PA28 is a 180,000-dalton protein that activates hydrolysis of small nonubiquitinated peptides by the 20 S proteasome. PA28 is composed of two homologous subunits, ␣ and ␤, arranged in alternating positions in a ringshaped oligomer with a likely stoichiometry of (␣␤) 3 . Our previous work demonstrated that the carboxyl terminus of the ␣ subunit was necessary for PA28 to bind to and activate the proteasome. The goals of this work were to define the exact structural basis for this effect and to determine the relative roles of the ␣ and ␤ subunits in proteasome activation. Each subunit and various mutants of the ␣ subunit were expressed in Escherichia coli and purified. PA28␣ stimulated the proteasome, but had a much greater K act than native heteromeric PA28. In contrast, PA28␤ was unable to stimulate the proteasome. Mutants of the ␣ subunit in which the carboxyl-terminal tyrosine residue was deleted or substituted with charged amino acids could neither bind to nor activate the proteasome. However, substitution of the carboxyl-terminal tyrosine with other amino acids resulted in proteins which could stimulate the proteasome to various extents. Tryptophan mutants stimulated the proteasome as well as did native PA28, whereas serine or phenylalanine mutants stimulated the proteasome much poorer than did wild type PA28␣. Deletion of the "KEKE" motif, a 28-amino acid domain near the amino terminus of PA28␣, had no effect on proteasome stimulatory activity. Hetero-oligomeric PA28 proteins were reconstituted from isolated wild type and mutant subunits. PA28 reconstituted from wild type subunits had structural and functional properties that were indistinguishable from those of the native hetero-oligomeric protein. PA28 molecules reconstituted from inactive ␣ subunits and wild type ␤ subunits remained inactive. However, PA28 molecules reconstituted from suboptimally active ␣ mutants and wild type ␤ subunits had the same activity as native heteromeric PA28. These results indicate that the ␤ subunit modulates PA28 activity, perhaps by influencing the affinity of PA28 for the proteasome.

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.

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The cross-conjugated 1,1-diethynylethylene derivatives 8b−10b were prepared from the corresponding bromides 16, 19, and 17 by Sonogashira coupling with trimethylsilylethyne and hydrolysis of the TMS-protected intermediates thus formed. Coupling of the tetrabromide 18 with (trimethylsilylethynyl)magnesium bromide in the presence of 1,3-[bis(diphenylphosphanyl)propane]nickel(II) chloride yielded the protected tetraalkyne 32, from which the 7,7,8,8-tetraethynyl-tetrahydro-p-quinodimethane 33 was liberated by fluoride treatment. Although 33 is a highly unstable cross-conjugated hydrocarbon, it could be converted into its tetraphenyl derivative by Sonogashira coupling with phenyl iodide. Both 32 and the corresponding tetraphenyl derivative were oxidized to the 7,7,8,8-tetraethynyl-dihydro-p-quinodimethane derivatives 35 and 36, respectively, on treatment with DDQ in dioxane. Further dehydrogenation of 35 to 34 failed, how-

The ubiqoitin system in plants. -Physiol. Plant. 97: 618-624,The small polypeptide ubiquitin participates in a variety of fundamental cellular events, such as cell differentiation, stress response, determination of steady state levels of regulatoi-y proteins, cell cycle control, regulation of transcription, and programmed cell death. Although the complex mechanisms of these processes are not fully understood, ubiquitinylation of regulatory proteins involved in those events is obviously essential. Target proteins can he covalently coupled with one or a few ubiquitin molecules, which is supposed to present a (reversible) post-translationai modification, Polyubiquitinylation, bowevei, marks proteins selectively for degradation by the 26S proteasome. The ubiquitin system has been studied mostly with animal systems or yeast, but all basic reactions of ubiqaitin appear in plants as well The scope of this review is to summarize several implications of recent studies directed towards the plant ubiquitin system.