A model for the quaternary structure of the proteasome activator PA28

Xiaoling Song, Joni D. Mott, Jan Von Kampen, Bikash Pramanik, Keiji Tanaka, Clive A. Slaughter, George N. DeMartino

Research output: Contribution to journalArticle

66 Citations (Scopus)

Abstract

PA28 is a protein activator of the 20S proteasome. It has a native molecular weight of approximately 200,000 and is composed of six 28,000- dalton subunits arranged in a ring-shaped complex. Purified preparations of PA28 contain two polypeptides, α and β, which are about 50% identical in primary structure. It has been unclear whether native PA28 consists of two distinct homohexameric proteins or of a single protein containing both and β subunits. To distinguish between these possibilities, we prepared antibodies that reacted specifically with either the α or β subunit and used these subunit-specific antibodies in two types of experiments designed to elucidate PA28 quaternary structure. In the first experiment, the α and β subunits were completely co-immunoprecipitated by each subunit-specific antibody, indicating that both subunits were part of a single protein complex. In the second experiment, PA28 was chemically cross-linked using bis(sulfosuccinimidyl)suberate. When the cross-linked products were immunoblotted after SDS-polyacrylamide gel electrophoresis, indistinguishable patterns were obtained with each subunit-specific antibody. These results confirm that the α and β subunits were part of the same protein complex. The pattern of cross-linked products also provided insight as to the relative abundance and arrangement of the subunits within the PA28 complex and indicated that the ring-shaped PA28 hexamer may be composed of alternating α and β subunits with a stoichiometry of (αβ)3. PA28 was inactivated by treatment with carboxypeptidase Y, which cleaved Tyr and Ile residues from the carboxyl terminus of the α subunit but had very little effect on the β subunit. This selective and limited proteolysis prevented binding of both α and β subunits to the proteasome and therefore provides additional evidence of the heterodimeric nature of PA28. These results indicate that a short carboxyl-terminal sequence of the α subunit is critical for binding of native PA28 to the proteasome. To learn about the relative functions of the and β subunits, PA28α was expressed in Escherichia coli and purified to homogeneity. Purified PA28α stimulated proteasome activity but required 5- 10-fold greater concentrations than the heterodimeric PA28 to achieve a given level of activity. These results suggest that the heterodimeric structure of PA28 is required for maximal proteasome activation.

Original languageEnglish (US)
Pages (from-to)26410-26417
Number of pages8
JournalJournal of Biological Chemistry
Volume271
Issue number42
DOIs
StatePublished - Oct 30 1996

Fingerprint

Proteasome Endopeptidase Complex
Antibodies
Proteins
Cathepsin A
Proteolysis
Experiments
Electrophoresis
Stoichiometry
Escherichia coli
Polyacrylamide Gel Electrophoresis
Molecular Weight
Chemical activation
Molecular weight
Peptides

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Song, X., Mott, J. D., Von Kampen, J., Pramanik, B., Tanaka, K., Slaughter, C. A., & DeMartino, G. N. (1996). A model for the quaternary structure of the proteasome activator PA28. Journal of Biological Chemistry, 271(42), 26410-26417. https://doi.org/10.1074/jbc.271.42.26410

A model for the quaternary structure of the proteasome activator PA28. / Song, Xiaoling; Mott, Joni D.; Von Kampen, Jan; Pramanik, Bikash; Tanaka, Keiji; Slaughter, Clive A.; DeMartino, George N.

In: Journal of Biological Chemistry, Vol. 271, No. 42, 30.10.1996, p. 26410-26417.

