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Hajime Tokuda

Publications and source records attributed to Hajime Tokuda.

12 recordsLinked to original sources
PubMed2003

Depletion of SecDF-YajC causes a decrease in the level of SecG: implication for their functional interaction.

SecA and an apparatus comprising SecYEG and SecDF-YajC complexes catalyze protein translocation across the Escherichia coli membrane. SecDF-YajC and SecG facilitate membrane insertion of SecA, which is the driving force for protein translocation. Here we report that SecDF-YajC depletion together with SecG depletion nearly completely inhibits protein translocation both in vivo and in vitro, although SecDF-YajC had been thought to be unnecessary for in vitro translocation. The level of SecG in membranes decreased to about half upon SecDF-YajC depletion and recovered to a normal level when SecDF-YajC was expressed. SecDF-YajC inhibited disulfide bond formation between two SecG molecules possessing a single cysteine residue. These results suggest functional interaction between SecDF-YajC and SecG.

Antigens, Bacterial↗
PubMed2003

Mechanism underlying the inner membrane retention of Escherichia coli lipoproteins caused by Lol avoidance signals.

Escherichia coli lipoproteins are localized to either the inner or outer membrane depending on the residue at position 2. The inner membrane retention signal, Asp at position 2 in combination with certain residues at position 3, functions as a Lol avoidance signal, i.e. the signal inhibits the recognition of lipoproteins by LolCDE that releases lipoproteins from the inner membrane. To understand the role of the residue at position 2, outer membrane-specific lipoproteins with Cys at position 2 were subjected to chemical modification followed by the release reaction in reconstituted proteoliposomes. Sulfhydryl-specific introduction of nonprotein molecules or a negative charge to Cys did not inhibit the LolCDE-dependent release. In contrast, oxidation of Cys to cysteic acid resulted in generation of the Lol avoidance signal, indicating that the Lol avoidance signal requires a critical length of negative charge at the second residue. Furthermore, not only modification of the carboxylic acid of Asp at position 2 but also that of the amine of phosphatidylethanolamine abolished the Lol avoidance function. Based on these results, the Lol avoidance mechanism is discussed.

Amino Acid Sequence↗
PubMed2003

A practical phasing procedure using the MAD method without the aid of XAFS measurements: successful solution in the structure determination of the outer-membrane lipoprotein carrier LolA.

A practical procedure for MAD phasing was successfully performed in the structure determination of the LolA protein, even though the XAFS data could not be measured owing to the overlap of the fluorescence spectra of the atoms that contribute significant signals for anomalous dispersion. The LolA protein, a periplasmic chaperone functioning as an outer-membrane lipoprotein carrier of the Lol system which mediates translocation of the water-insoluble outer-membrane lipoprotein across the periplasm in Gram-negative bacteria, was crystallized in two forms: orthorhombic (I222) and trigonal (P3(1)21 or P3(2)21). A multi-wavelength data set was collected from a platinum derivative of the orthorhombic crystals grown from a buffer solution containing zinc acetate and cacodylate (an arsenic compound), but XAFS measurements could not be performed because the energies of the fluorescence spectra of Zn atoms, As atoms and Pt atoms are in very close proximity. However, effective MAD data were collected with six wavelength data sets near the platinum absorption edge and the f' and f" values could subsequently be estimated from the data statistics and the peak height of the dispersive and anomalous difference Patterson maps. The subsequent MAD phasing gave a high-quality initial electron-density map which was sufficient to construct a complete molecular model of the LolA protein.

Arsenic↗
PubMed2003

Crystal structures of bacterial lipoprotein localization factors, LolA and LolB.

Lipoproteins having a lipid-modified cysteine at the N-terminus are localized on either the inner or the outer membrane of Escherichia coli depending on the residue at position 2. Five Lol proteins involved in the sorting and membrane localization of lipoprotein are highly conserved in Gram-negative bacteria. We determined the crystal structures of a periplasmic chaperone, LolA, and an outer membrane lipoprotein receptor, LolB. Despite their dissimilar amino acid sequences, the structures of LolA and LolB are strikingly similar to each other. Both have a hydrophobic cavity consisting of an unclosed beta barrel and an alpha-helical lid. The cavity represents a possible binding site for the lipid moiety of lipoproteins. Detailed structural differences between the two proteins provide significant insights into the molecular mechanisms underlying the energy-independent transfer of lipoproteins from LolA to LolB and from LolB to the outer membrane. Furthermore, the structures of both LolA and LolB determined from different crystal forms revealed the distinct structural dynamics regarding the association and dissociation of lipoproteins. The results are discussed in the context of the current model for the lipoprotein transfer from the inner to the outer membrane through a hydrophilic environment.

