ATP-stimulated degradation of endogenous proteins in cell free extracts of BHK 21/C13 fibroblasts A key role for the proteinase, macropain, in the ubiquitin-dependent degradation of short-lived proteins

George N. DeMartino, Marci L. McCullough, Jane F. Reckelhoff, Dorothy E. Cloall, Aaron Ciechanover, Michael J. McGuire

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13 Citations (Scopus)

Abstract

Baby hamster kidney (BHK) 21/C13 cell proteins, labeled with [35S]methionine, [14C]leucine or [3H]leucine in intact cells, were degraded in soluble, cell-free extracts by an ATP-stimulated process. The stimulatory effect of ATP appeared to require ATP hydrolysis and was mediated to a large extent by ubiquitin. Although the cell extracts contained endogenous ubiquitin, supplementation with exogeneous ubiquitin increased ATP-dependent proteolysis by up to 2-fold. Furthermore, antibodies against the E1 ubiquitin conjugating enzyme specifically inhibited both conjugation of [125I]ubiquitin to endogeneous proteins and ATP/ubiquitin-dependent proteolysis. Addition of purified E1 to antibody-treated extracts restored conjugation and proteolysis. Proteins containing the amino acid analogues canavanine and azatryptophan were also degraded in vitro by an ATP/ubiquitin-dependent process but at a rate up to 2-fold faster than normal proteins. These results indicate that soluble, cell-free extracts of BHK cells can selectively degrade proteins whose rate of degradation are increased in intact cells. Treatment of cell-free extracts with antibodies against the high molecular weight proteinase, macropain, also greatly inhibited the ATP/ubiquitin-dependent degradation of endogenous proteins. Proteolysis was specifically restored when purified macropain L was added to the antibody-treated extracts. Treatment of cell extracts with both anti-macropain and anti-E1 antibodies reduced ATP/ubiquitin-dependent proteolysis to the same extent as treatment with either antibody alone. Furthermore, proteolysis could be restored to the double antibody treated extracts only after addition of both purified E1 and macropain. These results provide strong evidence for an important role for macropain in the ATP/ubiquitin-dependent degradation of endogenous proteins in BHK cell extracts.

Original languageEnglish
Pages (from-to)299-308
Number of pages10
JournalBBA - General Subjects
Volume1073
Issue number2
DOIs
StatePublished - 4 Mar 1991

Fingerprint

Proteasome Endopeptidase Complex
Fibroblasts
Ubiquitin
Cell Extracts
Cricetinae
Proteolysis
Peptide Hydrolases
Adenosine Triphosphate
Kidney
Degradation
Antibodies
Proteins
Leucine
Canavanine
Cells
Ubiquitin-Conjugating Enzymes
Methionine
Anti-Idiotypic Antibodies
Hydrolysis
Molecular Weight

Keywords

  • ATP
  • Macropain
  • Multicatalytic proteinase
  • Proteasome
  • Protein degradation
  • Ubiquitin

Cite this

@article{af338fc534b544c6ac9f1edd107e67ac,
title = "ATP-stimulated degradation of endogenous proteins in cell free extracts of BHK 21/C13 fibroblasts A key role for the proteinase, macropain, in the ubiquitin-dependent degradation of short-lived proteins",
abstract = "Baby hamster kidney (BHK) 21/C13 cell proteins, labeled with [35S]methionine, [14C]leucine or [3H]leucine in intact cells, were degraded in soluble, cell-free extracts by an ATP-stimulated process. The stimulatory effect of ATP appeared to require ATP hydrolysis and was mediated to a large extent by ubiquitin. Although the cell extracts contained endogenous ubiquitin, supplementation with exogeneous ubiquitin increased ATP-dependent proteolysis by up to 2-fold. Furthermore, antibodies against the E1 ubiquitin conjugating enzyme specifically inhibited both conjugation of [125I]ubiquitin to endogeneous proteins and ATP/ubiquitin-dependent proteolysis. Addition of purified E1 to antibody-treated extracts restored conjugation and proteolysis. Proteins containing the amino acid analogues canavanine and azatryptophan were also degraded in vitro by an ATP/ubiquitin-dependent process but at a rate up to 2-fold faster than normal proteins. These results indicate that soluble, cell-free extracts of BHK cells can selectively degrade proteins whose rate of degradation are increased in intact cells. Treatment of cell-free extracts with antibodies against the high molecular weight proteinase, macropain, also greatly inhibited the ATP/ubiquitin-dependent degradation of endogenous proteins. Proteolysis was specifically restored when purified macropain L was added to the antibody-treated extracts. Treatment of cell extracts with both anti-macropain and anti-E1 antibodies reduced ATP/ubiquitin-dependent proteolysis to the same extent as treatment with either antibody alone. Furthermore, proteolysis could be restored to the double antibody treated extracts only after addition of both purified E1 and macropain. These results provide strong evidence for an important role for macropain in the ATP/ubiquitin-dependent degradation of endogenous proteins in BHK cell extracts.",
keywords = "ATP, Macropain, Multicatalytic proteinase, Proteasome, Protein degradation, Ubiquitin",
author = "DeMartino, {George N.} and McCullough, {Marci L.} and Reckelhoff, {Jane F.} and Cloall, {Dorothy E.} and Aaron Ciechanover and McGuire, {Michael J.}",
year = "1991",
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language = "English",
volume = "1073",
pages = "299--308",
journal = "Biochimica et Biophysica Acta - General Subjects",
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ATP-stimulated degradation of endogenous proteins in cell free extracts of BHK 21/C13 fibroblasts A key role for the proteinase, macropain, in the ubiquitin-dependent degradation of short-lived proteins. / DeMartino, George N.; McCullough, Marci L.; Reckelhoff, Jane F.; Cloall, Dorothy E.; Ciechanover, Aaron; McGuire, Michael J.

