Cargando…

Real-Time Fluorescence Detection of ERAD Substrate Retrotranslocation in a Mammalian In Vitro System

Secretory proteins unable to assemble into their native states in the endoplasmic reticulum (ER) are transported back or “retrotranslocated” into the cytosol for ER-associated degradation (ERAD). To examine the roles of different components in ERAD, one fluorescence-labeled ERAD substrate was encaps...

Descripción completa

Detalles Bibliográficos
Autores principales: Wahlman, Judit, DeMartino, George N., Skach, William R., Bulleid, Neil J., Brodsky, Jeffrey L., Johnson, Arthur E.
Formato: Texto
Lenguaje:English
Publicado: Cell Press 2007
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1890003/
https://www.ncbi.nlm.nih.gov/pubmed/17540174
http://dx.doi.org/10.1016/j.cell.2007.03.046
_version_ 1782133710582185984
author Wahlman, Judit
DeMartino, George N.
Skach, William R.
Bulleid, Neil J.
Brodsky, Jeffrey L.
Johnson, Arthur E.
author_facet Wahlman, Judit
DeMartino, George N.
Skach, William R.
Bulleid, Neil J.
Brodsky, Jeffrey L.
Johnson, Arthur E.
author_sort Wahlman, Judit
collection PubMed
description Secretory proteins unable to assemble into their native states in the endoplasmic reticulum (ER) are transported back or “retrotranslocated” into the cytosol for ER-associated degradation (ERAD). To examine the roles of different components in ERAD, one fluorescence-labeled ERAD substrate was encapsulated with selected lumenal factors inside mammalian microsomes. After mixing microsomes with fluorescence-quenching agents and selected cytosolic proteins, the rate of substrate efflux was monitored continuously in real time by the decrease in fluorescence intensity as cytosolic quenchers contacted dye-labeled substrates. The retrotranslocation kinetics of nonglycosylated pro-α factor were not significantly altered by replacing all lumenal proteins with only protein disulfide isomerase or all cytosolic proteins with only PA700, the 19S regulatory particle of the 26S proteasome. Retrotranslocation was blocked by antibodies against a putative retrotranslocation channel protein, derlin-1, but not Sec61α. In addition, pro-α factor photocrosslinked derlin-1, but not Sec61α. Thus, derlin-1 appears to be involved in pro-α factor retrotranslocation.
format Text
id pubmed-1890003
institution National Center for Biotechnology Information
language English
publishDate 2007
publisher Cell Press
record_format MEDLINE/PubMed
spelling pubmed-18900032007-06-11 Real-Time Fluorescence Detection of ERAD Substrate Retrotranslocation in a Mammalian In Vitro System Wahlman, Judit DeMartino, George N. Skach, William R. Bulleid, Neil J. Brodsky, Jeffrey L. Johnson, Arthur E. Cell Article Secretory proteins unable to assemble into their native states in the endoplasmic reticulum (ER) are transported back or “retrotranslocated” into the cytosol for ER-associated degradation (ERAD). To examine the roles of different components in ERAD, one fluorescence-labeled ERAD substrate was encapsulated with selected lumenal factors inside mammalian microsomes. After mixing microsomes with fluorescence-quenching agents and selected cytosolic proteins, the rate of substrate efflux was monitored continuously in real time by the decrease in fluorescence intensity as cytosolic quenchers contacted dye-labeled substrates. The retrotranslocation kinetics of nonglycosylated pro-α factor were not significantly altered by replacing all lumenal proteins with only protein disulfide isomerase or all cytosolic proteins with only PA700, the 19S regulatory particle of the 26S proteasome. Retrotranslocation was blocked by antibodies against a putative retrotranslocation channel protein, derlin-1, but not Sec61α. In addition, pro-α factor photocrosslinked derlin-1, but not Sec61α. Thus, derlin-1 appears to be involved in pro-α factor retrotranslocation. Cell Press 2007-06-01 /pmc/articles/PMC1890003/ /pubmed/17540174 http://dx.doi.org/10.1016/j.cell.2007.03.046 Text en . https://creativecommons.org/licenses/by/3.0/ Open Access under CC BY 3.0 (https://creativecommons.org/licenses/by/3.0/) license
spellingShingle Article
Wahlman, Judit
DeMartino, George N.
Skach, William R.
Bulleid, Neil J.
Brodsky, Jeffrey L.
Johnson, Arthur E.
Real-Time Fluorescence Detection of ERAD Substrate Retrotranslocation in a Mammalian In Vitro System
title Real-Time Fluorescence Detection of ERAD Substrate Retrotranslocation in a Mammalian In Vitro System
title_full Real-Time Fluorescence Detection of ERAD Substrate Retrotranslocation in a Mammalian In Vitro System
title_fullStr Real-Time Fluorescence Detection of ERAD Substrate Retrotranslocation in a Mammalian In Vitro System
title_full_unstemmed Real-Time Fluorescence Detection of ERAD Substrate Retrotranslocation in a Mammalian In Vitro System
title_short Real-Time Fluorescence Detection of ERAD Substrate Retrotranslocation in a Mammalian In Vitro System
title_sort real-time fluorescence detection of erad substrate retrotranslocation in a mammalian in vitro system
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1890003/
https://www.ncbi.nlm.nih.gov/pubmed/17540174
http://dx.doi.org/10.1016/j.cell.2007.03.046
work_keys_str_mv AT wahlmanjudit realtimefluorescencedetectionoferadsubstrateretrotranslocationinamammalianinvitrosystem
AT demartinogeorgen realtimefluorescencedetectionoferadsubstrateretrotranslocationinamammalianinvitrosystem
AT skachwilliamr realtimefluorescencedetectionoferadsubstrateretrotranslocationinamammalianinvitrosystem
AT bulleidneilj realtimefluorescencedetectionoferadsubstrateretrotranslocationinamammalianinvitrosystem
AT brodskyjeffreyl realtimefluorescencedetectionoferadsubstrateretrotranslocationinamammalianinvitrosystem
AT johnsonarthure realtimefluorescencedetectionoferadsubstrateretrotranslocationinamammalianinvitrosystem