Cargando…

Cellular Entry of the Diphtheria Toxin Does Not Require the Formation of the Open-Channel State by Its Translocation Domain

Cellular entry of diphtheria toxin is a multistage process involving receptor targeting, endocytosis, and translocation of the catalytic domain across the endosomal membrane into the cytosol. The latter is ensured by the translocation (T) domain of the toxin, capable of undergoing conformational ref...

Descripción completa

Detalles Bibliográficos
Autores principales: Ladokhin, Alexey S., Vargas-Uribe, Mauricio, Rodnin, Mykola V., Ghatak, Chiranjib, Sharma, Onkar
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5666346/
https://www.ncbi.nlm.nih.gov/pubmed/28937631
http://dx.doi.org/10.3390/toxins9100299
_version_ 1783275291678867456
author Ladokhin, Alexey S.
Vargas-Uribe, Mauricio
Rodnin, Mykola V.
Ghatak, Chiranjib
Sharma, Onkar
author_facet Ladokhin, Alexey S.
Vargas-Uribe, Mauricio
Rodnin, Mykola V.
Ghatak, Chiranjib
Sharma, Onkar
author_sort Ladokhin, Alexey S.
collection PubMed
description Cellular entry of diphtheria toxin is a multistage process involving receptor targeting, endocytosis, and translocation of the catalytic domain across the endosomal membrane into the cytosol. The latter is ensured by the translocation (T) domain of the toxin, capable of undergoing conformational refolding and membrane insertion in response to the acidification of the endosomal environment. While numerous now classical studies have demonstrated the formation of an ion-conducting conformation—the Open-Channel State (OCS)—as the final step of the refolding pathway, it remains unclear whether this channel constitutes an in vivo translocation pathway or is a byproduct of the translocation. To address this question, we measure functional activity of known OCS-blocking mutants with H-to-Q replacements of C-terminal histidines of the T-domain. We also test the ability of these mutants to translocate their own N-terminus across lipid bilayers of model vesicles. The results of both experiments indicate that translocation activity does not correlate with previously published OCS activity. Finally, we determined the topology of TH5 helix in membrane-inserted T-domain using W281 fluorescence and its depth-dependent quenching by brominated lipids. Our results indicate that while TH5 becomes a transbilayer helix in a wild-type protein, it fails to insert in the case of the OCS-blocking mutant H322Q. We conclude that the formation of the OCS is not necessary for the functional translocation by the T-domain, at least in the histidine-replacement mutants, suggesting that the OCS is unlikely to constitute a translocation pathway for the cellular entry of diphtheria toxin in vivo.
format Online
Article
Text
id pubmed-5666346
institution National Center for Biotechnology Information
language English
publishDate 2017
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-56663462017-11-09 Cellular Entry of the Diphtheria Toxin Does Not Require the Formation of the Open-Channel State by Its Translocation Domain Ladokhin, Alexey S. Vargas-Uribe, Mauricio Rodnin, Mykola V. Ghatak, Chiranjib Sharma, Onkar Toxins (Basel) Article Cellular entry of diphtheria toxin is a multistage process involving receptor targeting, endocytosis, and translocation of the catalytic domain across the endosomal membrane into the cytosol. The latter is ensured by the translocation (T) domain of the toxin, capable of undergoing conformational refolding and membrane insertion in response to the acidification of the endosomal environment. While numerous now classical studies have demonstrated the formation of an ion-conducting conformation—the Open-Channel State (OCS)—as the final step of the refolding pathway, it remains unclear whether this channel constitutes an in vivo translocation pathway or is a byproduct of the translocation. To address this question, we measure functional activity of known OCS-blocking mutants with H-to-Q replacements of C-terminal histidines of the T-domain. We also test the ability of these mutants to translocate their own N-terminus across lipid bilayers of model vesicles. The results of both experiments indicate that translocation activity does not correlate with previously published OCS activity. Finally, we determined the topology of TH5 helix in membrane-inserted T-domain using W281 fluorescence and its depth-dependent quenching by brominated lipids. Our results indicate that while TH5 becomes a transbilayer helix in a wild-type protein, it fails to insert in the case of the OCS-blocking mutant H322Q. We conclude that the formation of the OCS is not necessary for the functional translocation by the T-domain, at least in the histidine-replacement mutants, suggesting that the OCS is unlikely to constitute a translocation pathway for the cellular entry of diphtheria toxin in vivo. MDPI 2017-09-22 /pmc/articles/PMC5666346/ /pubmed/28937631 http://dx.doi.org/10.3390/toxins9100299 Text en © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Ladokhin, Alexey S.
Vargas-Uribe, Mauricio
Rodnin, Mykola V.
Ghatak, Chiranjib
Sharma, Onkar
Cellular Entry of the Diphtheria Toxin Does Not Require the Formation of the Open-Channel State by Its Translocation Domain
title Cellular Entry of the Diphtheria Toxin Does Not Require the Formation of the Open-Channel State by Its Translocation Domain
title_full Cellular Entry of the Diphtheria Toxin Does Not Require the Formation of the Open-Channel State by Its Translocation Domain
title_fullStr Cellular Entry of the Diphtheria Toxin Does Not Require the Formation of the Open-Channel State by Its Translocation Domain
title_full_unstemmed Cellular Entry of the Diphtheria Toxin Does Not Require the Formation of the Open-Channel State by Its Translocation Domain
title_short Cellular Entry of the Diphtheria Toxin Does Not Require the Formation of the Open-Channel State by Its Translocation Domain
title_sort cellular entry of the diphtheria toxin does not require the formation of the open-channel state by its translocation domain
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5666346/
https://www.ncbi.nlm.nih.gov/pubmed/28937631
http://dx.doi.org/10.3390/toxins9100299
work_keys_str_mv AT ladokhinalexeys cellularentryofthediphtheriatoxindoesnotrequiretheformationoftheopenchannelstatebyitstranslocationdomain
AT vargasuribemauricio cellularentryofthediphtheriatoxindoesnotrequiretheformationoftheopenchannelstatebyitstranslocationdomain
AT rodninmykolav cellularentryofthediphtheriatoxindoesnotrequiretheformationoftheopenchannelstatebyitstranslocationdomain
AT ghatakchiranjib cellularentryofthediphtheriatoxindoesnotrequiretheformationoftheopenchannelstatebyitstranslocationdomain
AT sharmaonkar cellularentryofthediphtheriatoxindoesnotrequiretheformationoftheopenchannelstatebyitstranslocationdomain