Intrinsic Nucleoside Diphosphate Kinase-like Activity Is a Novel Function of the 20 S Proteasome

Abstract

The eukaryotic 20 S proteasome is the prototype of a new family of the N-terminal nucleophil hydrolases and is composed of numerous low molecular mass subunits arranged in a stack of four rings, each containing seven different ␣-or ␤-subunits. Among the ␤-type subunits in the yeast proteasome, three proteolytically active ones were identified, although the functions of the other ␤-and ␣-type subunits remain to be clarified. We report here that the purified 20 S proteasome exhibits intrinsic nucleoside diphosphate (NDP) kinase-like activity. The proteasome exhibited a preference for ATP and dATP as phosphate donors, and a broad specificity for NDPs, other than GDP, as phosphate acceptors, unlike conventional NDP kinase, which catalyzes the transfer of ␥-phosphate between NDPs and nucleoside triphosphates. During the transfer of ␥-phosphate, the proteasome formed acid-labile phosphohistidine as autophosphorylated intermediates, and NDP-dependent dephosphorylation of the latter then occurred. These enzymatic properties are similar to those of the molecular chaperone, Hsp70, which also exhibits intrinsic NDP kinase-like activity, instead of ATPase activity. C5 among the ␤-type subunits and C8 among the ␣-type subunits were autophosphorylated during the ␥-phosphate transfer reaction and were photoaffinity labeled with 8-azido-[␣-32 P]ATP, suggesting that the C5 and C8 subunits of the proteasome are responsible for the NDP kinase-like activity.The 20 S proteasome, representing a new family of the Ntn 1 hydrolases (1, 2), is the central enzyme in protein degradation in both the cytosol and the nucleus and plays a role in the control of cellular processes, such as metabolism, the cell cycle, the immune response (by generating antigenic peptides), and the stress response (by removing abnormal proteins) (3). Eukaryotic proteasomes have multiple proteolytic activities that have been widely referred to as trypsin-like, chymotrypsin-like, and peptidylglutamyl-peptide hydrolase activities (1-6), and mammalian proteasomes have another two activities (7). These functions are linked to ubiquitin-and ATP-requiring and to ubiquitin-independent protein degradation pathways, both of which involve the 26 S proteasome, the core and proteolytic chamber of which are formed by the 20 S proteasome. It is generally thought that the proteolytic activity of the 26 S proteasome is regulated by 19 S "cap" regulatory complexes (8, 9), which contain ATPases and serve to unfold substrate proteins prior to translocation to the proteolytic 20 S core, although direct biochemical evidence of such a chaperone-like function of the 19 S cap is lacking (3, 6). The eukaryotic 20 S proteasome consists of 28 subunits with seven different ␣ and ␤ subunits (4, 10, 11), and that in animal cells contains additional nonessential subunits that are ␥-interferon-inducible and contribute to antigen processing (12). The crystal structures of the yeast and archaebacterium Thermoplasma acidophilum proteasomes revealed that the subunits are arranged in a particle compri...

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