The surface-associated elongation factor Tu is concealed for antibody binding on viable pneumococci and meningococci

Proteome analyses revealed that elongation factor-Tu (EF-Tu) is associated with cytoplasmic membranes of Gram-positive bacteria and outer membranes of Gram-negative bacteria. It is still debatable whether EF-Tu is located on the external side or the internal side of the membranes. Here, we have generated two new monoclonal antibodies (mAbs) and polyclonal rabbit antibodies against pneumococcal EF-Tu. These antibodies were used to investigate the amount of surface-exposed EF-Tu on viable bacteria using a flow cytometric analysis. The control antibodies recognizing the pneumococcal surface protein A and phosphorylcholine showed a significant binding to viable pneumococci. In contrast, anti-EF-Tu antibodies did not recognize pneumococcal EF-Tu. However, heat killing of pneumococci lacking capsular polysaccharides resulted in specific antibody binding to EF-Tu and, moreover, increased the exposure of recognized phosphorylcholine epitopes. Similarly, our EF-Tu-specific antibodies did not recognize EF-Tu of viable Neisseria meningitidis. However, pretreatment of meningococci with ethanol resulted in specific antibody binding to EF-Tu on outer membranes. Importantly, these treatments did not destroy the membrane integrity as analysed with control mAbs directed against cytoplasmic proteins. In conclusion, our flow cytrometric assays emphasize the importance of using viable bacteria and not heat-killed or ethanol-treated bacteria for surface-localization experiments of proteins, because these treatments modulate the cytoplasmic and outer membranes of bacteria and the binding results may not reflect the situation under physiological conditions.     

Uncoupler-induced relocation of elongation factor Tu to the outer membrane in an uncoupler-resistant mutant of Escherichia coli

Escherichia coli UV6, a mutant which is resistant to the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP), when grown in the presence of CCCP, but not in its absence, incorporated a new protein (Mr, 42 000) into the cell envelope. This protein was found in both cytoplasmic and outer-membrane fractions. In the outer membrane it was one of three or four most abundant proteins. The protein was tightly bound to the membranes and was not solubilized by several detergents. Solubilization was achieved with sodium lauroylsarcosinate (sarkosyl). The protein was purified close to homogeneity by affinity chromatography on a column of GDP-Sepharose. It was identified as elongation factor Tu (EF-Tu) on the basis of electrophoretic mobility, profiles of peptide fragments produced by proteolysis, and by its ability to bind to GDP-Sepharose. Disruption of cells in the presence of CCCP or incubation of envelopes with EF-Tu did not result in incorporation of EF-Tu into the membranes. It is suggested that this protein is incorporated into the outer membrane as a consequence of an alteration in the normal protein biosynthetic mechanisms of the mutant induced by the presence of CCCP.     

Immunocytochemical localization of the elongation factor Tu in E. coli cells

The localization of the elongation factor Tu (EF-Tu) in ultrathin cryosections of E. coli cells was determined with the electron microscope using a highly specific immunological labelling technique. EF-Tu is distributed almost homogeneously throughout the cytoplasm. Although it has often been suggested that EF-Tu could be part of a putative prokaryotic cytoskeleton, we did not find any evidence for supramolecular assemblies, such as fibres or filaments, containing a large amount of EF-Tu. EF-Tu was not observed in association with the outer cell membrane and periplasmic space. A topological relationship with the inner membrane is not apparent in our micrographs. In cells in which the EF-Tu level is raised significantly, the protein piles up in discrete cell regions.     

Yeast two-hybrid screening identifies binding partners of human Tom34 that have ATPase activity and form a complex with Tom34 in the cytosol

In the accompany paper (Mukhopadhyay, A., Avramova, L. V. and Weiner, H., Arch. Biochem. Biophys.), it was shown that Tom34, a previously proposed putative translocase of the mitochondrial outer membrane, binds to the mature region of a precursor protein and appears to be a cytosol protein. Here Tom34 was used as bait in a yeast two-hybrid screening to search for its potential binding partners. Two of the identified proteins were the ATPase-related valosin-containing protein (VCP) and the lysosomal H(+)-transporting ATPase member M (ATP6M). Tom34 was found primarily in the cytosol while VCP and ATP6M were found in the cytosol as well as in nonmitochondrial organelles. Tom34 formed a approximately 400-kDa complex with them in the cytosol. Tom34 was found to possess a weak ATPase activity that did not change when associated with VCP. The tetratricopeptide repeat (TPR) motif region of Tom34 (residue 201-256) was responsible for binding to the other proteins. Tom34 appears not to be a member of the mitochondrial outer membrane translocase family but might function as a chaperone-like protein during protein translocation.     

