Translocation of nascent non-signal sequence protein in heated Escherichia coli

Exposure of Escherichia coli to heat resulted in 1) selective inhibition of protein synthesis, 2) synthesis of heat shock proteins, and 3) altered subcellular distribution of newly synthesized proteins. Either 5 min or 1 h at 48 degrees C increases outer membrane proteins of Coomassie Blue-stained gels. After 1 h, there was a loss of stained proteins from the soluble fraction. Much greater changes in the distribution of radiolabeled (newly synthesized) proteins were observed, with marked increases in the number of outer membrane protein species and a corresponding loss of soluble fraction proteins. Three major species of radiolabeled proteins from heat-treated cells remain in the soluble fraction; these proteins have apparent Mr 56,000, 69,200, and 79,400. Cells were labeled with L-[35S] methionine at either 37 or 48 degrees C and chased with non-radiolabeled methionine before a temperature shift to either 48 or 37 degrees C, respectively. Only proteins synthesized at elevated temperature participated in translocation. It is suggested that heat disordering of membrane lipids promotes interlipidic connections between the inner and outer membrane providing pathways for protein movement to the outer membrane and may be the mechanism whereby a cell quickly responds to environmental temperature stress. The response does not require but may trigger synthesis of mRNA.     

Influence of tat mutations on the ribose-binding protein translocation in Escherichia coli

Proteins are exported across the bacterial cytoplasmic membrane either as unfolded precursors via the Sec machinery or in folded conformation via the Tat system. The ribose-binding protein (RBP) of Escherichia coli is a Sec-pathway substrate. Intriguingly, it exhibits fast folding kinetics and its export is independent of SecB, a general chaperone protein dedicated for protein secretion. In this study, we found that the quantity of RBP was significantly reduced in the periplasm of tat mutants, which was restored by in trans expression of the tatABC genes. Pulse-chase experiments showed that significant amount of wild-type RBP was processed in a secY mutant in the presence of azide (SecA inhibitor), whereas the processing of a slow folding RBP derivative was almost completely blocked under the same conditions. These results would suggest that under the Sec-defective conditions the export of a portion of folded RBP could be rescued by the Tat system.     

Influence of stringent and relaxed response on excretion of recombinant proteins and fatty acid composition in Escherichia coli

In contrast to stringent (relA⁺) cells of Escherichia coli, relaxed (relA) cells excreted recombinant proteins (-lactamase, interferon 1) into the culture medium during amino acid limitation. Comparative analyses of overall fatty acid composition in relA⁺ cells and relA cells were performed and revealed that, in wild-type cells, drastic alterations occurred during the stringent response. The portion of saturated fatty acids (C16:0) and the fractions of cyclopropane fatty acids (C17_cyc and C19_cyc) increased whereas the portions of unsaturated fatty acids (C16:1 and C18:1) decreased. In cells of the relaxed mutant, no significant changes in the overall composition of the fatty acids were observed after the onset amino acid limitation. These results indicate that a change in fatty acid composition of membrane lipids after starvation of cells may be responsible for the prevention of loss of cellular proteins into the culture medium in stringent controlled cells of Escherichia coli.     

The conserved third transmembrane segment of YidC contacts nascent Escherichia coli inner membrane proteins

Escherichia coli YidC is a polytopic inner membrane protein that plays an essential and versatile role in the biogenesis of inner membrane proteins. YidC functions in Sec-dependent membrane insertion but acts also independently as a separate insertase for certain small membrane proteins. We have used a site-specific cross-linking approach to show that the conserved third transmembrane segment of YidC contacts the transmembrane domains of both nascent Sec-dependent and -independent substrates, indicating a generic recognition of insertion intermediates by YidC. Our data suggest that specific residues of the third YidC transmembrane segment alpha-helix is oriented toward the transmembrane domains of nascent inner membrane proteins that, in contrast, appear quite flexibly positioned at this stage in biogenesis.     