Amino Acid Sequence↗
PubMed2003

Crystallization and preliminary crystallographic study of the outer-membrane lipoprotein receptor LolB, a member of the lipoprotein localization factors.

The Lol system mediates the translocation of the water-insoluble outer-membrane lipoprotein across the periplasm of Gram-negative bacteria depending on the sorting signal. The outer-membrane lipoprotein receptor LolB (21.2 kDa) is a member of the Lol system. A soluble mutant of LolB (mLolB) from Escherichia coli was crystallized in two forms. Monoclinic crystals diffract X-rays to 1.9 A resolution and belong to space group P2(1), with unit-cell parameters a = 37.2, b = 112.4, c = 47.8 A, beta = 111.4 degrees. The V(M) value is most likely to be 2.2 A(3) Da(-1), assuming the presence of two molecules in the asymmetric unit. Hexagonal crystals diffract X-rays to 2.2 A resolution and belong to space group P6(3)22, with unit-cell parameters a = b = 71.4, c = 133.9 A. The V(M) value is determined as 2.3 A(3) Da(-1), assuming a single molecule in the asymmetric unit. A four-wavelength data set was collected from a monoclinic crystal of selenomethionylated mLolB in order to perform MAD phasing. The quality of the initial electron-density map was sufficient to build a molecular model.

Bacterial Outer Membrane Proteins↗
PubMed2003

A mutation in the membrane subunit of an ABC transporter LolCDE complex causing outer membrane localization of lipoproteins against their inner membrane-specific signals.

Lipoproteins in Gram-negative bacteria are anchored to the inner or outer membrane via fatty acids attached to the N-terminal cysteine. The residue at position 2 determines the membrane specificity. An ATP binding cassette transporter LolCDE complex releases lipoproteins with residues other than aspartate at position 2 from the inner membrane, whereas those with aspartate at position 2 are rejected by LolCDE and therefore remain in the inner membrane. For further understanding of this rejection mechanism, a novel strategy was developed to select mutants in which lipoproteins with aspartate at position 2 are released. The isolated mutants carried an alanine to proline mutation at position 40 of LolC, a membrane subunit of the LolCDE complex. A significant portion of an inner membrane lipoprotein, L10P(DQ), was localized to the outer membrane when the LolC mutant was expressed. Periplasmic chaperone LolA formed a complex with the released L10P(DQ), which was subsequently incorporated into the outer membrane in a LolB-dependent manner, indicating that neither LolA nor LolB rejects lipoproteins with aspartate at position 2. The amount of the LolC mutant co-purified with LolD and LolE after membrane solubilization was reduced significantly. Taken together, these results indicate that the mutation causes destabilization of the LolCDE complex and concomitantly prevents the accurate recognition of lipoprotein-sorting signals.

ATP-Binding Cassette Transporters↗
PubMed2002

Dominant negative mutant of a lipoprotein-specific molecular chaperone, LolA, tightly associates with LolCDE.

Periplasmic molecular chaperone LolA and the inner membrane ATP binding cassette transporter LolCDE are essential for ATP-dependent release of outer membrane-directed lipoproteins from the inner membrane of Escherichia coli. A LolA(F47E) mutant carrying a Phe to Glu mutation at position 47 was defective in the release of lipoproteins from spheroplasts and proteoliposomes reconstituted with LolCDE. When incubated with proteoliposomes containing LolCDE, LolA remained in the supernatant whereas LolA(F47E) bound to proteoliposomes. This tight association of LolA(F47E) with LolCDE caused a dominant negative phenotype in vivo, suggesting that the LolA-LolCDE interaction is critical for lipoprotein release.

ATP-Binding Cassette Transporters↗
PubMed2002

Aminoacylation of the N-terminal cysteine is essential for Lol-dependent release of lipoproteins from membranes but does not depend on lipoprotein sorting signals.

Lipoproteins are present in a wide variety of bacteria and are anchored to membranes through lipids attached to the N-terminal cysteine. The Lol system of Escherichia coli mediates the membrane-specific localization of lipoproteins. Aspartate at position 2 functions as a Lol avoidance signal and causes the retention of lipoproteins in the inner membrane, whereas lipoproteins having residues other than aspartate at position 2 are released from the inner membrane and localized to the outer membrane by the Lol system. Phospholipid:apolipoprotein transacylase, Lnt, catalyzes the last step of lipoprotein modification, converting apolipoprotein into mature lipoprotein. To reveal the importance of this aminoacylation for the Lol-dependent membrane localization, apolipoproteins were prepared by inhibiting lipoprotein maturation. Lnt was also purified and used to convert apolipoprotein into mature lipoprotein in vitro. The release of these lipoproteins was examined in proteoliposomes. We show here that the aminoacylation is essential for the Lol-dependent release of lipoproteins from membranes. Furthermore, lipoproteins with aspartate at position 2 were found to be aminoacylated both in vivo and in vitro, indicating that the lipoprotein-sorting signal does not affect lipid modification.