Precursor for elongation factor Tu from Escherichia coli

The tufA gene, one of two genes in Escherichia coli encoding elongation factor Tu (EF-Tu), was cloned into a ColE1-derived plasmid downstream of the lac promoter-operator. In cells carrying this plasmid, the synthesis of EF-Tu was increased four- to fivefold upon the addition of isopropyl-beta-D-thiogalactopyranoside (an inducer of the lac promoter). This condition led to the synthesis of a novel protein, called pTu, which comigrated with EF-Tu on a sodium dodecyl sulfate-polyacrylamide gel but could be separated on an isoelectric focusing gel, since pTu is slightly more basic than EF-Tu. The synthesis of pTu could also be induced by the synthesis of a hybrid protein containing just the amino-terminal half of the EF-Tu protein. Genetic data suggest that pTu is the product of the tufA and tufB genes. The pTu protein was shown to be related to EF-Tu by gel electrophoresis of tryptic peptides. Pulse-chase experiments suggest that pTu is a precursor of EF-Tu. Interestingly, in a classic membrane fractionation procedure, EF-Tu was found in the cytosolic fraction, whereas pTu was partitioned with the outer membrane.     

Mgr2 promotes coupling of the mitochondrial presequence translocase to partner complexes

Many mitochondrial proteins are synthesized with N-terminal presequences in the cytosol. The presequence translocase of the inner mitochondrial membrane (TIM23) translocates preproteins into and across the membrane and associates with the matrix-localized import motor. The TIM23 complex consists of three core components and Tim21, which interacts with the translocase of the outer membrane (TOM) and the respiratory chain. We have identified a new subunit of the TIM23 complex, the inner membrane protein Mgr2. Mitochondria lacking Mgr2 were deficient in the Tim21-containing sorting form of the TIM23 complex. Mgr2 was required for binding of Tim21 to TIM23(CORE), revealing a binding chain of TIM23(CORE)-Mgr2/Tim21-respiratory chain. Mgr2-deficient yeast cells were defective in growth at elevated temperature, and the mitochondria were impaired in TOM-TIM23 coupling and the import of presequence-carrying preproteins. We conclude that Mgr2 is a coupling factor of the presequence translocase crucial for cell growth at elevated temperature and for efficient protein import.     

A 70-kd protein of the yeast mitochondrial outer membrane is targeted and anchored via its extreme amino terminus

The major 70-kd protein of the yeast mitochondrial outer membrane is made on cytosolic ribosomes and imported into the outer membrane without proteolytic cleavage. We have attempted to identify the sequences which target the protein to the mitochondria and which permanently anchor it to the lipid bilayer of the outer membrane. By manipulating the cloned gene we have deleted 13 different regions throughout the polypeptide; in addition, we have fused amino-terminal regions of different length to beta-galactosidase. Each altered gene was introduced into yeast and the intracellular fate of the corresponding polypeptide product was determined by subcellular fractionation. All the information for targeting and anchoring the 70-kd protein (617 amino acids) was contained within the amino-terminal 41 amino acids. When this entire region was deleted, the protein was recovered with the cytosol fraction. However, several restricted deletions within this amino-terminal region appeared to affect targeting and anchoring differentially: most of the altered protein remained in the cytosol but a small fraction was misrouted into the mitochondrial matrix space. We suggest that targeting is mediated by a region which includes the 11 amino-terminal amino acids whereas the permanent membrane anchor is provided by a typical transmembrane sequence between residues 9 and 38.     