Reconstitution of translocation activity for secretory proteins from solubilized components of Escherichia coli

The protein translocation system of Escherichia coli was solubilized and reconstituted, using the octylglucoside dilution method, into liposomes prepared from E. coli phospholipids. SecA, ATP, phospholipids and membrane proteins were found to be essential for the translocation of a model secretory protein, uncleavable OmpF-Lpp. Phospholipids were found to play roles not only in liposome formation but also in the stabilization of membrane proteins during the octylglucoside extraction. The effects of IgGs specific to five distinct regions of the SecY molecule on protein translocation into proteoliposomes were examined. IgGs specific to the amino- and carboxyl-terminal regions of the SecY molecule strongly inhibited the translocation activity, indicating the participation of SecY in the translocation. Generation of a proton motive force due to the simultaneous reconstitution of F0F1-ATPase was also observed in the presence of ATP. An ATP-generating system consisting of creatine phosphate and creatine kinase significantly enhanced the formation of the proton motive force and the protein translocation activity of the proteoliposomes. Collapse of the proton motive force thus generated partially inhibited the translocation.     

Kinetic analysis of the translocation of fluorescent precursor proteins into Escherichia coli membrane vesicles

Protein secretion in Escherichia coli is mediated by translocase, a multi-subunit membrane protein complex with SecA as ATP-driven motor protein and the SecYEG complex as translocation pore. A fluorescent assay was developed to facilitate kinetic studies of protein translocation. Single cysteine mutants of proOmpA were site-specific labeled with fluorescent dyes, and the SecA and ATP-dependent translocation into inner membrane vesicles and SecYEG proteoliposomes was monitored by means of protease accessibility and in gel fluorescent imaging. The translocation of fluorescently labeled proOmpA was largely independent on the position and the size of the fluorescent label (up to a size of 13-16 A). A fluorophore at the +4 position blocked translocation, but inhibition was completely relieved in the PrlA4 mutant. The kinetics of translocation of the fluorescently labeled proOmpA could be directly monitored by means of fluorescence quenching. Inner membrane vesicles containing wild-type SecYEG were found to translocate proOmpA with a turnover of 4.5 molecules proOmpA/SecYEG complex/min and an apparent K(m) of 180 nm, whereas the PrlA4 mutant showed an almost 10-fold increase in turnover rate and a 3-fold increase of the apparent K(m) for proOmpA translocation.     

Lipid involvement in protein translocation in Escherichia coli

Signal peptides play an essential role in protein translocation. This review summarizes the current knowledge of the structure of signal peptides and signal peptide-lipid interactions and addresses the possibility that signal peptide-lipid interactions initiate membrane translocation of precursor proteins. A new model for protein translocation in Escherichia coli is proposed, which includes as central features conformational changes of the signal peptide and signal-peptide-induced local changes in membrane organization (non-bilayer lipids).     

Proton translocation in cytochrome-deficient mutants of Escherichia coli.

Cytochrome-deficient cells of a strain of Escherichia coli lacking 5-amino-levulinate synthetase have been used to study proton translocation associated with the reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase region of the electron transport chain. Menadione was used as electron acceptor, and mannitol was used as the substrate for the generation of intracellular NADH. The effects of iron deficiency on NADH- and D-lactate-menadione reductase activities were studied in iron-deficient cells of a mutant strain unable to synthesize the iron chelator enterochelin; both activities were reduced. The NADH- menadione reductase activity in cytochrome-deficient cells was associated with proton translocation and could be coupled to the uptake of proline. However proton translocation associated with the NADH-menadione reductase activity was prevented by a mutation in an unc gene. It was concluded that there is no proton translocation associated with the NADH-dehydrogenase region of the electron transport chain in E. coli and that the proton translocation obtained with mannitol as substrate is due to the activity of membrane-bound adenosine triphosphatase.     

Outer-membrane damage in sublethally heated Escherichia coli K-12.

Exponentially grown cells of Escherichia coli K-12 heated at 48 degrees C in potassium phosphate buffer at pH 7.0 were structrually injured before death. During heating for 60 min about 20% of the cellular lipopolysaccharide (LPS) was released from the outer membrane into the heating medium. Removal of 30% of the cellular LPS, by washing the cells in buffer containing ethylenediaminetetraacetic acid (EDTA), caused no significant increase in the rate of death and structural injury produced by heating. The addition of EDTA to the heating medium produced only a slight increase in the rate of thermal death but a large increase in the rate of structural injury. By a combination of heating at 48 degrees C and washing with EDTA, a maximum of 50% of the LPS was released from cells. These results taken together suggest that structural injury and loss of LPS are not the direct causes of death. The addition of 5 m M Mg2+ to the heating medium protected the cells from death and structural injury caused by heating at 48 degrees C.     