Research output: Contribution to journalArticle

Song, X, Mott, JD, Von Kampen, J, Pramanik, B, Tanaka, K, Slaughter, CA & DeMartino, GN 1996, 'A model for the quaternary structure of the proteasome activator PA28', Journal of Biological Chemistry, vol. 271, no. 42, pp. 26410-26417. https://doi.org/10.1074/jbc.271.42.26410
Song, Xiaoling ; Mott, Joni D. ; Von Kampen, Jan ; Pramanik, Bikash ; Tanaka, Keiji ; Slaughter, Clive A. ; DeMartino, George N. / A model for the quaternary structure of the proteasome activator PA28. In: Journal of Biological Chemistry. 1996 ; Vol. 271, No. 42. pp. 26410-26417.
@article{d9605790933c4cf5a987828b720496f9,
title = "A model for the quaternary structure of the proteasome activator PA28",
abstract = "PA28 is a protein activator of the 20S proteasome. It has a native molecular weight of approximately 200,000 and is composed of six 28,000- dalton subunits arranged in a ring-shaped complex. Purified preparations of PA28 contain two polypeptides, α and β, which are about 50{\%} identical in primary structure. It has been unclear whether native PA28 consists of two distinct homohexameric proteins or of a single protein containing both and β subunits. To distinguish between these possibilities, we prepared antibodies that reacted specifically with either the α or β subunit and used these subunit-specific antibodies in two types of experiments designed to elucidate PA28 quaternary structure. In the first experiment, the α and β subunits were completely co-immunoprecipitated by each subunit-specific antibody, indicating that both subunits were part of a single protein complex. In the second experiment, PA28 was chemically cross-linked using bis(sulfosuccinimidyl)suberate. When the cross-linked products were immunoblotted after SDS-polyacrylamide gel electrophoresis, indistinguishable patterns were obtained with each subunit-specific antibody. These results confirm that the α and β subunits were part of the same protein complex. The pattern of cross-linked products also provided insight as to the relative abundance and arrangement of the subunits within the PA28 complex and indicated that the ring-shaped PA28 hexamer may be composed of alternating α and β subunits with a stoichiometry of (αβ)3. PA28 was inactivated by treatment with carboxypeptidase Y, which cleaved Tyr and Ile residues from the carboxyl terminus of the α subunit but had very little effect on the β subunit. This selective and limited proteolysis prevented binding of both α and β subunits to the proteasome and therefore provides additional evidence of the heterodimeric nature of PA28. These results indicate that a short carboxyl-terminal sequence of the α subunit is critical for binding of native PA28 to the proteasome. To learn about the relative functions of the and β subunits, PA28α was expressed in Escherichia coli and purified to homogeneity. Purified PA28α stimulated proteasome activity but required 5- 10-fold greater concentrations than the heterodimeric PA28 to achieve a given level of activity. These results suggest that the heterodimeric structure of PA28 is required for maximal proteasome activation.",
author = "Xiaoling Song and Mott, {Joni D.} and {Von Kampen}, Jan and Bikash Pramanik and Keiji Tanaka and Slaughter, {Clive A.} and DeMartino, {George N.}",
year = "1996",
month = "10",
day = "30",
doi = "10.1074/jbc.271.42.26410",
language = "English (US)",
volume = "271",
pages = "26410--26417",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "42",

}

TY - JOUR

T1 - A model for the quaternary structure of the proteasome activator PA28

AU - Song, Xiaoling

AU - Mott, Joni D.

AU - Von Kampen, Jan

AU - Pramanik, Bikash

AU - Tanaka, Keiji

AU - Slaughter, Clive A.

AU - DeMartino, George N.

