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, 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.
Highlights
** To whom correspondence should be addressed: Dept. of Physiology, The University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235
Proteasomes in higher eukaryotes demonstrate a greater subunit complexity and in a given source are composed of 14 different gene products, 7 of which are homologous to the archaebacterial ␣ subunit and 7 of which are homologous to the archaebacterial  subunit [8, 9]
The data reported here provide strong support for a heterodimeric model of PA28 quaternary structure. This model of PA28 structure prompted us to examine the biochemical nature of the carboxyl-terminal modification of PA28 that results in loss of binding to the proteasome, a phenomenon identified before PA28 was recognized to contain the distinct ␣ and  subunits
Summary
** To whom correspondence should be addressed: Dept. of Physiology, The University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235. Another protein, called PA28, activates the proteasome’s hydrolysis of small peptides but does not promote its degradation of ubiquitinated proteins [16, 17] These distinct regulatory proteins do not share common subunits [18, 19], they each interact with the proteasome by binding to its terminal ␣ rings. Recent work in our laboratory demonstrated that purified preparations of PA28 contain two polypeptides, termed ␣ and  [22] These proteins represent distinct gene products but are about 50% identical to one another in primary structure [23]. The data reported here provide strong support for a heterodimeric model of PA28 quaternary structure This model of PA28 structure prompted us to examine the biochemical nature of the carboxyl-terminal modification of PA28 that results in loss of binding to the proteasome, a phenomenon identified before PA28 was recognized to contain the distinct ␣ and  subunits.
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