In: BBA - General Subjects, Vol. 1073, No. 2, 04.03.1991, p. 299-308.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - ATP-stimulated degradation of endogenous proteins in cell free extracts of BHK 21/C13 fibroblasts A key role for the proteinase, macropain, in the ubiquitin-dependent degradation of short-lived proteins

AU - DeMartino, George N.

AU - McCullough, Marci L.

AU - Reckelhoff, Jane F.

AU - Cloall, Dorothy E.

AU - Ciechanover, Aaron

AU - McGuire, Michael J.

PY - 1991/3/4

Y1 - 1991/3/4

N2 - Baby hamster kidney (BHK) 21/C13 cell proteins, labeled with [35S]methionine, [14C]leucine or [3H]leucine in intact cells, were degraded in soluble, cell-free extracts by an ATP-stimulated process. The stimulatory effect of ATP appeared to require ATP hydrolysis and was mediated to a large extent by ubiquitin. Although the cell extracts contained endogenous ubiquitin, supplementation with exogeneous ubiquitin increased ATP-dependent proteolysis by up to 2-fold. Furthermore, antibodies against the E1 ubiquitin conjugating enzyme specifically inhibited both conjugation of [125I]ubiquitin to endogeneous proteins and ATP/ubiquitin-dependent proteolysis. Addition of purified E1 to antibody-treated extracts restored conjugation and proteolysis. Proteins containing the amino acid analogues canavanine and azatryptophan were also degraded in vitro by an ATP/ubiquitin-dependent process but at a rate up to 2-fold faster than normal proteins. These results indicate that soluble, cell-free extracts of BHK cells can selectively degrade proteins whose rate of degradation are increased in intact cells. Treatment of cell-free extracts with antibodies against the high molecular weight proteinase, macropain, also greatly inhibited the ATP/ubiquitin-dependent degradation of endogenous proteins. Proteolysis was specifically restored when purified macropain L was added to the antibody-treated extracts. Treatment of cell extracts with both anti-macropain and anti-E1 antibodies reduced ATP/ubiquitin-dependent proteolysis to the same extent as treatment with either antibody alone. Furthermore, proteolysis could be restored to the double antibody treated extracts only after addition of both purified E1 and macropain. These results provide strong evidence for an important role for macropain in the ATP/ubiquitin-dependent degradation of endogenous proteins in BHK cell extracts.

AB - Baby hamster kidney (BHK) 21/C13 cell proteins, labeled with [35S]methionine, [14C]leucine or [3H]leucine in intact cells, were degraded in soluble, cell-free extracts by an ATP-stimulated process. The stimulatory effect of ATP appeared to require ATP hydrolysis and was mediated to a large extent by ubiquitin. Although the cell extracts contained endogenous ubiquitin, supplementation with exogeneous ubiquitin increased ATP-dependent proteolysis by up to 2-fold. Furthermore, antibodies against the E1 ubiquitin conjugating enzyme specifically inhibited both conjugation of [125I]ubiquitin to endogeneous proteins and ATP/ubiquitin-dependent proteolysis. Addition of purified E1 to antibody-treated extracts restored conjugation and proteolysis. Proteins containing the amino acid analogues canavanine and azatryptophan were also degraded in vitro by an ATP/ubiquitin-dependent process but at a rate up to 2-fold faster than normal proteins. These results indicate that soluble, cell-free extracts of BHK cells can selectively degrade proteins whose rate of degradation are increased in intact cells. Treatment of cell-free extracts with antibodies against the high molecular weight proteinase, macropain, also greatly inhibited the ATP/ubiquitin-dependent degradation of endogenous proteins. Proteolysis was specifically restored when purified macropain L was added to the antibody-treated extracts. Treatment of cell extracts with both anti-macropain and anti-E1 antibodies reduced ATP/ubiquitin-dependent proteolysis to the same extent as treatment with either antibody alone. Furthermore, proteolysis could be restored to the double antibody treated extracts only after addition of both purified E1 and macropain. These results provide strong evidence for an important role for macropain in the ATP/ubiquitin-dependent degradation of endogenous proteins in BHK cell extracts.

KW - ATP

KW - Macropain

KW - Multicatalytic proteinase

KW - Proteasome

KW - Protein degradation

KW - Ubiquitin

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DO - 10.1016/0304-4165(91)90135-4

M3 - Article

VL - 1073

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EP - 308

JO - Biochimica et Biophysica Acta - General Subjects

JF - Biochimica et Biophysica Acta - General Subjects

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ER -