Transmembrane protein topology mapping by the substituted cysteine accessibility method (SCAMTM): Application to lipid-specific membrane protein topogenesis

Bogdanov, Mikhail; Zhang, Wei; Xie, Jun; Dowhan, William

doi:10.1016/j.ymeth.2004.11.002

Cited by 139 publications

(185 citation statements)

References 145 publications

(380 reference statements)

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“…To test these predictions, we used a cysteine accessibility approach. This method is based on the accessibility of cysteine residues to 3-(N-maleimidylpropionyl) biocytin (MPB), a sulfhydryl reagent that readily passes the OM but only inefficiently the IM of Gram-negative bacteria (27). Based on TMH predictions, cysteine substitutions were introduced into the cysteine-less PorL protein at various positions.…”

Section: Resultsmentioning

confidence: 99%

Characterization of the Porphyromonas gingivalis Type IX Secretion Trans-envelope PorKLMNP Core Complex

Vincent¹,

Canestrari²,

Leone³

et al. 2017

Journal of Biological Chemistry

125

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Edited by Thomas SöllnerThe transport of proteins at the cell surface of Bacteroidetes depends on a secretory apparatus known as type IX secretion system (T9SS). This machine is responsible for the cell surface exposition of various proteins, such as adhesins, required for gliding motility in Flavobacterium, S-layer components in Tannerella forsythia, and tooth tissue-degrading enzymes in the oral pathogen Porphyromonas gingivalis. Although a number of subunits of the T9SS have been identified, we lack details on the architecture of this secretion apparatus. Here we provide evidence that five of the genes encoding the core complex of the T9SS are co-transcribed and that the gene products are distributed in the cell envelope. Protein-protein interaction studies then revealed that these proteins oligomerize and interact through a dense network of contacts.

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Section: Resultsmentioning

confidence: 99%

Characterization of the Porphyromonas gingivalis Type IX Secretion Trans-envelope PorKLMNP Core Complex

Vincent¹,

Canestrari²,

Leone³

et al. 2017

Journal of Biological Chemistry

125

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“…The exact topology of SaeS in the cytoplasmic membrane has not been reported. To address this, we used the substituted cysteine accessibility method (SCAM) (19), which is a technique that allows membrane protein topology to be determined with single amino acid resolution and has recently been adapted for use in S. aureus (20). SCAM requires protein constructs that contain only one cysteine residue, which is labeled with a maleimide ring conjugated to biotin (MPB).…”

Section: Resultsmentioning

confidence: 99%

Differential regulation of staphylococcal virulence by the sensor kinase SaeS in response to neutrophil-derived stimuli

Flack

Zurek

Meishery

et al. 2014

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

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Two-component systems (TCSs) are highly conserved across bacteria and are used to rapidly sense and respond to changing environmental conditions. The human pathogen Staphylococcus aureus uses the S. aureus exoprotein expression (sae) TCS to sense host signals and activate transcription of virulence factors essential to pathogenesis. Despite its importance, the mechanism by which the histidine kinase SaeS recognizes specific host stimuli is unknown. After mutagenizing the predicted extracellular loop of SaeS, we discovered one methionine residue (M31) was essential for the ability of S. aureus to transcribe sae target genes, including hla, lukAB/lukGH, and hlgA. This single M31A mutation also significantly reduced cytotoxicity in human neutrophils to levels observed in cells following interaction with ΔsaeS. Another important discovery was that mutation of two aromatic anchor residues (W32A and F33A) disrupted the normal basal signaling of SaeS in the absence of inducing signals, yet both mutant kinases had appropriate activation of effector genes following exposure to neutrophils. Although the transcriptional profile of aromatic mutation W32A was consistent with that of WT in response to human α-defensin 1, mutant kinase F33A did not properly transcribe the γ-toxin genes in response to this stimulus. Taken together, our results provide molecular evidence for how SaeS recognizes host signals and triggers activation of select virulence factors to facilitate evasion of innate immunity. These findings have important implications for signal transduction in prokaryotes and eukaryotes due to conservation of aromatic anchor residues across both of these domains and the important role they play in sensor protein structure and function.host-pathogen interactions | membrane proteins

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“…To verify this unusual hydropathic arrangement experimentally, we examined the water accessibility of YidC TM residues using intact living cells and the N-ethylmaleimide (NEM) reactivity assay. NEM is membranepermeable and alkylates the thiol group of a Cys residue of protein in a water-dependent reaction (23)(24)(25), enabling us to assess the water availability of a specific site of the target protein in intact cells by strategically placing a Cys residue.…”

Section: Resultsmentioning

confidence: 99%

Hydrophilic microenvironment required for the channel-independent insertase function of YidC protein

Shimokawa-Chiba

Kumazaki

Tsukazaki

et al. 2015

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

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The recently solved crystal structure of YidC protein suggests that it mediates membrane protein insertion by means of an intramembrane cavity rather than a transmembrane (TM) pore. This concept of protein translocation prompted us to characterize the native, membrane-integrated state of YidC with respect to the hydropathic nature of its TM region. Here, we show that the cavityforming region of the stage III sporulation protein J (SpoIIIJ), a YidC homolog, is indeed open to the aqueous milieu of the Bacillus subtilis cells and that the overall hydrophilicity of the cavity, along with the presence of an Arg residue on several alternative sites of the cavity surface, is functionally important. We propose that YidC functions as a proteinaceous amphiphile that interacts with newly synthesized membrane proteins and reduces energetic costs of their membrane traversal.

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Transmembrane protein topology mapping by the substituted cysteine accessibility method (SCAMTM): Application to lipid-specific membrane protein topogenesis

Cited by 139 publications

References 145 publications

Characterization of the Porphyromonas gingivalis Type IX Secretion Trans-envelope PorKLMNP Core Complex

Characterization of the Porphyromonas gingivalis Type IX Secretion Trans-envelope PorKLMNP Core Complex

Differential regulation of staphylococcal virulence by the sensor kinase SaeS in response to neutrophil-derived stimuli

Hydrophilic microenvironment required for the channel-independent insertase function of YidC protein

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