Solubilization and reconstitution of membrane energy‐transducing systems of <i>Escherichia coli</i>

Tsuchiya, Tomofusa; Misawa, Atsuko; MIYAKE, Yumi; Yamasaki, Kyoko; Niiya, S

doi:10.1016/0014-5793(82)80141-5

Cited by 21 publications

(8 citation statements)

References 14 publications

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“…Purification of Cytochrome o. Sequential extraction of E. coli membranes with urea and cholate provides a significant in situ purification of the lac carrier protein (2, 3) and octyl glucoside has been shown to extract NNN',N'-tetramethylphenylenediamine (TMPD) oxidase activity (22). These procedures were combined to partially purify and solubilize cytochrome o oxidase from the membrane of a cytochrome d-deficient mutant (14) ( Table 1).…”

Section: Resultsmentioning

confidence: 99%

Reconstitution of active transport in proteoliposomes containing cytochrome o oxidase and lac carrier protein purified from Escherichia coli.

Matsushita¹,

Patel²,

Gennis³

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

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Most active transport across the bacterial cell membrane is driven by a proton electrochemical gradient (AJAH+, interior negative and alkaline) generated via electron transfer through a membrane-bound respiratory chain. This phenomenon is now reproduced in vitro with proteoliposomes containing only two proteins purified from the membrane of Escherichia coli. An o-type cytochrome oxidase was extracted from membranes of a cytochrome d terminal oxidase mutant with octyl 8-D-glucopyranoside after sequential treatment with urea and cholate and was purified to homogeneity by ion-exchange chromatography. The purified oxidase contains four polypeptides (Mrs 66,000, 35,000, 22,000, and1.7,000), two b-type cytochromes (b558 and b563), and 16-17 nmol of heme b per mg of protein, and it catalyzes the oxidation of ubiquinol and other electron donors with specific activities 20-to 30-fold higher than crude membranes. The lac carrier protein was purified as described. Proteoliposomes were formed in the presence of the oxidase and lac carrier protein by detergent dilution, followed by freeze-thaw/sonication. The system generates a A/AH+ (interior negative and alkaline) with ubiquinol as electron donor and the magnitude of AI.H+ is dependent on the concentration of cytochrome o in the proteoliposomes. Furthermore, the proteoliposomes transport lactose against a concentration gradient to an extent that is commensurate with the magnitude of AILH+ generated. The results provide powerful additional support for the "chemiosmotic hypothesis" and demonstrate that purified lac carrier protein retains the ability to function in a physiological manner.

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Section: Resultsmentioning

confidence: 99%

Reconstitution of active transport in proteoliposomes containing cytochrome o oxidase and lac carrier protein purified from Escherichia coli.

Matsushita¹,

Patel²,

Gennis³

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

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“…This Na' requirement appears not to be restricted to glutaconyl-CoA decarboxylase containing organisms, since Clostridium t e t a n o~o r p h u m H 1 and Escherichia coli [25] also grow on glutamate only in the presence of Na'. Probably the transport systems for glutamate are driven by N a + gradients [26]. Thereby anaerobic bacteria with Na+-pumping decarboxylases should have advantages over those which have t o spend a considerable amount of their A T P for those processes.…”

Section: Discussionmentioning

confidence: 99%

Purification, characterisation and reconstitution of glutaconyl‐CoA decarboxylase, a biotin‐dependent sodium pump from anaerobic bacteria

Semmler

1983

European Journal of Biochemistry

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Glutaconyl‐CoA decarboxylase from Acidaminococcus fermentans is a biotin enzyme, which is integrated into membranes. It is activated by Triton X‐100 and inhibited by avidin. The results obtained by a combination of both agents indicate that biotin and the substrate‐binding site are located on the same side of the membrane. The decarboxylase was solubilized with Triton X‐100 and purified by affinity chromatography on monomeric avidin‐Sepharose. The enzyme is composed of three types of polypeptides: the group of α chains (Mr 120000–140000) containing the biotin, the β chain (60000) and an apparently hydrophobic γ chain (35000). Sodium ions specifically protected the latter chain from tryptic digestion. It was supposed, therefore, that this chain might function as the Na+ channel. The β and γ chains but not the α chain could be labelled by N‐ethyl‐[14C]maleimide. Similar decarboxylases but with much smaller biotin peptides (Mr 15000–20000) were isolated from Peptococcus aerogenes and Clostridium symbiosum. The decarboxylases from all three organisms could be reconstituted to active sodium pumps by incubation with phospholipid vesicles and octylglucoside followed by dilution. The Na+ uptake catalysed by the enzyme from A. fermentans was completely inhibited by monensin and activated twofold by valinomycin/K+ indicating an electrogenic Na+ pump. The coupling between Na+ transport and decarboxylation was not tight. During the reaction the ratio decreased from initially 1 to 0.2. The three organisms mentioned above and Clostridium tetanomorphum without glutaconyl‐CoA decarboxylase are able to ferment glutamate and require 10 mM Na+ for rapid growth. There is no correlation between the concentration of monensin necessary to inhibit growth and the presence of decarboxylase in these organisms.

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“…The membrane fraction used for the reconstitution of proteoliposomes was prepared from RR1, an uric' strain. The reconstitution of H f -translocating (F,F,) ATPase, products of the unc genes, has been reported using the octylglucoside dilution method [33]. We, therefore, determined the extent of ATP hydrolysis during the translocation reaction.…”

Section: An Atp-generating System Considerably Enhanced Translocationmentioning

confidence: 99%

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

Tokuda

Shiozuka

Mizushima

1990

European Journal of Biochemistry

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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 carboxylterminal 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 FoF1-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.The establishment of in vitro assay systems for the translocation of secretory proteins in Escherichia coli facilitated the detailed analysis of the translocation mechanism. It now seems certain that protein translocation across the cytoplasmic membrane of E. coli generally requires both ATP and a proton motive force [l -81. We have shown that ATP is essential but that the proton motive force is not obligatory for the translocation of various model proteins [6] and OmpA [6, 81, one of the major proteins of the outer membrane. Several proteins have been demonstrated to be involved in protein translocation in E. coli [9]. The roles of SecA [lo-121 and SecB [13, 141, both of which have been purified from the cytosolic fraction, in protein translocation have been well studied in vitro. On the other hand, involvement of SecY [I 51 and SecE [16], both of which are membrane proteins, in protein translocation is suggested from genetic studies. Inhibition of the translocation reaction by anti-SecY antisera was also observed recently [17, 181. Moreover, it is not known whether or not other membrane components are required for protein translocation. In order to elucidate the overall mechanism of protein translocation, it is essential to identify the membrane components necessary for protein translocation and to clarify their functions biochemically. Establishment of a reconstitution system for protein translocation is, therefore, essential.Various membrane proteins have been successfully reconstituted into liposomes through the use of the octylglucoside

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Solubilization and reconstitution of membrane energy‐transducing systems of Escherichia coli

Cited by 21 publications

References 14 publications

Reconstitution of active transport in proteoliposomes containing cytochrome o oxidase and lac carrier protein purified from Escherichia coli.

Reconstitution of active transport in proteoliposomes containing cytochrome o oxidase and lac carrier protein purified from Escherichia coli.

Purification, characterisation and reconstitution of glutaconyl‐CoA decarboxylase, a biotin‐dependent sodium pump from anaerobic bacteria

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

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