Shoji Mizushima

SecD is involved in the release of translocated secretory proteins from the cytoplasmic membrane of Escherichia coli.

The SecD protein is one of the components that has been suggested from genetic studies to be involved in the protein secretion across the cytoplasmic membrane of Escherichia coli. We examined the effect of anti‐SecD IgG on protein secretion using spheroplasts. Inhibition of the secretion of OmpA and maltose‐binding protein (MBP) by this IgG was observed with concomitant accumulation of their precursor and mature forms in spheroplasts. This effect was specific to anti‐SecD IgG. Anti‐SecE and anti‐SecY IgGs, of which the epitopes are located at the periplasmic domains of SecE and SecY, respectively, did not interfere with the secretion. Time‐course experiments investigating the processing of proMBP and the release of MBP from spheroplasts revealed that anti‐SecD IgG interfered with the release of the translocated mature MBP. The mature form of MBP thus accumulated was sensitive to trypsin, which was externally added to spheroplasts, whereas MBP released into the medium was resistant to trypsin as the native MBP is. The precursor form of MBP accumulated in spheroplasts was also trypsin resistant. We conclude that SecD is directly involved in protein secretion and important for the release of proteins that have been translocated across the cytoplasmic membrane.

A novel membrane protein involved in protein translocation across the cytoplasmic membrane of Escherichia coli.

A novel factor, which is a membrane component of the protein translocation machinery of Escherichia coli, was discovered. This factor was found in the trichloracetic acid‐soluble fraction of solubilized cytoplasmic membrane. The factor was purified to homogeneity by ion exchange column chromatographies and found to be a hydrophobic protein with a molecular mass of approximately 12 kDa. The factor caused > 20‐fold stimulation of the protein translocation when it was reconstituted into proteoliposomes together with SecE and SecY. SecE, SecY, SecA and ATP were essential for the factor‐dependent stimulation of the activity. The factor stimulated the translocation of all three precursor proteins examined, including authentic proOmpA. Stimulation of the translocation of proOmpF‐Lpp, a model presecretory protein, was especially remarkable, since no translocation was observed unless proteoliposomes were reconstituted with the factor. Partial amino acid sequence of the purified factor was determined. An antibody raised against a synthetic peptide of this sequence inhibited the protein translocation into everted membrane vesicles, indicating that the factor is playing an important role in protein translocation into membrane vesicles. The partial amino acid sequence was found to coincide with that deduced from the reported DNA sequence of the upstream region of the leuU gene. Cloning and sequencing of the upstream region revealed the presence of a new open reading frame, which encodes a hydrophobic protein of 11.4 kDa. We propose that the factor is a general component of the protein translocation machinery of E. coli.

SecA, an essential component of the secretory machinery of Escherichiacoli, exists as homodimer

Size exclusion chromatography of the cytosolic fraction of SecA-overproducing cells of Escherichia coli suggested that SecA, an essential component of the secretory machinery, exists as an oligomer. The subunit structure of SecA was then studied using a purified specimen. Estimation of the molecular mass by means of ultracentrifugation and chemical crosslinking analysis revealed that SecA exists as a homodimer. The purified SecA was denatured in 6 M guanidine-HCl and renatured to a dimer, which was fully active in terms of translocation, even in the presence of 1 mM dithiothreitol. It is suggested that the dimeric structure is not critically maintained by disulfide bonding between the two subunits, each of which contains four cysteine residues.

SecE-dependent overproduction of SecY in Escherichia coli: Evidence for interaction between two components of the secretory machinery

The secY and secE genes were individually cloned and placed under the control of the tac promoter on plasmids. Induction with isopropyl-β-D-thiogalactopyranoside resulted in the overproduction of SecE, but not that of SecY. The simultaneous induced expression of both genes in the same cells resulted in the overproduction of SecY together with that of SecE. SecY and SecE thus overproduced were localized in the cytoplasmic membrane as those expressed at the normal levels were. It is suggested that SecY and SecE interact with each other in the cytoplasmic membrane. The numbers of the SecY and SecE molecules per cell were estimated.The secY and secE genes were individually cloned and placed under the control of the tac promoter on plasmids. Induction with isopropyl‐β‐D‐thiogalactopyranoside resulted in the overproduction of SecE, but not that of SecY. The simultaneous induced expression of both genes in the same cells resulted in the overproduction of SecY together with that of SecE. SecY and SecE thus overproduced were localized in the cytoplasmic membrane as those expressed at the normal levels were. It is suggested that SecY and SecE interact with each other in the cytoplasmic membrane. The numbers of the SecY and SecE molecules per cell were estimated.

Isolation and characterization of two outer membrane preparations from Escherichia coli

A method was developed for releasing specifically a part of outer membrane during spheroplast formation. A highly purified outer membrane (outer membrane I) was obtained from the spheroplast medium by isopycnic sucrose gradient centrifugation. The remaining outer membrane (outer membrane II) and cytoplasmic membrane was also isolated from the spheroplasts by the isopycnic centrifugation. Two outer membrane preparations were different from the cytoplasmic membrane in protein composition, enzyme localization, phospholipid composition, lipopolysaccharide content and electron micrographs. Although outer membranes I and II were almost the same in various respects, they seemed to be different from each other under electron microscope and in cardiolipin content. It is suggested that the outer membrane I and the outer membrane II, at least a part of the outer membrane II, are integrated in a different fashion in the outer-most layer of Escherichia coli cell surface.