New Insights into the Protein Import Machinery of the Chloroplast's Outer Envelope

A large number of plastid localized proteins are post-translationally imported as precursor proteins from the cytosol into the organelle. Recognition and translocation is accomplished by a subset of chloroplast envelope proteins, which were identified by different but complementary methods. The o uter e nvelope p roteins OEP 86, OEP 75, OEP 70 (a heat shock cognate 70 homologue) and OEP 34 are clearly involved in the import event and can be isolated as one functionally active translocation unit. For three of these proteins cDNA clones have been very recently obtained, namely OEP 86, OEP 75 and OEP 34. OEP 86 seems to be a precursor protein receptor which could be regulated by GTP binding and ATP-dependent phosphorylation-dephosphorylation. OEP 75 is part of the translocation pore traversing the membrane in multiple β-sheets. OEP 34 is tightly associated with OEP 75. It represents a new type of GTP-binding protein which possesses endogenous GTPase activity. Multiple GTP binding and hydrolysis cycles as well as protein phosphorylation-dephosphorylation events might, therefore, regulate the interaction of a precursor protein with the translocation machinery of the outer envelope, making it very distinct from the mitochondrial outer membrane system. Further proteins of the inner envelope membrane, namely IEP 97 and IEP 36, have been implied to function in the translocation event. These recent data allow not only identification of the players in the game but also speculation about mechanisms and regulation of translocation.     

Functional definition of outer membrane proteins involved in preprotein import into mitochondria

The role of plant mitochondrial outer membrane proteins in the process of preprotein import was investigated, as some of the principal components characterized in yeast have been shown to be absent or evolutionarily distinct in plants. Three outer membrane proteins of Arabidopsis thaliana mitochondria were studied: TOM20 (translocase of the outer mitochondrial membrane), METAXIN, and mtOM64 (outer mitochondrial membrane protein of 64 kD). A single functional Arabidopsis TOM20 gene is sufficient to produce a normal multisubunit translocase of the outer membrane complex. Simultaneous inactivation of two of the three TOM20 genes changed the rate of import for some precursor proteins, revealing limited isoform subfunctionalization. Inactivation of all three TOM20 genes resulted in severely reduced rates of import for some but not all precursor proteins. The outer membrane protein METAXIN was characterized to play a role in the import of mitochondrial precursor proteins and likely plays a role in the assembly of beta-barrel proteins into the outer membrane. An outer mitochondrial membrane protein of 64 kD (mtOM64) with high sequence similarity to a chloroplast import receptor was shown to interact with a variety of precursor proteins. All three proteins have domains exposed to the cytosol and interacted with a variety of precursor proteins, as determined by pull-down and yeast two-hybrid interaction assays. Furthermore, inactivation of one resulted in protein abundance changes in the others, suggesting functional redundancy. Thus, it is proposed that all three components directly interact with precursor proteins to participate in early stages of mitochondrial protein import.     

Specificity of elongation factor EF-TU for hydrophobic peptides

The elongation factor EF-Tu carries aminoacyl-tRNAs to the A-site of the ribosome during the elongation process of protein biosynthesis. We, and others, have recently reported that the Escherichia coli EF-Tu interacts with unfolded and denatured proteins and behaves like a chaperone in protein folding and protection against protein thermal denaturation. In this study, we have identified EF-Tu binding sites in protein substrates by screening cellulose-bound peptides scanning the sequences of several proteins. The binding motifs recognized by EF-Tu in protein substrates are also recognized by the chaperone DnaK and mainly consist of hydrophobic clusters. EF-Tu interacts as efficiently as DnaK with the membrane spanning sequence of the membrane protein phospholemman and with the signal sequence of alkaline phosphatase. It interacts less efficiently with several other hydrophobic clusters of lysozyme and alkaline phosphatase, which are also DnaK substrates and fails to bind to several DnaK binding sites. Our results suggest that EF-Tu, like DnaK, interacts albeit more weakly with the hydrophobic regions of substrate protein and are consistent with the hypothesis that it possesses chaperone properties.     

Overproduction of Pex5p stimulates import of alcohol oxidase and dihydroxyacetone synthase in a Hansenula polymorpha Pex14 null mutant

Hansenula polymorpha Deltapex14 cells are affected in peroxisomal matrix protein import and lack normal peroxisomes. Instead, they contain peroxisomal membrane remnants, which harbor a very small amount of the major peroxisomal matrix enzymes alcohol oxidase (AO) and dihydroxyacetone synthase (DHAS). The bulk of these proteins is, however, mislocated in the cytosol. Here, we show that in Deltapex14 cells overproduction of the PTS1 receptor, Pex5p, leads to enhanced import of the PTS1 proteins AO and DHAS but not of the PTS2 protein amine oxidase. The import of the PTS1 protein catalase (CAT) was not stimulated by Pex5p overproduction. The difference in import behavior of AO and CAT was not related to their PTS1, since green fluorescent protein fused to the PTS1 of either AO or CAT were both not imported in Deltapex14 cells overproducing Pex5p. When produced in a wild type control strain, both proteins were normally imported into peroxisomes. In Deltapex14 cells overproducing Pex5p, Pex5p had a dual location and was localized in the cytosol and bound to the outer surface of the peroxisomal membrane. Our results indicate that binding of Pex5p to the peroxisomal membrane and import of certain PTS1 proteins can proceed in the absence of Pex14p.     

