to bacterial homologs, as if the genes had been received via lateral transfer from bacteria, whereas the upstream flanking regions have no bacterial markers, but instead have typical archaeal promoters, which are like those of eukaryotes. Furthermore, the chaperonin system in all archaea studied to the present, including those that possess a bacterial-like chaperone machine, is similar to that of the eukaryotic-cell cytosol. Thus, two chaperoning systems that are designed to interact with a compatible partner, e.g., the bacterial chaperone machine physiologically interacts with the bacterial but not with the eucaryal chaperonins, coexist in archaeal cells in spite of their apparent functional incompatibility. It is difficult to understand how these hybrid characteristics of the archaeal chaperoning system became established and work, if one bears in mind the classical ideas learned from studying bacteria and eukaryotes. No doubt, archaea are intriguing organisms that offer an opportunity to find novel molecules and mechanisms that will, most likely, enhance our understanding of the stress response and the protein folding and refolding processes in the three phylogenetic domains.     

Co- and post-translational processing of the hevein preproprotein of latex of the rubber tree (Hevea brasiliensis)

Hevein is a chitin-binding protein of 43 amino acids found in the lutoid body-enriched fraction of rubber tree latex. A hevein cDNA clone (HEV1) (Broekaert, W., Lee, H.-i., Kush, A., Nam, C.-H., and Raikhel, N. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7633-7637) encodes a putative signal sequence of 17 amino acids followed by a polypeptide of 187 amino acids. Interestingly, this polypeptide has two distinct domains: an amino-terminal domain of 43 amino acids, corresponding to mature hevein, and a carboxyl-terminal domain of 144 amino acids. To investigate the mechanisms involved in processing of the protein encoded by HEV1, three domain-specific antisera were raised against fusion proteins harboring the amino-terminal domain (N domain), carboxyl-terminal domain (C domain), and both domains (NC domain). Translocation experiments using an in vitro translation system show that the first 17-amino acid sequence encoded by the cDNA functions as a signal peptide. Immunoblot analysis of proteins extracted from lutoid bodies demonstrates that a 5-kDa protein comigrated with purified mature hevein and cross-reacted with N domain- and NC domain-specific antibodies. A 14-kDa protein was recognized by C domain- and NC domain-specific antibodies. A 20-kDa protein was cross-reactive with all three antibodies. Microsequencing data further suggest that the 5-kDa (amino-terminal domain) and 14-kDa (carboxyl-terminal domain) proteins are post-translational cleavage products of the 20-kDa polypeptide (both domains) which corresponds to the proprotein encoded by HEV1. In addition, it was found that the amino-terminal domain could provide chitin-binding properties to a fusion protein bearing it either amino terminally or carboxyl terminally.     

A Protein Related to Eucaryal and Bacterial DNA-Motor Proteins in the Hyperthermophilic Archaeon Sulfolobus acidocaldarius

We have isolated a new gene encoding a putative 103-kDa protein from the hyperthermophilic archaeon Sulfolobus acidocaldarius. Analysis of the deduced amino-acid sequence shows an extended central domain, predicted to form coiled-coil structures, and two terminal domains that display purine NTPase motifs. These features are reminiscent of mechanochemical motor proteins which use the energy of ATP hydrolysis to move specific cellular components. Comparative analysis of the amino-acid sequence of the terminal domains and predicted structural organization of this putative purine NTPase show that it is related both to eucaryal proteins from the ``SMC family' involved in the condensation of chromosomes and to several bacterial and eucaryal proteins involved in DNA recombination/repair. Further analyses revealed that these proteins are all members of the so called ``UvrA-related NTP-binding proteins superfamily' and form a large subgroup of motor-like NTPases involved in different DNA processing mechanisms. The presence of such protein in Archaea, Bacteria, and Eucarya suggests an early origin of DNA-motor proteins that could have emerged and diversified by domain shuffling. Received: 29 June 1996 / Accepted: 28 February 1997     

Tracking genetically engineered bacteria: monoclonal antibodies against surface determinants of the soil bacterium Pseudomonas putida 2440.

