Proteasome activator 11S REG or PA28: Recombinant REGα/REGβ hetero- oligomers are heptamers

Zhiguo Zhang, Andrew Krutchinsky, Scott Endicott, Claudio Realini, Martin Rechsteiner, Kenneth G. Standing

Research output: Contribution to journalArticle

107 Citations (Scopus)

Abstract

The proteasome activator 11S REG or PA28 is a conical molecule composed of two homologous subunits, REGα and REGβ. Recombinant REGα forms a heptamer, whereas recombinant REGβ is a monomer. When mixed with REGβ, a monomeric REGα mutant (N50Y) forms an active hetero-oligomer in which the molar ratio of REGβ to REGα(N50Y) is close to 1.3. This apparent stoichiometry is consistent with the REGα(N50Y)/REGβ hetero-oligomer being a heptamer composed of three α and four β subunits. Chemical cross-linking of the α/β oligomers revealed the presence of REGα-REGβ and REGβ-REGβ dimers, but REGα-REGα dimers were not detected. The mass of the REGα(N50Y)/REGβ hetero-oligomer determined by electrospray ionization time- of-flight mass spectrometry (ESI-TOF MS) is 194 871 ± 40 Da in good agreement with the theoretical mass of 194 856 Da for an α3β4 heptamer. Hexamers were not observed in the mass spectrum. For wild-type REG subunits coexpressed in bacteria cells at an apparent β/α molar ratio of ~1.2, the resulting hetero-oligomers observed by ESI-TOF MS were again predominantly α3β4 heptamers, with trace amounts of α4β3 heptamers also present. On the other hand, the mass spectrum contained a mixture of α7, α6β1, α5β2, and α4β3 heptamers when the REGβ/REGα ratio was 0.1. Thus, formation of heptamers is an intrinsic property of recombinant REGα and REGβ subunits. on the basis of these results, we propose that 11S RG purified directly from eukaryotic cells is also heptameric, likely α3β4 or a mixture of α3β4 and α4β3 species.

Original languageEnglish (US)
Pages (from-to)5651-5658
Number of pages8
JournalBiochemistry
Volume38
Issue number17
DOIs
StatePublished - Apr 27 1999
Externally publishedYes

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Proteasome Endopeptidase Complex
Oligomers
Mass Spectrometry
Eukaryotic Cells
Electrospray ionization
Dimers
Mass spectrometry
Bacteria
Stoichiometry
Monomers
Molecules

ASJC Scopus subject areas

  • Biochemistry

Cite this

Zhang, Z., Krutchinsky, A., Endicott, S., Realini, C., Rechsteiner, M., & Standing, K. G. (1999). Proteasome activator 11S REG or PA28: Recombinant REGα/REGβ hetero- oligomers are heptamers. Biochemistry, 38(17), 5651-5658. https://doi.org/10.1021/bi990056+

Proteasome activator 11S REG or PA28 : Recombinant REGα/REGβ hetero- oligomers are heptamers. / Zhang, Zhiguo; Krutchinsky, Andrew; Endicott, Scott; Realini, Claudio; Rechsteiner, Martin; Standing, Kenneth G.

In: Biochemistry, Vol. 38, No. 17, 27.04.1999, p. 5651-5658.

Research output: Contribution to journalArticle

Zhang, Z, Krutchinsky, A, Endicott, S, Realini, C, Rechsteiner, M & Standing, KG 1999, 'Proteasome activator 11S REG or PA28: Recombinant REGα/REGβ hetero- oligomers are heptamers', Biochemistry, vol. 38, no. 17, pp. 5651-5658. https://doi.org/10.1021/bi990056+
Zhang Z, Krutchinsky A, Endicott S, Realini C, Rechsteiner M, Standing KG. Proteasome activator 11S REG or PA28: Recombinant REGα/REGβ hetero- oligomers are heptamers. Biochemistry. 1999 Apr 27;38(17):5651-5658. https://doi.org/10.1021/bi990056+
Zhang, Zhiguo ; Krutchinsky, Andrew ; Endicott, Scott ; Realini, Claudio ; Rechsteiner, Martin ; Standing, Kenneth G. / Proteasome activator 11S REG or PA28 : Recombinant REGα/REGβ hetero- oligomers are heptamers. In: Biochemistry. 1999 ; Vol. 38, No. 17. pp. 5651-5658.
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abstract = "The proteasome activator 11S REG or PA28 is a conical molecule composed of two homologous subunits, REGα and REGβ. Recombinant REGα forms a heptamer, whereas recombinant REGβ is a monomer. When mixed with REGβ, a monomeric REGα mutant (N50Y) forms an active hetero-oligomer in which the molar ratio of REGβ to REGα(N50Y) is close to 1.3. This apparent stoichiometry is consistent with the REGα(N50Y)/REGβ hetero-oligomer being a heptamer composed of three α and four β subunits. Chemical cross-linking of the α/β oligomers revealed the presence of REGα-REGβ and REGβ-REGβ dimers, but REGα-REGα dimers were not detected. The mass of the REGα(N50Y)/REGβ hetero-oligomer determined by electrospray ionization time- of-flight mass spectrometry (ESI-TOF MS) is 194 871 ± 40 Da in good agreement with the theoretical mass of 194 856 Da for an α3β4 heptamer. Hexamers were not observed in the mass spectrum. For wild-type REG subunits coexpressed in bacteria cells at an apparent β/α molar ratio of ~1.2, the resulting hetero-oligomers observed by ESI-TOF MS were again predominantly α3β4 heptamers, with trace amounts of α4β3 heptamers also present. On the other hand, the mass spectrum contained a mixture of α7, α6β1, α5β2, and α4β3 heptamers when the REGβ/REGα ratio was 0.1. Thus, formation of heptamers is an intrinsic property of recombinant REGα and REGβ subunits. on the basis of these results, we propose that 11S RG purified directly from eukaryotic cells is also heptameric, likely α3β4 or a mixture of α3β4 and α4β3 species.",
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