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Koreaki Ito

Publications and source records attributed to Koreaki Ito.

At least 37 records · Page 2Linked to original sources
PubMed2004

Characterization of the menaquinone-dependent disulfide bond formation pathway of Escherichia coli.

In the protein disulfide-introducing system of Escherichia coli, plasma membrane-integrated DsbB oxidizes periplasmic DsbA, the primary disulfide donor. Whereas the DsbA-DsbB system utilizes the oxidizing power of ubiquinone (UQ) under aerobic conditions, menaquinone (MK) is believed to function as an immediate electron acceptor under anaerobic conditions. Here, we characterized MK reactivities with DsbB. In the absence of UQ, DsbB was complexed with MK8 in the cell. In vitro studies showed that, by binding to DsbB in a manner competitive with UQ, MK specifically oxidized Cys41 and Cys44 of DsbB and activated its catalytic function to oxidize reduced DsbA. In contrast, menadione used in earlier studies proved to be a more nonspecific oxidant of DsbB. During catalysis, MK8 underwent a spectroscopic transition to develop a visible violet color (lambdamax = 550 nm), which required a reduced state of Cys44 as shown previously for UQ color development (lambdamax = 500 nm) on DsbB. In an in vitro reaction system of MK8-dependent oxidation of DsbA at 30 degrees C, two reaction components were observed, one completing within minutes and the other taking >1 h. Both of these reaction modes were accompanied by the transition state of MK, for which the slower reaction proceeded through the disulfide-linked DsbA-DsbB(MK) intermediate. The MK-dependent pathway provides opportunities to further dissect the quinone-dependent DsbA-DsbB redox reactions.

Antifibrinolytic Agents↗
PubMed2004

Translation arrest of SecM is essential for the basal and regulated expression of SecA.

The SecM protein of Escherichia coli contains an arrest sequence (F(150)XXXXWIXXXXGIRAGP(166)), which interacts with the ribosomal exit tunnel to halt translation elongation beyond Pro-166. This inhibition is reversed by active export of the nascent SecM chain. Here, we studied the physiological roles of SecM. Arrest-alleviating mutations in the arrest sequence reduced the expression of secA, a downstream gene on the same mRNA. Among such mutations, the arrest-abolishing P166A substitution mutation on the chromosomal secM gene proved lethal unless the mutant cells are complemented with excess SecA. Whereas secretion defect due either to azide addition, a secY mutation, or low temperature leads to up-regulated SecA biosynthesis, this regulation was lost by a secM mutation, which synergistically retarded growth of cells with lowered secretion activity. Finally, an arrest-alleviating rRNA mutation affecting the constricted part of the exit tunnel lowered the basal level of SecA as well as its secretion defect-induced up-regulation. Thus, the arrest sequence of SecM has at least two roles in SecA translation. First, the transient elongation arrest in normal cells is required for the synthesis of SecA at levels sufficient to support cell growth. Second, the prolonged SecM elongation arrest under conditions of unfavorable protein secretion is required for the enhanced expression of SecA to cope with such conditions.

Adenosine Triphosphatases↗
PubMed2004

Intraribosomal regulation of expression and fate of proteins.

Our studies of SecM (secretion monitor) in E. coli have revealed that some amino acid sequences can interact with ribosomal interior components, particularly with gate components of the exit tunnel, thereby interfering with their own translation elongation. Such translation arrest can be regulated by interaction of the N-terminal portion of the nascent polypeptide with other cellular components outside the ribosome. These properties of nascent proteins can in turn provide regulatory mechanisms by which the expression of genetic information at different levels is regulated.

Adenosine Triphosphatases↗
PubMed2004

Control of SecA and SecM translation by protein secretion.

SecA, the protein translocation ATPase of E. coli is subject to secretion-defect-response control. SecM (secretion monitor) encoded by the 5' region of the secM-secA mRNA is involved in this regulation. SecM translation is subject to transient elongation arrest at Pro166, which is prolonged when export of the nascent SecM is blocked. An "arrest sequence", FXXXXWIXXXXGIRAGP, was identified at a carboxy-terminal region of SecM that interacts with the ribosomal exit tunnel. Presumably, the stalled ribosome disrupts the secondary structure of the secM-secA mRNA such that the Shine-Dalgarno sequence for translation of secA is exposed. Mutation studies established that the SecM elongation arrest is required for the viability of E. coli as well as for constitutive (in secretion-proficient cells) and upregulated (in secretion compromised cells) expression of SecA. Furthermore, evidence suggests that elongation-arresting SecM has a role of upregulating the functionality of newly synthesized SecA molecules, presumably by bringing the mRNA to the vicinity of the membrane/Sec translocation apparatus. These results are discussed in relation to the versatile nature of SecA in its localization and structure.