A novel outer membrane lipoprotein, LolB (HemM), involved in the LolA (p20)-dependent localization of lipoproteins to the outer membrane of Escherichia coli.

The Escherichia coli major outer membrane lipoprotein (Lpp) is released from the inner membrane into the periplasm as a complex with a carrier protein, LolA (p20), and is then specifically incorporated into the outer membrane. An outer membrane protein playing a critical role in Lpp incorporation was identified, and partial amino acid sequences of the protein, named LolB, were identical to those of HemM, which has been suggested to play a role in 5-aminolevulinic acid synthesis in the cytosol. In contrast to this suggested role, the deduced amino acid sequence of HemM implied that the gene encodes a novel outer membrane lipoprotein. Indeed, an antibody raised against highly purified LolB revealed its outer membrane localization, and inhibited in vitro Lpp incorporation into the outer membrane. Furthermore, LolB was found to be synthesized as a precursor with a signal sequence and then processed to a lipid-modified mature form. An E.coli strain possessing chromosomal hemM under the control of the lac promoter-operator required IPTG for growth, indicating that hemM (lolB) is an essential gene. Outer membrane prepared from LolB-depleted cells did not incorporate Lpp. When the Lpp-LolA complex was incubated with a water-soluble LolB derivative, Lpp was transferred from LolA to LolB. Based on these results, the outer membrane localization pathway for E.coli lipoprotein is discussed with respect to the functions of LolA and LolB.     

Photoaffinity labeling of three renal cyclic 3',5'-adenosine monophosphate-binding proteins.

Evidence is presented for the presence of multiple cyclic AMP binding components in the plasma membrane and cytosol fractions of porcine renal cortex and medulla. N6-(Ethyl-2-diazomalonyl)-3',5'-adenosine monophosphate, a photoaffinity label for cyclic AMP binding sites, exhibits non-covalent binding characteristics similar to cyclic AMP in membrane and soluble fractions. Binding data for either compound to the plasma membrane fraction yields biphasic Scatchard plots while triphasic plots are obtained with the dialyzed cytosol. When covalently labeled fractions are separated on SDS-polyacrylamide gel electrophoresis, the cyclic AMP photoaffinity label is found on 49 000 and 130 000 dalton components in each kidney fraction. DEAE-cellulose and gel filtration chromatography of the labeled cortical cytosol fraction establishes that the three components suggested by the binding data correspond to two 49 000 dalton species and a 130 000 component. The 49 000 species have higher affinities for cyclic AMP than the 130 000 component (Ka(1) = 2.0 . 10(9), Ka(2) = 1.7 . 10(8), Ka(3) = 1.0 . 10(7)). The 49 000 components are associated with protein kinase activity while the 130 000 component does not exhibit protein kinase, adenosine deaminase, or cyclic nucleotide phosphodiesterase activity. Immunologic results and effects of phosphorylation and cyclic GMP on cyclic AMP binding further suggest that the 49 000 components are regulatory subunits of cyclic AMP-dependent protein kinases. Cyclic AMP binding to the 130 000 component is markedly inhibited by adenosine and adenine nucleotides, but not cyclic GMP. Thus, this component may reflect an aspect of adenosine control or metabolism which may or may not be a cyclic AMP-related cellular function.     

Localization and characterization of three different beta-adrenergic receptors expressed in Escherichia coli

