Abstract
The activity of the intracellular protease, the proteasome, is modulated by a number of specific regulatory proteins. One such regulator, PA700, is a 700,000-Da multisubunit protein that activates hydrolytic activities of the proteasome via a mechanism that involves the ATP-dependent formation of a proteasome-PA700 complex. Four subunits of PA700 have been shown previously to be members of a protein family that contains a consensus sequence for ATP binding, and purified PA700 expresses ATPase activity. We report here the identification, purification, and initial characterization of a new modulator of the proteasome. The modulator has no direct effect on the activity of the proteasome, but enhances PA700 activation of the proteasome by up to 8-fold. This activation is associated with the formation of a proteasome/PA700-containing complex that is significantly larger than that formed in its absence. The modulator has a native Mr of approximately 300,000, as determined by gel filtration chromatography, and is composed of three electrophoretically distinct subunits with Mr values of 50,000, 42,000, and 27,000 (p50, p42, and p27, respectively). Amino acid sequence analysis of the subunits shows that p50 and p42 are members of the same ATP-binding protein family found in PA700. The p50 subunit is identical to TBP1, a protein previously reported to interact with human immunodeficiency virus Tat protein (Nelbock, P., Dillion, P. J., Perkins, A., and Rosen, C. A. (1990) Science 248, 1650-1653), while the p42 subunit seems to be a new member of the family. The p27 subunit has no significant sequence similarity to any previously described protein. Both p50 and p42, but not p27, were also identified as components of PA700, increasing the number of ATP-binding protein family members in this complex to six. Thus, p50 and p42 are subunits common to two protein complexes that regulate the proteasome. The PA700-dependent proteasome activator represents a new member of a growing list of proteins that regulate proteasome activity.
Highlights
The proteasome is a 700,000-Da multicatalytic protease that participates in a number of proteolytically mediated intracellular processes, including the constitutive turnover of many
The proteasome can be isolated as part of a larger protein complex (Mr Ն 1,500,000) referred to as the “26 S protease” [11]
Because it seemed reasonable to assume that some proteasome regulatory proteins might act in concert or might themselves be regulated by other proteins, we have designed assays to test the effects of cell extracts on the proteasome in the presence of previously identified regulators
Summary
The proteasome is a 700,000-Da multicatalytic protease that participates in a number of proteolytically mediated intracellular processes, including the constitutive turnover of many. At least four of the ϳ20 electrophoretically distinct subunits of PA700 are homologous to one another and are members of a large protein family that contains a consensus sequence for ATP binding [15,16,17] Some of these same proteins have been identified as components of the purified 26 S protease, providing additional strong evidence that the proteasomePA700 complex is similar, if not identical, to the 26 S protease [13, 15, 16]. Many of these “ATPase” subunits of PA700 have been identified independently as proteins involved in processes with no obvious relationships to proteasome function [17].
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