Stable incorporation of ATPase subunits into 19 S regulatory particle of human proteasome requires nucleotide binding and C-terminal tails

Seung Hoon Lee, Joo Hong Moon, Sungjoo Kim Yoon, Jong-Bok Yoon

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

The 26 S proteasome is a large multi-subunit protein complex that degrades ubiquitinated proteins in eukaryotic cells. Proteasome assembly is a complex process that involves formation of sixand seven-membered ring structures from homologous subunits. Here we report that the assembly of hexameric Rpt ring of the 19 S regulatory particle (RP) requires nucleotide binding but not ATP hydrolysis. Disruption of nucleotide binding to an Rpt subunit by mutation in the Walker A motif inhibits the assembly of the Rpt ring without affecting heterodimer formation with its partner Rpt subunit. Coexpression of the base assembly chaperones S5b and PAAF1with mutant Rpt1 and Rpt6, respectively, relieves assembly inhibition of mutant Rpts by facilitating their interaction with adjacent Rpt dimers. The mutation in the Walker B motif which impairs ATP hydrolysis does not affect Rpt ring formation. Incorporation of a Walker B mutant Rpt subunit abrogates the ATPase activity of the 19 S RP, suggesting that failure of the mutant Rpt to undergo the conformational transition from an ATP-bound to an ADP-bound state impairs conformational changes in the other five wild-type Rpts in the Rpt ring. In addition, we demonstrate that the C-terminal tails of Rpt subunits possessing core particle (CP)-binding affinities facilitate the cellular assembly of the 19 SRP, implying that the 20 S CP may function as a template for base assembly in human cells. Taken together, these results suggest that the ATP-bound conformational state of an Rpt subunit with the exposed C-terminal tail is competent for cellular proteasome assembly.

Original languageEnglish
Pages (from-to)9269-9279
Number of pages11
JournalJournal of Biological Chemistry
Volume287
Issue number12
DOIs
Publication statusPublished - 2012 Mar 16

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Proteasome Endopeptidase Complex
Adenosine Triphosphatases
Tail
Nucleotides
Adenosine Triphosphate
Hydrolysis
Ubiquitinated Proteins
Mutation
Protein Subunits
Eukaryotic Cells
Adenosine Diphosphate
Dimers
Cells

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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title = "Stable incorporation of ATPase subunits into 19 S regulatory particle of human proteasome requires nucleotide binding and C-terminal tails",
abstract = "The 26 S proteasome is a large multi-subunit protein complex that degrades ubiquitinated proteins in eukaryotic cells. Proteasome assembly is a complex process that involves formation of sixand seven-membered ring structures from homologous subunits. Here we report that the assembly of hexameric Rpt ring of the 19 S regulatory particle (RP) requires nucleotide binding but not ATP hydrolysis. Disruption of nucleotide binding to an Rpt subunit by mutation in the Walker A motif inhibits the assembly of the Rpt ring without affecting heterodimer formation with its partner Rpt subunit. Coexpression of the base assembly chaperones S5b and PAAF1with mutant Rpt1 and Rpt6, respectively, relieves assembly inhibition of mutant Rpts by facilitating their interaction with adjacent Rpt dimers. The mutation in the Walker B motif which impairs ATP hydrolysis does not affect Rpt ring formation. Incorporation of a Walker B mutant Rpt subunit abrogates the ATPase activity of the 19 S RP, suggesting that failure of the mutant Rpt to undergo the conformational transition from an ATP-bound to an ADP-bound state impairs conformational changes in the other five wild-type Rpts in the Rpt ring. In addition, we demonstrate that the C-terminal tails of Rpt subunits possessing core particle (CP)-binding affinities facilitate the cellular assembly of the 19 SRP, implying that the 20 S CP may function as a template for base assembly in human cells. Taken together, these results suggest that the ATP-bound conformational state of an Rpt subunit with the exposed C-terminal tail is competent for cellular proteasome assembly.",
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Stable incorporation of ATPase subunits into 19 S regulatory particle of human proteasome requires nucleotide binding and C-terminal tails. / Lee, Seung Hoon; Moon, Joo Hong; Yoon, Sungjoo Kim; Yoon, Jong-Bok.

In: Journal of Biological Chemistry, Vol. 287, No. 12, 16.03.2012, p. 9269-9279.

Research output: Contribution to journalArticle

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T1 - Stable incorporation of ATPase subunits into 19 S regulatory particle of human proteasome requires nucleotide binding and C-terminal tails

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N2 - The 26 S proteasome is a large multi-subunit protein complex that degrades ubiquitinated proteins in eukaryotic cells. Proteasome assembly is a complex process that involves formation of sixand seven-membered ring structures from homologous subunits. Here we report that the assembly of hexameric Rpt ring of the 19 S regulatory particle (RP) requires nucleotide binding but not ATP hydrolysis. Disruption of nucleotide binding to an Rpt subunit by mutation in the Walker A motif inhibits the assembly of the Rpt ring without affecting heterodimer formation with its partner Rpt subunit. Coexpression of the base assembly chaperones S5b and PAAF1with mutant Rpt1 and Rpt6, respectively, relieves assembly inhibition of mutant Rpts by facilitating their interaction with adjacent Rpt dimers. The mutation in the Walker B motif which impairs ATP hydrolysis does not affect Rpt ring formation. Incorporation of a Walker B mutant Rpt subunit abrogates the ATPase activity of the 19 S RP, suggesting that failure of the mutant Rpt to undergo the conformational transition from an ATP-bound to an ADP-bound state impairs conformational changes in the other five wild-type Rpts in the Rpt ring. In addition, we demonstrate that the C-terminal tails of Rpt subunits possessing core particle (CP)-binding affinities facilitate the cellular assembly of the 19 SRP, implying that the 20 S CP may function as a template for base assembly in human cells. Taken together, these results suggest that the ATP-bound conformational state of an Rpt subunit with the exposed C-terminal tail is competent for cellular proteasome assembly.

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