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 alpha- or beta-subunits. Among the beta-type subunits in the yeast proteasome, three proteolytically active ones were identified, although the functions of the other beta- and alpha-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 gamma-phosphate between NDPs and nucleoside triphosphates. During the transfer of gamma-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 beta-type subunits and C8 among the alpha-type subunits were autophosphorylated during the gamma-phosphate transfer reaction and were photoaffinity labeled with 8-azido-[alpha-(32)P]ATP, suggesting that the C5 and C8 subunits of the proteasome are responsible for the NDP kinase-like activity.
Highlights
The 20 S proteasome, representing a new family of the Ntn1 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, and the stress response [3]
To exclude the possibility of contaminating proteins in our 20 S proteasome preparation with ATP synthesis and ATP hydrolysis activities, such as conventional nucleoside diphosphate (NDP) kinase [27,28,29] and Hsp70 [16], the purified proteasome was applied to a Mono Q anion-exchange column, which is able to separate conventional NDP kinase protein with a molecular mass of 16 kDa from Hsp70 and/or Hsc70 [16, 27]
Conventional NDP kinase and Hsp70, which are eluted with approximately 0.05 M and 0.1 M KCl, respectively, in 25 mM Tris-HCl buffer, pH 7.2, containing 0.1 mM EDTA and 0.5 mM dithiothreitol, was not detected in the 20 S proteasome preparation, and the proteasome with the activities of ATP synthesis, ATP hydrolysis, and peptidases was eluted with the much higher concentration of KCl of 0.2– 0.3 M on that chromatography
Summary
N-terminal nucleophil; AMP-PNP, 5Ј-adenylyl-,␥-imidodiphosphate; ATP␥S, adenosine 5Ј-3-O-(thiotriphosphate); DEPC, diethylpyrocarbonate; Boc, N-tert-butyloxycarbonyl; MCA, 4-methyl-coumaryl-7-amide; Z, carbobenzoxyl; Suc, succinyl; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; Mes, 2-morpholineethanesulfonic acid; Ches, 2-(cyclohexylamino)ethanesulfonic acid; Caps, 3-cyclohexylamino propanesulfonic acid. It is generally thought that the proteolytic activity of the 26 S proteasome is regulated by S “cap” regulatory complexes [8, 9], which contain ATPases and serve to unfold substrate proteins prior to translocation to the proteolytic S core, direct biochemical evidence of such a chaperone-like function of the S cap is lacking [3, 6]. We found the intrinsic NDP kinase-like activity, but not ATPase activity, of the molecular chaperone, Hsp70 [16], which catalyzes the transfer of ␥-phosphoryl groups from NTPs to NDPs. The interaction of Hsp with protein or peptide substrates is regulated by the activities of ATP hydrolysis and ADP-ATP exchange. We first report that the eukaryotic 20 S proteasome exhibits a novel function, i.e. intrinsic NDP/NTP exchange activity other than proteolytic activity, the enzymatic properties being similar to those of the NDP kinase-like activity of Hsp. We discuss the possible role of this NDP kinase-like activity in the proteolytic function of the proteasome
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