PY - 1996/10/30

Y1 - 1996/10/30

N2 - PA28 is a protein activator of the 20S proteasome. It has a native molecular weight of approximately 200,000 and is composed of six 28,000- dalton subunits arranged in a ring-shaped complex. Purified preparations of PA28 contain two polypeptides, α and β, which are about 50% identical in primary structure. It has been unclear whether native PA28 consists of two distinct homohexameric proteins or of a single protein containing both and β subunits. To distinguish between these possibilities, we prepared antibodies that reacted specifically with either the α or β subunit and used these subunit-specific antibodies in two types of experiments designed to elucidate PA28 quaternary structure. In the first experiment, the α and β subunits were completely co-immunoprecipitated by each subunit-specific antibody, indicating that both subunits were part of a single protein complex. In the second experiment, PA28 was chemically cross-linked using bis(sulfosuccinimidyl)suberate. When the cross-linked products were immunoblotted after SDS-polyacrylamide gel electrophoresis, indistinguishable patterns were obtained with each subunit-specific antibody. These results confirm that the α and β subunits were part of the same protein complex. The pattern of cross-linked products also provided insight as to the relative abundance and arrangement of the subunits within the PA28 complex and indicated that the ring-shaped PA28 hexamer may be composed of alternating α and β subunits with a stoichiometry of (αβ)3. PA28 was inactivated by treatment with carboxypeptidase Y, which cleaved Tyr and Ile residues from the carboxyl terminus of the α subunit but had very little effect on the β subunit. This selective and limited proteolysis prevented binding of both α and β subunits to the proteasome and therefore provides additional evidence of the heterodimeric nature of PA28. These results indicate that a short carboxyl-terminal sequence of the α subunit is critical for binding of native PA28 to the proteasome. To learn about the relative functions of the and β subunits, PA28α was expressed in Escherichia coli and purified to homogeneity. Purified PA28α stimulated proteasome activity but required 5- 10-fold greater concentrations than the heterodimeric PA28 to achieve a given level of activity. These results suggest that the heterodimeric structure of PA28 is required for maximal proteasome activation.

AB - PA28 is a protein activator of the 20S proteasome. It has a native molecular weight of approximately 200,000 and is composed of six 28,000- dalton subunits arranged in a ring-shaped complex. Purified preparations of PA28 contain two polypeptides, α and β, which are about 50% identical in primary structure. It has been unclear whether native PA28 consists of two distinct homohexameric proteins or of a single protein containing both and β subunits. To distinguish between these possibilities, we prepared antibodies that reacted specifically with either the α or β subunit and used these subunit-specific antibodies in two types of experiments designed to elucidate PA28 quaternary structure. In the first experiment, the α and β subunits were completely co-immunoprecipitated by each subunit-specific antibody, indicating that both subunits were part of a single protein complex. In the second experiment, PA28 was chemically cross-linked using bis(sulfosuccinimidyl)suberate. When the cross-linked products were immunoblotted after SDS-polyacrylamide gel electrophoresis, indistinguishable patterns were obtained with each subunit-specific antibody. These results confirm that the α and β subunits were part of the same protein complex. The pattern of cross-linked products also provided insight as to the relative abundance and arrangement of the subunits within the PA28 complex and indicated that the ring-shaped PA28 hexamer may be composed of alternating α and β subunits with a stoichiometry of (αβ)3. PA28 was inactivated by treatment with carboxypeptidase Y, which cleaved Tyr and Ile residues from the carboxyl terminus of the α subunit but had very little effect on the β subunit. This selective and limited proteolysis prevented binding of both α and β subunits to the proteasome and therefore provides additional evidence of the heterodimeric nature of PA28. These results indicate that a short carboxyl-terminal sequence of the α subunit is critical for binding of native PA28 to the proteasome. To learn about the relative functions of the and β subunits, PA28α was expressed in Escherichia coli and purified to homogeneity. Purified PA28α stimulated proteasome activity but required 5- 10-fold greater concentrations than the heterodimeric PA28 to achieve a given level of activity. These results suggest that the heterodimeric structure of PA28 is required for maximal proteasome activation.

UR - http://www.scopus.com/inward/record.url?scp=0029921985&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0029921985&partnerID=8YFLogxK

U2 - 10.1074/jbc.271.42.26410

DO - 10.1074/jbc.271.42.26410

M3 - Article

C2 - 8824298

AN - SCOPUS:0029921985

VL - 271

SP - 26410

EP - 26417

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 42

ER -