Document Detail


A model for the quaternary structure of the proteasome activator PA28.
MedLine Citation:
PMID:  8824298     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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, alpha and beta, 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 alpha and beta subunits. To distinguish between these possibilities, we prepared antibodies that reacted specifically with either the alpha or beta subunit and used these subunit-specific antibodies in two types of experiments designed to elucidate PA28 quaternary structure. In the first experiment, the alpha and beta 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 alpha and beta 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 alpha and beta subunits with a stoichiometry of (alphabeta)3. PA28 was inactivated by treatment with carboxypeptidase Y, which cleaved Tyr and Ile residues from the carboxyl terminus of the alpha subunit but had very little effect on the beta subunit. This selective and limited proteolysis prevented binding of both alpha and beta 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 alpha subunit is critical for binding of native PA28 to the proteasome. To learn about the relative functions of the alpha and beta subunits, PA28alpha was expressed in Escherichia coli and purified to homogeneity. Purified PA28alpha 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.
Authors:
X Song; J D Mott; J von Kampen; B Pramanik; K Tanaka; C A Slaughter; G N DeMartino
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, Non-P.H.S.; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  The Journal of biological chemistry     Volume:  271     ISSN:  0021-9258     ISO Abbreviation:  J. Biol. Chem.     Publication Date:  1996 Oct 
Date Detail:
Created Date:  1996-11-26     Completed Date:  1996-11-26     Revised Date:  2009-11-19    
Medline Journal Info:
Nlm Unique ID:  2985121R     Medline TA:  J Biol Chem     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  26410-7     Citation Subset:  IM; S    
Affiliation:
Department of Physiology, The University of Texas Southwestern Medical Center, Dallas, Texas 75235, USA.
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MeSH Terms
Descriptor/Qualifier:
Animals
Antibodies
Carboxypeptidases / metabolism
Cathepsin A
Cattle
Electrophoresis, Polyacrylamide Gel
Mass Spectrometry
Models, Chemical*
Molecular Weight
Protein Conformation*
Proteins / chemistry*
Grant Support
ID/Acronym/Agency:
DK46181/DK/NIDDK NIH HHS; HL06296/HL/NHLBI NIH HHS
Chemical
Reg. No./Substance:
0/Antibodies; 0/Proteins; EC 3.4.-/Carboxypeptidases; EC 3.4.16.5/Cathepsin A

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