Assessment of potential risks involved in the release of genetically engineered microorganisms is facilitated by the availability of monoclonal antibodies (MAbs), a tool potentially able to monitor specific organisms. We raised a bank of MAbs against the soil bacterium Pseudomonas putida 2440, which is a host for modified TOL plasmids and other recombinant plasmids. Three MAbs, 7.3B, 7.4D, and 7.5D, were highly specific and recognized only P. putida bacteria. Furthermore, we developed a semiquantitative dot blot assay that allowed us to detect as few as 100 cells per spot. A 40-kDa cell surface protein was the target for MAbs 7.4D and 7.5D. Detection of the cell antigen depended on the bacterial growth phase and culture medium. The O antigen of lipopolysaccharide seems to be the target for MAb 7.3B, and its in vivo detection was independent of the bacterial growth phase and culture medium. MAb 7.3B was used successfully to track P. putida (pWW0) released in unsterile lake mesocosms.     

Isolation of chimaeric forms of elongation factor EF-Tu by affinity chromatography

Six different recombinant chimaeric forms of a three-domain protein, proteosynthetic elongation factor Tu (EF-Tu), composed of domains of EF-Tu of mesophilic (Escherichia coli) and thermophilic (Bacillus stearothermophilus) origin as well as free N-terminal domains of EF-Tu, and the whole recombinant EF-Tus of both organisms were prepared and isolated by the GST (glutathione S-transferase) fusion technology. Several modifications in the standard isolation and purification procedures are described that proved necessary to obtain the proteins in a purified and undegraded form.     

The crystal structure of elongation factor G complexed with GDP, at 2.7 A resolution.

Elongation factor G (EF-G) catalyzes the translocation step of protein synthesis in bacteria, and like the other bacterial elongation factor, EF-Tu--whose structure is already known--it is a member of the GTPase superfamily. We have determined the crystal structure of EF-G--GDP from Thermus thermophilus. It is an elongated molecule whose large, N-terminal domain resembles the G domain of EF-Tu, except for a 90 residue insert, which covers a surface that is involved in nucleotide exchange in EF-Tu and other G proteins. The tertiary structures of the second domains of EF-G and EF-Tu are nearly identical, but the relative placement of the first two domains in EF-G--GDP resembles that seen in EF-Tu--GTP, not EF-Tu--GDP. The remaining three domains of EF-G look like RNA binding domains, and have no counterparts in EF-Tu.     

Identification and Characterization of Sulfated Carbohydrate-Binding Protein from Lactobacillus reuteri

We previously purified a putative sulfated-galactosylceramide (sulfatide)-binding protein with a molecular weight of 47 kDa from the cell surface of Lactobacillus reuteri JCM1081. The aim of this study was to identify the 47-kDa protein, examine its binding to sulfated glycolipids and mucins, and evaluate its role in bacterial adhesion to mucosal surfaces. By cloning and sequencing analysis, the 47-kDa protein was identified as elongation factor-Tu (EF-Tu). Adhesion properties were examined using 6×Histidine-fused EF-Tu (His₆-EF-Tu). Surface plasmon resonance analysis demonstrated pH-dependent binding of His₆-EF-Tu to sulfated glycolipids, but not to neutral or sialylated glycolipids, suggesting that a sulfated galactose residue was responsible for EF-Tu binding. Furthermore, His₆-EF-Tu was found to bind to porcine gastric mucin (PGM) by enzyme-linked immunosorbent assay. Binding was markedly reduced by sulfatase treatment of PGM and in the presence of acidic and desialylated oligosaccharide fractions containing sulfated carbohydrate residues prepared from PGM, demonstrating that sulfated carbohydrate moieties mediated binding. Histochemical staining revealed similar localization of His₆-EF-Tu and high iron diamine staining in porcine mucosa. These results indicated that EF-Tu bound PGM via sulfated carbohydrate moieties. To characterize the contribution of EF-Tu to the interaction between bacterial cells and PGM, we tested whether anti-EF-Tu antibodies could inhibit the interaction. Binding of L. reuteri JCM1081 to PGM was significantly blocked in a concentration-dependent matter, demonstrating the involvement of EF-Tu in bacterial adhesion. In conclusion, the present results demonstrated, for the first time, that EF-Tu bound sulfated carbohydrate moieties of sulfated glycolipids and sulfomucin, thereby promoting adhesion of L. reuteri to mucosal surfaces.     

Monoclonal antibody recognition and function of a DnaK (HSP70) epitope found in gram-negative bacteria.