Adenosine Triphosphatases↗
PubMed2004

Mutational analysis of transmembrane regions 3 and 4 of SecY, a central component of protein translocase.

The SecYEG heterotrimeric membrane protein complex functions as a channel for protein translocation across the Escherichia coli cytoplasmic membrane. SecY is the central subunit of the SecYEG complex and contains 10 transmembrane segments (TM1 to TM10). Previous mutation studies suggested that TM3 and TM4 are particularly important for SecY function. To further characterize TM3 and TM4, we introduced a series of cysteine-scanning mutations into these segments. With one exception (an unstable product), all the mutant proteins complemented the cold-sensitive growth defect of the secY39 mutant. A combination of this secY mutation and the secG deletion resulted in synthetic lethality, and the TM3 and TM4 SecY cysteine substitution mutations were examined for their ability to complement this lethality. Although they were all positive for complementation, some of the complemented cells exhibited significant retardation of protein export. The substitution-sensitive residues in TM3 can be aligned to one side of the alpha-helix, and those in TM4 revealed a tendency for residues closer to the cytosolic side of the membrane to be more severely affected. Disulfide cross-linking experiments identified a specific contact point for TM3 and SecG TM2 as well as for TM4 and SecG TM1. Thus, although TM3 and TM4 do not contain any single residue that is absolutely required, they include functionally important helix surfaces and specific contact points with SecG. These results are discussed in light of the structural information available for the SecY complex.

Adenosine Triphosphatases↗
PubMed2003

YaeL proteolysis of RseA is controlled by the PDZ domain of YaeL and a Gln-rich region of RseA.

sigmaE is an alternative sigma factor involved in a pathway of extracytoplasmic stress responses in Escherichia coli. Under normal growth conditions, sigmaE activity is down-regulated by the membrane-bound anti-sigmaE protein, RseA. Extracytoplasmic stress signals induce degradation of RseA by two successive proteolytic events: DegS-catalyzed first cleavage at a periplasmic site followed by YaeL-mediated second proteolysis at an intramembrane region. Normally, the second reaction (site-2 proteolysis) only occurs after the first cleavage (site-1 cleavage). Here, we show that YaeL variants with the periplasmic PDZ domain deleted or mutated allows unregulated cleavage of RseA and consequent sigmaE activation. It was also found that a glutamine-rich region in the periplasmic domain of RseA was required for the avoidance of the YaeL-mediated proteolysis in the absence of site-1 cleavage. These results indicate that multiple negative elements both in the enzyme (PDZ domain) and in the substrate (glutamine-rich region) determine the strict dependence of the site-2 proteolysis on the site-1 cleavage.

Amino Acid Sequence↗
PubMed2003

DsbB elicits a red-shift of bound ubiquinone during the catalysis of DsbA oxidation.

DsbB is an Escherichia coli plasma membrane protein that reoxidizes the Cys30-Pro-His-Cys33 active site of DsbA, the primary dithiol oxidant in the periplasm. Here we describe a novel activity of DsbB to induce an electronic transition of the bound ubiquinone molecule. This transition was characterized by a striking emergence of an absorbance peak at 500 nm giving rise to a visible pink color. The ubiquinone red-shift was observed stably for the DsbA(C33S)-DsbB complex as well as transiently by stopped flow rapid scanning spectroscopy during the reaction between wild-type DsbA and DsbB. Mutation and reconstitution experiments established that the unpaired Cys at position 44 of DsbB is primarily responsible for the chromogenic transition of ubiquinone, and this property correlates with the functional arrangement of amino acid residues in the neighborhood of Cys44. We propose that the Cys44-induced anomaly in ubiquinone represents its activated state, which drives the DsbB-mediated electron transfer.

Amino Acids↗
PubMed2003

Nearest neighbor analysis of the SecYEG complex. 1. Identification of a SecY-SecG interface.

Integral membrane components SecY, SecE, and SecG of protein translocase form a complex in the Escherichia coli plasma membrane. To characterize subunit interactions of the SecYEG complex, a series of SecY variants having a single cysteine in its cytoplasmic (C1-C6) or periplasmic (P1-P5) domain were subjected to site-specific cross-linking experiments using bifunctional agents with thiol-amine reactivity. Experiments using inverted membrane vesicles revealed specific cross-linkings between a cysteine residue placed in the C2 or C3 domain of SecY and the cytosolic lysine (Lys26) near the first transmembrane segment of SecG. These SecY Cys residues also formed a disulfide bond with an engineered cytosolic cysteine at position 28 of SecG. Thus, the C2-C3 region of SecY is in the proximity of the N-terminal half of the SecG cytoplasmic loop. Experiments using spheroplasts revealed the physical proximity of P2 (SecY) and the C-terminal periplasmic region of SecG. In addition, mutations in secG were isolated as suppressors against a cold-sensitive mutation (secY104) affecting the TM4-C3 boundary of SecY. These results collectively suggest that a C2-TM3-P2-TM4-C3 region of SecY serves as an interface with SecG.