Phospholipids as a possible instrument for translocation of nascent proteins across biological membranes

The interaction of phospholipids with precursor proteins, particularly with the mitochondrial precursor protein apocytochromec is reviewed and integrated with other aspects of protein insertion and translocation, leading to a model for (apo)cytochrome c import into mitochondria, in which phospholipids play a dominant role.     

High-affinity calcium-binding proteins in Escherichia coli

Crude extracts of Escherichia coli contain at least three heat stable proteins of Mr, 33,000, 47,000, and 60,000, which bind 45Ca2+ in buffers containing micromolar calcium and physiological salt concentrations. Fractions containing these proteins neither activated the calmodulin-dependent enzyme, NAD kinase, nor inhibited the activity of this enzyme in the presence of brain calmodulin. Radioimmunoassay of crude extracts for calmodulin indicated the presence of a calmodulin-like antigen. Crude extracts also contain proteins that interact with 2-trifluoromethyl-10H-(3'-aminopropyl)phenothiazine-Sepharose in a calcium-dependent manner, but proteins eluted from this resin did not bind calcium with high affinity.     

大肠杆菌周质蛋白提取工艺的改进

介绍一种简捷高效的大肠杆菌周质蛋白提取工艺,即用含一定浓度溶菌酶的细胞裂解缓冲液一步提取周质蛋白,与传统的高渗和低渗两步提取法相比,不仅操作简单快捷,并且显著的提高了大肠杆菌周质蛋白的提取率.     

Variations on stoichiometry of ribosomal proteins in Escherichia coli

Experiments are described in which the Stoichiometry of the ribosomal proteins before and after ribosome release from mRNA is compared. Polysomes labeled with ³H (or ¹⁴C) and run-off 70 S particles (Subramanian el al., 1969) labeled with ¹⁴C (or ³H) were separately isolated, mixed, and the ribosomal proteins extracted and fractionated by two-dimensional gel electrophoresis. The measurement of the isotopic ratios shows that 47 proteins out of the 53 investigated are present in the same amount in polysomes as in run-off ribosomes indicating that they remain with the ribosome during the release step. Proteins S₁, S₂, S₆, S₂₁, $\text{L}{}_{\mathrm{<mtext>7</mtext>}}\text{L}{}_{\mathrm{<mtext>12</mtext>}}$ (Wittmann et al., 1971), however, show higher amounts in polysomes than in run-off ribosomes. The significance of these results is discussed.     

Overexpression of archaeal proteins in Escherichia coli

Six archaeal proteins containing a high number of Escherichia coli rare codons in their genes were not well expressed in E. coli. These genes showed a five to twenty-fold increase in production when expressed in the presence of a plasmid harboring and expressing the argU and ileX genes encoding rare tRNAs (tRNA arg(de)AGA/AGG and tRNA ile(de)AUA. © Rapid Science Ltd. 1998     

Acid shock proteins of Escherichia coli

Synthesis of total cellular proteins of Escherichia coli was studied after transfer of cultures from pH 6.9 to pH 4.3. Proteins induced by such an external pH shift down were identified by mono- and bi-dimensional electrophoresis. 30 to 45 min after an acid shift, a group of at least sixteen polypeptides was markedly induced. Four of these polypeptides corresponded to the well known heat shock proteins GroEL, DnaK, HtpG and HtpM. Their pH induction was RpoH-dependent. Three other pH-induced proteins were previously identified as stress proteins induced either by osmolarity or aerobiosis or low temperature (proteins 32 (defined in this paper), C70.0 and C62.7). Seven other proteins were specifically induced after an acid shift and were called acid shock proteins (ASP). The induction of one of these proteins was RpoH-dependent, whereas that of others was RpoH-independent.