2015
DOI: 10.1016/j.bbamem.2014.09.013
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Membrane translocation assay based on proteolytic cleavage: Application to diphtheria toxin T domain

Abstract: The function of diphtheria toxin translocation (T) domain is to transfer the catalytic domain across the endosomal membrane upon acidification. The goal of this study was to develop and apply an in vitro functional assay for T domain activity, suitable for investigation of structure-function relationships of translocation across lipid bilayers of various compositions. Traditionally, T domain activity in vitro is estimated by measuring either conductance in planar lipid bilayers or the release of fluorescent ma… Show more

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Cited by 6 publications
(14 citation statements)
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“…Although membrane leakage is a very sensitive way of detecting protein-membrane interactions, our previous studies with various mutants indicate that it provides a less specific measure of physiological activity (26), especially when compared to the N-terminus translocation assay (48). Here, we applied this protease-based method, which we previously developed to study T domain activity, to examine the translocational activity of both proteins as a function of pH.…”
Section: Resultsmentioning
confidence: 99%
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“…Although membrane leakage is a very sensitive way of detecting protein-membrane interactions, our previous studies with various mutants indicate that it provides a less specific measure of physiological activity (26), especially when compared to the N-terminus translocation assay (48). Here, we applied this protease-based method, which we previously developed to study T domain activity, to examine the translocational activity of both proteins as a function of pH.…”
Section: Resultsmentioning
confidence: 99%
“…The N-terminus translocation assay was performed using thrombin-loaded large unilamellar vesicles (LUVs) made of a 3:1 molar ratio of POPC and POPG, as described previously (48). The recombinantly expressed T domain in pET15b plasmid contains an N-terminal His-tag with thrombin cleavage-site treatment.…”
Section: N-terminus Translocation Assaymentioning
confidence: 99%
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“…This view is strongly supported by the high WT-like activity of OCS-blocking mutants H322Q, H323Q, and H372Q observed in both (a) N-terminus translocation in mutants of the T-domain relative to N-terminus translocation of the WT protein at pH 5.8. The assay is based on proteolytic cleavage of N-terminal segment preloaded into lipid vesicles followed by SDS-PAGE [49]. The data are normalized to the WT and are plotted against previously published OCS activity [38].…”
Section: Ocs: Critical Intermediate or Byproduct Of Translocationmentioning
confidence: 99%
“…In contrast, Pathway 1 indicates that the OCS is formed after the translocation, and passageway through the bilayer is formed via an unknown, possibly transient conformation. In order to distinguish between the two pathways, we will use new and published data to compare how various mutations affect the following measures of T-domain activity: (a) conductance in planar bilayers (i.e., formation of the OCS) [37,38,[45][46][47][48], (b) N-terminus translocation in vesicles [37,49], and ultimately (c) cell death assay based on inhibition of protein synthesis in vivo [37,46,48,50]. The starting structure on top corresponds to the crystal structure of the toxin at neutral pH [42], and consists of the C-domain (red), T-domain (helices color-coded according to OCS topology), and R-domain (green).…”
Section: Comparing the Two Translocation Pathwaysmentioning
confidence: 99%