Mutational Analysis of Transmembrane Regions 3 and 4 of SecY, a Central Component of Protein Translocase

Mori, Hiroyuki; Shimokawa, Naofumi; Satoh, Yasuyuki; Ito, Koreaki

doi:10.1128/jb.186.12.3960-3969.2004

Cited by 11 publications

(8 citation statements)

References 29 publications

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“…Alkylation of the denatured protein by malPEG in this second step would indicate that the cysteines were not accessible to AMS in spheroplasts in the first step; lack of alkylation would indicate that they were accessible to AMS in spheroplasts. This approach, previously used by Mori et al (2004), is very useful for determining cysteine accessibility, and we refer to it as counter‐alkylation.…”

Section: Resultsmentioning

confidence: 99%

Redox-active cysteines of a membrane electron transporter DsbD show dual compartment accessibility

Cho

Porat

et al. 2007

EMBO J

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The membrane-embedded domain of the unusual electron transporter DsbD (DsbDb) uses two redox-active cysteines to catalyze electron transfer between thioredoxin-fold polypeptides on opposite sides of the bacterial cytoplasmic membrane. How the electrons are transferred across the membrane is unknown. Here, we show that DsbDb displays an inherent functional and structural symmetry: first, the two cysteines of DsbDb can be alkylated from both the cytoplasm and the periplasm. Second, when the two cysteines are disulfide-bonded, cysteine scanning shows that the C-terminal halves of the cysteine-containing transmembrane segments 1 and 4 are exposed to the aqueous environment while the N-terminal halves are not. Third, proline residues located pseudo-symmetrically around the two cysteines are required for redox activity and accessibility of the cysteines. Fourth, mixed disulfide complexes, apparent intermediates in the electron transfer process, are detected between DsbDb and thioredoxin molecules on each side of the membrane. We propose a model where the two redox-active cysteines are located at the center of the membrane, accessible on both sides of the membrane to the thioredoxin proteins.

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Section: Resultsmentioning

confidence: 99%

Redox-active cysteines of a membrane electron transporter DsbD show dual compartment accessibility

Cho

Porat

et al. 2007

EMBO J

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“…On the 3D structure model of SecY (2,38), the side chains of Ser-111 (C2), Tyr-248 (C4), Ser-262 (C4), Lys-347 (C5), and Lys-434 (C6) all are located at or near the tips of the cytoplasmic protrusions (Fig. 6A).…”

Section: Discussionmentioning

confidence: 99%

Different modes of SecY–SecA interactions revealed by site-directed in vivo photo-cross-linking

Mori

Ito

2006

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

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116

110

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Benzoyl-phenylalanine introduced into specific SecY positions at the second, fourth, fifth, and sixth cytoplasmic domains allowed UV cross-linking with SecA. Cross-linked products exhibited two distinct electrophoretic mobilities. SecA cross-linking at the most C-terminal cytoplasmic region (C6) was specifically enhanced in the presence of NaN3, which arrests the ATPase cycle, and this enhancement was canceled by cis placement of some secY mutations that affect SecY-SecA cooperation. In vitro experiments showed directly that SecA approaches C6 when it is engaging in ATPdependent preprotein translocation. On the basis of these findings, we propose that the C6 tail of SecY interacts with the working form of SecA, whereas C4 -C5 loops may offer constitutive SecA-binding sites.protein translocation ͉ translocon

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“…The formation of protein disulfides in the membrane has also been used to analyze the interactions between two different proteins. The interaction between SecE and SecY was studied by cysteine scanning (33), as was the interaction between SecG and SecY (25). Intramolecular contacts between two transmembrane regions were found in the Tar receptor protein (28), the lactose permease (34), and the leader peptidase (35).…”

Section: Discussionmentioning

confidence: 99%

Cysteine Residues in the Transmembrane Regions of M13 Procoat Protein Suggest that Oligomeric Coat Proteins Assemble onto Phage Progeny

Nagler

Kühn

2007

J Bacteriol

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The M13 phage assembles in the inner membrane of Escherichia coli. During maturation, about 2,700 copies of the major coat protein move from the membrane onto a single-stranded phage DNA molecule that extrudes out of the cell. The major coat protein is synthesized as a precursor, termed procoat protein, and inserts into the membrane via a Sec-independent pathway. It is processed by a leader peptidase from its leader (signal) peptide before it is assembled onto the phage DNA. The transmembrane regions of the procoat protein play an important role in all these processes. Using cysteine mutants with mutations in the transmembrane regions of the procoat and coat proteins, we investigated which of the residues are involved in multimer formation, interaction with the leader peptidase, and formation of M13 progeny particles. We found that most single cysteine residues do not interfere with the membrane insertion, processing, and assembly of the phage. Treatment of the cells with copper phenanthroline showed that the cysteine residues were readily engaged in dimer and multimer formation. This suggests that the coat proteins assemble into multimers before they proceed onto the nascent phage particles. In addition, we found that when a cysteine is located in the leader peptide at the ؊6 position, processing of the mutant procoat protein and of other exported proteins is affected. This inhibition of the leader peptidase results in death of the cell and shows that there are distinct amino acid residues in the M13 procoat protein involved at specific steps of the phage assembly process.

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Mutational Analysis of Transmembrane Regions 3 and 4 of SecY, a Central Component of Protein Translocase

Cited by 11 publications

References 29 publications

Redox-active cysteines of a membrane electron transporter DsbD show dual compartment accessibility

Redox-active cysteines of a membrane electron transporter DsbD show dual compartment accessibility

Different modes of SecY–SecA interactions revealed by site-directed in vivo photo-cross-linking

Cysteine Residues in the Transmembrane Regions of M13 Procoat Protein Suggest that Oligomeric Coat Proteins Assemble onto Phage Progeny

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