After fusion with the N-proximal portion of the outer membrane protein LamB, three beta-adrenergic receptors, the human beta 1- and beta 2- and turkey beta 1-adrenergic receptor, were expressed in Escherichia coli with retention of their own specific pharmacological properties. Molecular characterization and localization of the three receptors in bacteria and comparison of the behaviour of each hybrid protein are reported. The bacteria were lysed and fractionated on a sucrose gradient. Saturable [125I]iodocyanopindolol binding activity was found associated mainly with the inner membrane fraction, suggesting that the receptor is correctly folded in this membrane. Binding activity was also found in the outer membrane fraction but varied according to the receptor type. Photoaffinity labeling experiments revealed that the receptors exhibit binding activity only after proteolytic removal of the LamB moiety from the fusion protein. The three hybrid proteins, detected in immunoblots by anti-peptide antibodies, were found mainly in the outer membrane fraction. Each of them exhibited different susceptibility to intrinsic bacterial proteolytic enzymes; sites of proteolytic cleavage were localized by the use of anti-peptide antibodies. The functional expression in E. coli of three beta-adrenergic receptors with similar structure but different amino acid sequences suggests that this expression system may be a general feature among similar receptors of the family of G-protein-coupled receptors. The level of expressed binding activity of a given receptor will be within the control of proteolytic degradation processes, depending on the primary sequence of the receptor. Constructions of new hybrid proteins, in combination with expression in protease mutants of E. coli, should help in controlling such processes.     

The affinity of elongation factor Tu for an aminoacyl-tRNA is modulated by the esterified amino acid

When different mutations were introduced into the anticodon loop and at position 73 of YFA2, a derivative of yeast tRNA(Phe), a single tRNA body was misacylated with 13 different amino acids. The affinities of these misacylated tRNAs for Thermus thermophilus elongation factor Tu (EF-Tu).GTP were determined using a ribonuclease protection assay. A range of 2.5 kcal/mol in the binding energies was observed, clearly demonstrating that EF-Tu specifically recognizes the side chain of the esterified amino acid. Furthermore, this specificity can be altered by introducing a mutation in the amino acid binding pocket on the surface of EF-Tu. Also, when discussed in conjunction with the previously determined specificity of EF-Tu for the tRNA body, these experiments further demonstrate that EF-Tu uses thermodynamic compensation to bind cognate aminoacyl-tRNAs similarly.     

Mitochondrial import receptors for precursor proteins.

The specific targeting of precursor proteins synthesized in the cytosol to various cell organelles is a central aspect of intracellular protein traffic. Several hundred different proteins are imported from the cytosol into the mitochondria. Recent studies have identified the mitochondrial outer membrane proteins MOM19, MOM72, MOM38 (approximately ISP42) and p32 which have a role in initial steps of protein import. The first three components are present in a multi-subunit complex that catalyses recognition and membrane insertion of precursor proteins.     

The First Steps of Protein Import into Mitochondria

Protein import into mitochondria is initiated by the recognition and binding of precursor proteins by import components in the cytosol, on the mitochondrial surface, and in the mitochondrial outer membrane. Following their synthesis on cytoplasmic ribosomes, some precursor proteins interact with molecular chaperones in the cytosol which function in maintaining the precursor protein in an import-competent state and may also aid in the delivery of the precursor to the mitochondria. A multisubunit protein import receptor then recognises and binds precursor proteins before feeding them into the outer membrane import site. Some proteins are sorted from the import site into the outer membrane, but most precursor proteins travel through the outer membrane import site into the mitochondria, where the later steps of protein import take place.     

Lipid fluidity-dependent biosynthesis and assembly of the outer membrane proteins of E. coli.

The reduction of the membrane lipids of E. coli to a nonfluid state resulted in the accumulation in the cell envelope of a high molecular weight precursor of the protoIG protein, a major outer membrane protein. The protoIG protein was as sensitive to trypsin as the mature toIG protein assembled in the outer membrane. In contrast to the toIG protein, however, the accumulated protoIG protein was easily released from the envelope fraction by both sodium lauryl sarcosinate extraction and sonication. This indicated that the precursor protein was loosely associated with the cell membrane. When a fluid lipid state was restored, the protoIG protein was processed to the mature form which was then correctly assembled in the outer membrane. These results suggest that the protoIG protein produced under nonfluid lipid conditions was properly translocated across the cytoplasmic membrane, but could not be assembled in the outer membrane due either to the reversible inhibition of the processing of the ProtoIG to the toIG protein or to the lack of interaction with a specific outer membrane component(s). Reduced lipid fluidity also caused various alterations in the biosynthesis and assembly of other membrane proteins. In addition to the toIG protein, a large number of new proteins were accumulated in the membrane. Alternatively, the matrix protein as well as the promatrix protein were not detected in the cell envelope. On the other hand, the lipoprotein was normally produced, processed, modified and assembled in the outer membrane. These results indicate that the outer membrane proteins are synthesized and assembled according to several different mechanisms, on which the physical state of the membrane has various effects.