The isolation and characterization of a monoclonal antibody (MAb 2G5) specific for the bacterial DnaK (HSP70) protein is described. The 2G5 MAb was initially selected because of its ability to bind to DnaK under denaturing conditions. Isotype analyses indicated that 2G5 was an immunoglobulin G2a. Dose-response Western blot (immunoblot) experiments with purified but unconcentrated 2G5 permitted detection of 10 ng of pure DnaK protein. The DnaK epitope was determined by Western blot analysis of a series of truncated DnaK fragments overproduced in Escherichia coli using 5' and 3' dnaK-deleted expression plasmids. The epitope mapped to a 22-amino-acid region spanning DnaK residues 288 and 310. Phylogenetic distribution of the epitope was examined by Western blot analysis of a wide variety of bacterial species and indicated that the epitope was uniquely present in gram-negative organisms. The proximity of the epitope to the presumed DnaK ATP-binding pocket suggested that MAb binding might inhibit DnaK ATPase activity. In vitro analysis supported this prediction and demonstrated that MAb-mediated inhibition of ATPase activity was antibody specific and occurred at stoichiometric molar ratios of MAb to DnaK. Possible mechanisms to explain the ability of the 2G5 MAb to inhibit DnaK activity are discussed.     

Monoclonal antibody specific for Listeria monocytogenes associated with a 66-kilodalton cell surface antigen.

A monoclonal antibody (MAb), EM-7G1, specific for Listeria monocytogenes was developed by using a previously developed MAb, C11E9 (A. K. Bhunia, P. H. Ball, A. T. Fuad, B. W. Kurz, J. W. Emerson, and M. G. Johnson, Infect. Immun. 59:3176-3184, 1991), to mask epitopes shared by L. monocytogenes and Listeria innocua in a 66-kDa cell surface protein. MAb EM-7G1 was an immunoglobulin subclass G1 antibody with kappa light chains. This MAb reacted with all 34 strains of L. monocytogenes tested and showed no cross-reaction with other Listeria spp. or other gram-positive or gram-negative organisms tested by enzyme-linked immunosorbent assay, dot blotting, and colony blotting. A second MAb, EM-6E11, reacted with all Listeria spp. tested but no other bacteria. In a Western blot (immunoblot) assay, EM-7G1 reacted with a crude cell surface protein of 66 kDa with a pI value of 6.7, while EM-6E11 reacted with two protein bands of 43 and 94 to 97 kDa with pI values of 4.0 and 4.3, respectively. Results with trypsin or pronase treatments indicated that the cell antigen reacting with EM-7G1 was on the surface of L. monocytogenes V7 and Scott A cells.     

Biochemical relationship of phosphoenolpyruvate carboxylases (PEPCs) from thermophilic archaea

An archaeal phosphoenolpyruvate carboxylase (PEPC) was purified from an acidophilic extreme thermophile, Sulfolobus acidocaldarius. The native enzyme was a homotetramer of 260±20 kDa molecular mass composed of 60±5 kDa subunits. The enzyme appeared to have a temperature optimum of 90°C and a pH optimum of 8.0. The activity of S. acidocaldarius phosphoenolpyruvate carboxylase was inhibited by l-aspartate and l-malate, but not enhanced by any metabolites. In comparison to the enzymatic and molecular properties of all other phosphoenolpyruvate carboxylases including another archaeal entity from the hyperthermophilic methanogen Methanothermus sociabilis, the archaeal phosphoenolpyruvate carboxylases were quite different from bacterial and eucaryal counterparts, and their small size and the lack of positively allosteric regulation were likely to be peculiar to the enzyme of the domain Archaea.     

An "elongated" translation elongation factor Tu for truncated tRNAs in nematode mitochondria

We have found the gene for a translation elongation factor Tu (EF-Tu) homologue in the genome of the nematode Caenorhabditis elegans. Because the corresponding protein was detected immunologically in a nematode mitochondrial (mt) extract, it could be regarded as a nematode mt EF-Tu. The protein possesses an extension of about 57 amino acids (we call this domain 3') at the C terminus, which is not found in any other known EF-Tu. Because most nematode mt tRNAs lack a T stem, domain 3' may be related to this feature. The nematode EF-Tu bound to nematode T stem-lacking tRNA, but bacterial EF-Tu was unable to do so. A series of domain exchange experiments strongly suggested that domains 3 and 3' are essential for binding to T stem-lacking tRNAs. This finding may constitute a novel example of the co-evolution of a structurally simplified RNA and the cognate RNA-binding protein, the latter having apparently acquired an additional domain to compensate for the lack of a binding site(s) on the RNA.