ATP-Binding Cassette Transporters↗
PubMed2002

Elucidation of the function of lipoprotein-sorting signals that determine membrane localization.

Escherichia coli lipoproteins are anchored to the inner or outer membrane depending on the residue at position 2. Aspartate at this position makes lipoproteins specific to the inner membrane, whereas other residues cause the release of lipoproteins from the inner membrane in a manner dependent on both ATP binding cassette (ABC) transporter LolCDE and molecular chaperone LolA, followed by LolB-dependent localization in the outer membrane. The function of lipoprotein-sorting signals was examined in proteoliposomes reconstituted from LolCDE and lipoproteins. The release of outer membrane-specific lipoproteins was inhibited on reconstitution with other outer membrane-specific, but not inner membrane-specific, lipoproteins. Outer membrane-specific lipoproteins stimulated ATP hydrolysis by LolCDE whereas inner membrane-specific ones did not. LolA was not required for the stimulation of ATP hydrolysis. These results revealed a previously undocumented function of aspartate at position 2, i.e., lipoproteins having this signal avoid being recognized by LolCDE, thereby remaining in the inner membrane.

Adenosine Triphosphate↗
PubMed2002

Membrane topology inversion of SecG detected by labeling with a membrane-impermeable sulfhydryl reagent that causes a close association of SecG with SecA.

SecG stimulates protein translocation in Escherichia coli by facilitating the membrane insertion-deinsertion cycle of SecA. SecG was previously shown to undergo membrane topology inversion, since SecA-dependent protein translocation renders the membrane-protected region of SecG sensitive to external proteases. To examine this topology inversion in more detail without protease-treatment, SecG derivatives with a single cysteine residue at various positions were labeled in the presence and absence of protein translocation with a membrane impermeable SH reagent, 4-acetamido-4'-maleimidylstilbene-2-2'-disulfonic acid (AMS). Treatment of spheroplasts with AMS revealed that a cysteine residue in the cytoplasmic region of SecG could be labeled from the periplasm side only in the presence of protein translocation, whereas a cytoplasmic protein, elongation factor, Tu, remained unlabeled. Treatment of inverted membrane vesicles with AMS also revealed that cysteine residues in the periplasmic region were labeled from the cytoplasmic side of membranes only when protein translocation was in progress. This labeling required ATP, SecA and a precursor protein, and became more efficient as the position of the cysteine residue became closer to the C-terminus. Crosslinking analyses revealed that the interaction between SecG and SecA in membranes markedly increases when SecA and SecG undergo membrane-insertion and topology inversion, respectively. Thus, the two most dynamic components of the translocation machinery were found for the first time to interact with each other when both undergo conformational changes.

Adenosine Triphosphatases↗
PubMed2002

Disruption of lolCDE, encoding an ATP-binding cassette transporter, is lethal for Escherichia coli and prevents release of lipoproteins from the inner membrane.

ATP-binding cassette transporter LolCDE was previously identified, by using reconstituted proteoliposomes, as an apparatus catalyzing the release of outer membrane-specific lipoproteins from the inner membrane of Escherichia coli. Mutations resulting in defective LolD were previously shown to be lethal for E. coli. The amino acid sequences of LolC and LolE are similar to each other, but the necessity of both proteins for lipoprotein release has not been proved. Moreover, previous reconstitution experiments did not clarify whether or not LolCDE is the sole apparatus for lipoprotein release. To address these issues, a chromosomal lolC-lolD-lolE null mutant harboring a helper plasmid that carries the lolCDE genes and a temperature-sensitive replicon was constructed. The mutant failed to grow at a nonpermissive temperature because of the depletion of LolCDE. In addition to functional LolD, both LolC and LolE were required for growth. At a nonpermissive temperature, the outer membrane lipoproteins were mislocalized in the inner membrane since LolCDE depletion inhibited the release of lipoproteins from the inner membrane. Furthermore, both LolC and LolE were essential for the release of lipoproteins. On the other hand, LolCDE depletion did not affect the translocation of a lipoprotein precursor across the inner membrane and subsequent processing to the mature lipoprotein. From these results, we conclude that the LolCDE complex is an essential ABC transporter for E. coli and the sole apparatus mediating the release of outer membrane lipoproteins from the inner membrane.

ATP-Binding Cassette Transporters↗