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

Judit Wahlman, George N. DeMartino, William R. Skach, Neil J. Bulleid, Jeffrey L. Brodsky, Arthur E Johnson

Research output: Contribution to journalArticle

100 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)943-955
Number of pages13
JournalCell
Volume129
Issue number5
DOIs
StatePublished - Jun 1 2007

Fingerprint

Fluorescence
Degradation
Substrates
Microsomes
Proteins
Endoplasmic Reticulum-Associated Degradation
Protein Disulfide-Isomerases
Fluorescent Dyes
Endoplasmic Reticulum
Cytosol
Quenching
Coloring Agents
In Vitro Techniques
Kinetics
Antibodies

Keywords

  • CELLBIO
  • PROTEINS

ASJC Scopus subject areas

  • Cell Biology
  • Molecular Biology

Cite this

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.

In: Cell, Vol. 129, No. 5, 01.06.2007, p. 943-955.

Research output: Contribution to journalArticle

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. In: Cell. 2007 ; Vol. 129, No. 5. pp. 943-955.
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