Separation and properties of outer and cytoplasmic membranes in Escherichia coli

Abstract Two kinds of membrane were resolved when spheroplast membranes of Escherichia coli K12 were dialyzed against EDTA and then subjected to linear sucrose gradient centrifugation. The less dense band, which is known to be composed mainly of lipoprotein, has a “unit membrane” structure and contains a relatively large amount of ATPase (EC 3.6.1.3) and components of the electron-transfer system. The denser one contained a relatively large amount of carbohydrate and NADH dehydrogenase (EC 1.6.99.3) without any other respiratory components, it has a complex-coiled structure similar to the oute membranes described by Birdsell and Cota-Robles . This membrane fraction was found—by immunological studies—to be located on the outside of the cell surface.

Molecular analysis of mutant ompR genes exhibiting different phenotypes as to osmoregulation of the ompF and ompC genes of Escherichia coli.

SummaryExpression of the ompF and ompC genes coding for major outer membrane proteins is osmoregulated by solutes, such as sucrose and NaCl, in the growth medium. The OmpR protein, a positive regulator of these genes, is involved in the osmoregulation (Dairi et al. 1985; Nara et al. 1984). In the present work, five mutant ompR genes exhibiting different phenotypes of osmoregulation were cloned and sequenced. Three of them, ompR1, ompR2 and ompR20, were previously isolated mutants. The others, ompR3 and ompR4, were isolated in the present work. The ompR1 mutation resulted in the deletion of 19 amino acids near the C-terminus of the OmpR protein. The ompR3 and ompR4 mutations resulted in Arg15 to Cys and Arg71 to Thr conversions, respectively, at the N-terminal portion, whereas the ompR20 and ompR2 mutations resulted in Arg150 to Cys and Val207 to Met conversions, respectively, at the C-terminal portion. Based on these results, the structure and function of the OmpR protein are discussed in relation to the mechanism of osmoregulation.

Secretion into the culture medium of a foreign gene product from Escherichia coli: use of the ompF gene for secretion of human beta-endorphin.

The ompF gene codes for a major outer membrane protein of Escherichia coli. A plasmid was constructed in which the structural gene for human beta‐endorphin is preceded by the upstream region of the ompF gene consisting of the promoter region and the coding regions for the signal peptide and the N terminus of the OmpF protein. When the plasmid was introduced into E. coli N99, and OmpF‐beta‐endorphin fused peptide was synthesized and secreted into the culture medium through both the cytoplasmic and outer membranes. The OmpF signal peptide was cleaved correctly during the secretion, indicating that the export of the fused protein across the cytoplasmic membrane was dependent on the signal peptide. The secretion into the culture medium was apparently selective. Neither beta‐lactamase nor alkaline phosphatase (both are periplasmic proteins) appeared in the culture medium in significant amounts. The mode of passage of the fused peptide across the outer membrane is discussed.

Overproduction, purification and characterization of SecD and SecF, integral membrane components of the protein translocation machinery of Escherichia coli

SecD and SecF proteins were overproduced by means of recombinant DNA technology. Immunoblot and amino-acid sequencing analysis revealed that the overproduced proteins are SecD and SecF. The SecD- or SecF-overproduced membrane fraction was subjected to differential solubilization. The SecD protein was then purified through ion-exchange and size-exclusion chromatographies. The SecF protein was purified through size exclusion chromatography. Proteoliposomes reconstituted from the purified SecD and SecF together with SecE and SecY were used to analyze the translocation activity. SecD and SecF did not exhibit significant effects on the translocation activity of proteoliposomes. The amounts of SecD and SecF in overproducers were determined densitometrically on a stained SDS gel and their overproduction (fold) was determined by means of immunoblot analysis. Then the number of these molecules in one normal cell were estimated. From these numbers, together with those of other Sec proteins, the number of the translocation machinery existing in one Escherichia coli cell was inferred to be around 500.

Isolation of outer membrane proteins of Escherichia coli and their characterization on polyacrylamide gel

Proteins from the outer membrane of Escherichia coli were studied on a ureadodecyl sulfate polyacrylamide gel by electrophoresis. A polyacrylamide gel containing sodium dodecyl sulfate and urea gave an excellent resolution of outer membrane proteins. Seventeen protein bands were reproducibly observed on a gel. By use of Sephadex G-200, DEAE-cellulose and polyacrylamide gel, eight proteins were purified to near homogeneity. Five of them were found to be heat-modifiable proteins. The behavior of these purified proteins was studied on a polyacrylamide gel under three different electrophoretic conditions, which had been used for the analysis of cell envelope proteins. Thus correspondence was made between these purified proteins and envelope proteins reported by other investigators.