IDENTIFICATION, PURIFICATION, AND CHARACTERIZATION OF A PA700-DEPENDENT ACTIVATOR OF THE PROTEASOME

The activity of the intracellular protease, the proteasome, is modulat ed by a number of specific regulatory proteins. One such regulator, PA 700, is a 700,000-Da multisubunit protein that activates hydrolytic ac tivities of the proteasome via a mechanism that involves the ATP-depen dent formation of a proteasome-PA700 complex. Four subunits of PA700 h ave been shown previously to be members of a protein family that conta ins 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 mo dulator has no direct effect on the activity of the proteasome, but en hances PA700 activation of the proteasome by up to 8-fold. This activa tion 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 M(r) of similar to 300,000, as determined by gel filtration chromatography, and is composed of three electrophor etically distinct subunits with M(r) 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-bindi ng protein family found in PA700. The p50 subunit is identical to TBP1 , a protein previously reported to interact with human immunodeficienc y virus Tat protein (Nelbock, P., Billion, P. J., Perkins, A., and Ros en, 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 s equence similarity to any previously described protein. Both p50 and p 42, but not p27, were also identified as components of PA700, increasi ng the number of ATP-binding protein family members in this complex to six. Thus, p50 and p42 are subunits common to two protein complexes t hat regulate the proteasome. The PA700-dependent proteasome activator represents a new member of a growing list of proteins that regulate pr oteasome activity.