Amino Acid Sequence↗
PubMed2003

Nearest neighbor analysis of the SecYEG complex. 2. Identification of a SecY-SecE cytosolic interface.

Although the importance of interactions involving both the cytosolic and transmembrane regions of SecY and SecE has been documented, no information has been available for the physical contact sites of these translocase subunits in their cytosolic domains. We now carried out site-specific cross-linking experiments to identify SecY and SecE regions that are physically close. Cysteines introduced into SecY residue 244 in the fourth cytosolic domain (C4) as well as into residues 354-356 and 362 in the C5 domain could be cross-linked with natural or engineered residues at positions 79 and 81 in the central part of the cytosolic loop of SecE. These cross-linkages were abolished by the Gly240 mutation in the SecY C4 region as well as by prlG alterations in SecE transmembrane segment 3, known to compromise SecY-SecE interaction. We suggest that the cytosolic and intramembrane interactions bring these two subunits together, forming a functionally crucial SecYE interface involving the SecY C5 region and the conserved cytosolic segment of SecE.

Amino Acid Sequence↗
PubMed2003

Reconstitution of membrane proteolysis by FtsH.

Escherichia coli FtsH is a membrane-bound and ATP-dependent protease responsible for degradation of several membrane proteins. The FtsH action is processive and presumably involves dislocation of the substrate from the membrane to the cytosol. Although elucidation of its molecular mechanism requires an in vitro reaction system, in vitro activities of this enzyme against membrane protein substrates have only been assayed using detergent-solubilized components. Here we report on the construction of in vitro reaction systems for FtsH-catalyzed membrane protein degradation. A combination of two inverted membrane vesicles or of two proteoliposomes, one bearing the enzyme and the other bearing a substrate, was fused by polyethylene glycol 3350 treatment. Addition of ATP then resulted in degradation of the substrate. It was shown that FtsH can function in the process of membrane proteins degradation without aid from any other cellular factors.

ATP-Dependent Proteases↗
PubMed2003

Characterization of mutations in the GTP-binding domain of IF2 resulting in cold-sensitive growth of Escherichia coli.

The infB gene encodes translation initiation factor IF2. We have determined the entire sequence of infB from two cold-sensitive Escherichia coli strains IQ489 and IQ490. These two strains have been isolated as suppressor strains for the temperature-sensitive secretion mutation secY24. The mutations causing the suppression phenotype are located within infB. The only variations from the wild-type (wt) infB found in the two mutant strains are a replacement of Asp409 with Glu in strain IQ489 and an insertion of Gly between Ala421 and Gly422 in strain IQ490. Both positions are located in the GTP-binding G-domain of IF2. A model of the G-domain of E.coli IF2 is presented in. Physiological quantities of the recombinant mutant proteins were expressed in vivo in E.coli strains from which the chromosomal infB gene has been inactivated. At 42 degrees C, the mutants sustained normal cell growth, whereas a significant decrease in growth rate was found at 25 degrees C for both mutants as compared to wt IF2 expressed in the control strain. Circular dichroism spectra were recorded of the wt and the two mutant proteins to investigate the structural properties of the proteins. The spectra are characteristic of alpha-helix dominated structure, and reveal a significant different behavior between the wt and mutant IF2s with respect to temperature-induced conformational changes. The temperature-induced conformational change of the wt IF2 is a two-state process. In a ribosome-dependent GTPase assay in vitro the two mutants showed practically no activity at temperatures below 10 degrees C and a reduced activity at all temperatures up to 45 degrees C, as compared to wt IF2. The results indicate that the amino acid residues, Asp409 and Gly422, are located in important regions of the IF2 G-domain and demonstrate the importance of GTP hydrolysis in translation initiation for optimal cell growth.

Amino Acid Sequence↗
PubMed2003

Fluorescence resonance energy transfer analysis of protein translocase. SecYE from Thermus thermophilus HB8 forms a constitutive oligomer in membranes.

