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 E. Johnson

doi:10.1016/j.cell.2007.03.046

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 E. Johnson

Biochemistry & Molecular Biology

Research output: Contribution to journal › Article › peer-review

114 Scopus citations

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 language	English (US)
Pages (from-to)	943-955
Number of pages	13
Journal	Cell
Volume	129
Issue number	5
DOIs	https://doi.org/10.1016/j.cell.2007.03.046
State	Published - Jun 1 2007

Funding

We are grateful to Y. Miao and Y. Shao for outstanding technical assistance and S. Saksena for photocrosslinking assistance. This work was supported by NIH grants GM26494 (A.E.J.), DK46181 (G.N.D.), GM53457 and DK51818 (W.R.S.), and GM75061 (J.L.B.); Wellcome Trust grant #074081 (N.J.B.); and the Robert A. Welch Foundation (A.E.J., chair grant BE-0017; G.N.D., grant I-1500).

Funders	Funder number
Author National Institutes of Health National Institutes of Health National Institutes of Health National Institutes of Health The Bev Hartig Huntington's Disease Foundation National Institutes of Health	GM53457, GM75061, DK51818, GM26494
National Institute of Diabetes and Digestive and Kidney Diseases	R01DK046181
Welch Foundation	BE-0017, I-1500
Wellcome Trust	074081

Keywords

CELLBIO
PROTEINS

ASJC Scopus subject areas

General Biochemistry, Genetics and Molecular Biology

Access to Document

10.1016/j.cell.2007.03.046

Cite this

@article{4cbe91de2f1e4359942d32bef2bf211f,

title = "Real-Time Fluorescence Detection of ERAD Substrate Retrotranslocation in a Mammalian In Vitro System",

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.",

keywords = "CELLBIO, PROTEINS",

author = "Judit Wahlman and DeMartino, \{George N.\} and Skach, \{William R.\} and Bulleid, \{Neil J.\} and Brodsky, \{Jeffrey L.\} and Johnson, \{Arthur E E.\}",

note = "Funding Information: We are grateful to Y. Miao and Y. Shao for outstanding technical assistance and S. Saksena for photocrosslinking assistance. This work was supported by NIH grants GM26494 (A.E.J.), DK46181 (G.N.D.), GM53457 and DK51818 (W.R.S.), and GM75061 (J.L.B.); Wellcome Trust grant \#074081 (N.J.B.); and the Robert A. Welch Foundation (A.E.J., chair grant BE-0017; G.N.D., grant I-1500). ",

year = "2007",

month = jun,

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doi = "10.1016/j.cell.2007.03.046",

language = "English (US)",

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AU - DeMartino, George N.

AU - Skach, William R.

AU - Bulleid, Neil J.

AU - Brodsky, Jeffrey L.

AU - Johnson, Arthur E E.

N1 - Funding Information: We are grateful to Y. Miao and Y. Shao for outstanding technical assistance and S. Saksena for photocrosslinking assistance. This work was supported by NIH grants GM26494 (A.E.J.), DK46181 (G.N.D.), GM53457 and DK51818 (W.R.S.), and GM75061 (J.L.B.); Wellcome Trust grant #074081 (N.J.B.); and the Robert A. Welch Foundation (A.E.J., chair grant BE-0017; G.N.D., grant I-1500).

PY - 2007/6/1

Y1 - 2007/6/1

N2 - 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.

AB - 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.

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