Functional studies of the ATP-binding subunits of PA700, an activator of 20S proteasome

Olga P. Zagnitko, George N. DeMartino, Clive A. Slaughter

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The 20S proteasome is a widely occurring intracellular protease of 700 kDa whose activity is modulated by a number of specific regulatory proteins. One such regulator, termed PA700, is a 700 kDa multisubunit protein that activates the proteasomal degradation of small peptides and ubiquitinated proteins. This activation is an ATP-dependent process that is closely linked to the formation of proteasome/PA700 complex. Purified PA700 is an ATPase with Km 25 μM.Six subunits of PA700 are the members of the AAA(ATPases Associated with a variety of cellular Activities) protein family containing a consensus sequence for ATP binding. We report here an investigation of the role of individual subunits of bovine PA700 in ATP-binding using a sitedirected labeling technique in which [a-32P]ATP is cross-linked to the protein by UV-irradiation. Several subunits bound ATP. The number of labeled subunits and the extent of labeling was dependent upon the nucleotide concentrations. At 10 μM ATP, the two subunits, p45 and p48, identified as close homologs of the transcriptional factors SUGl and TBP7, respectively, showed the strongest levels of photolabeling. Curves of extent of labeling vs. ATP concentrations indicated that p48 subunit was saturated at this concentration of ATP. Interestingly, only labeling of p48 is absolutely dependent on the presence of Mg2+. Photolabeling of both subunits was effectively prevented by 5μM ADP, although the 50% inhibition of ATPase activity of PA700 required 0.5mM ADP.These data suggest that different AAA-family members make quantitatively different contributions to the activity of PA700 and possibly fulfill different functions within the complex.

Original languageEnglish (US)
JournalFASEB Journal
Issue number6
StatePublished - Dec 1 1996
Externally publishedYes

ASJC Scopus subject areas

  • Agricultural and Biological Sciences (miscellaneous)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Cell Biology


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