SecY and SecE are the two principal translocase subunits that create a channel-like pathway for the transit of preprotein across the bacterial cytoplasmic membrane. Here we report the cloning, expression, and purification of the SecYE complex (TSecYE) from a thermophilic bacterium, Thermus thermophilus HB8. Purified TSecYE can be reconstituted into proteoliposomes that function in T. thermophilus SecA (TSecA) dependent preprotein translocation. After the mixing of TSecYE derivatives labeled with either a donor or an acceptor fluorophore during reconstitution, fluorescence resonance energy transfer experiments demonstrated that 2 or more units of TSecYE in the lipid bilayer associate to form a largely non-exchangeable oligomeric structure.

Amino Acid Sequence↗
PubMed2003

Biochemical characterization of a mutationally altered protein translocase: proton motive force stimulation of the initiation phase of translocation.

Protein translocation across the Escherichia coli plasma membrane is facilitated by concerted actions of the SecYEG integral membrane complex and the SecA ATPase. A secY mutation (secY39) affects Arg357, an evolutionarily conserved and functionally important residue, and impairs the translocation function in vivo and in vitro. In this study, we used the "superactive" mutant forms of SecA, which suppress the SecY39 deficiency, to characterize the mutationally altered SecY39EG translocase. It was found that SecY39-mediated preprotein translocation exhibited absolute dependence on the proton motive force. The proton motive force-dependent step proved to lie before signal peptide cleavage. We suggest that the proton motive force assists in the initiation phase of protein translocation.

Adenosine Triphosphatases↗
PubMed2003

A SecE mutation that modulates SecY-SecE translocase assembly, identified as a specific suppressor of SecY defects.

The SecY39(Cs) (cold-sensitive) alteration of Arg357 results in a defect of translocation initiation. As a means to dissect the Sec translocation machinery, we isolated mutations that act as suppressors of the secY39 defect. A specific secE mutation, designated secE105, was thus isolated. This mutation proved to be identical with the prlG2 mutation and to suppress a number of cold-sensitive secY mutations. However, other prlG mutations did not effectively suppress the secY defects. Evidence indicates that the Ser105-to-Pro alteration in the C-terminal transmembrane segment of SecE weakens SecY-SecE association. In vitro analyses showed that the SecE(S105P) alteration preferentially stimulates the initial phase of translocation. It is suggested that the S105P alteration affects the SecYEG channel such that it is more prone to open and to accept the translocation initiation domain of a preprotein molecule.

Adenosine Triphosphatases↗
PubMed2002

Superactive SecY variants that fulfill the essential translocation function with a reduced cellular quantity.

The fifth and the sixth cytoplasmic regions (C5 and C6) of SecY are important for the SecA-driven preprotein translocation reaction. A cold-sensitive mutation, secY205 (Tyr-429 --> Asp), in C6 impairs the ATP- and precursor-dependent SecA insertion into the membrane. We now identified second site mutations that suppressed the defect. Cis-placement of these mutations proved to suppress mutations at another essential residue (Arg-357) of SecY as well. Thus, they tolerate the otherwise defective SecY alterations in the same molecule. Two alterations (Ile-195 to Ser in TM5 region and Ile-408 to Leu in TM10 region) were found to make the translocation channel more active, because it enabled cells to survive with reduced content of the SecYE complex. These mutations only very weakly suppressed a signal sequence defect of the lambda receptor protein. The mutant SecYEG translocase exhibited higher than normal activity in vitro, being accompanied by striking independence of the proton motive force as well as by stabilization of a bound and active SecA species against urea treatment. These results have been interpreted in terms of balance shifts between channel closing and channel opening alterations in the SecYEG translocase.

Amino Acid Sequence↗
PubMed2002

YaeL (EcfE) activates the sigma(E) pathway of stress response through a site-2 cleavage of anti-sigma(E), RseA.

Escherichia coli YaeL (EcfE) is a homolog of human site-2 protease (S2P), a membrane-bound zinc metalloprotease involved in regulated intramembrane proteolysis. We have shown previously that YaeL, having essential metalloprotease active site motifs in the cytoplasmic domain, is indispensable for viability. Here, we obtained rpoE, encoding an extracytoplasmic stress response sigma factor (sigma(E)), as a multicopy suppressor against the yaeL disruption. Whereas sigma(E) is thought to be activated by regulated cleavage of RseA on the periplasmic side by the DegS protease, we found that a degradation intermediate of RseA consisting of the transmembrane and the cytoplasmic domains accumulated in the YaeL-depleted cells. This intermediate was degraded on expression of YaeL but not of its metalloprotease motif mutants. Cells depleted of YaeL were incapable of activating a sigma(E)-dependent promoter in response to an envelope stress. It is suggested that sigma(E) activation involves two successive proteolytic cleavages: first, at a periplasmic site by DegS; second, at a cytoplasmic or intramembrane site by YaeL. Thus, YaeL is positively required for the sigma(E) extracytoplasmic stress response.

Amino Acid Motifs↗