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 ATPdependent 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 M r of ϳ300,000, as determined by gel filtration chromatography, and is composed of three electrophoretically 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-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.
The nin1‐1 mutant of Saccharomyces cerevisiae cannot perform the G1/S and G2/M transitions at restrictive temperatures. At such temperatures, nin1‐1 strains fail to activate histone H1 kinase after release from alpha factor‐imposed G1 block and after release from hydroxyurea‐imposed S block. The nin1‐1 mutation shows synthetic lethality with certain cdc28 mutant alleles such as cdc28‐IN. Two lines of evidence indicate that Nin1p is a component of the 26S proteasome complex: (i) Nin1p, as well as the known component of the 26S proteasome, shifted to the 26S proteasome peak in the glycerol density gradient after preincubation of crude extract with ATP‐Mg2+, and (ii) nin1‐1 cells accumulated polyubiquitinated proteins under restrictive conditions. These results suggest that activation of Cdc28p kinase requires proteolysis. We have cloned a human cDNA encoding a regulatory subunit of the 26S proteasome, p31, which was found to be a homolog of Nin1p.
SummaryInterferon (IFN) y induces replacements of the proteasomal subunits X and Y by LMP7 and LMP2, respectively, resulting in an alteration of the proteolytic specificity. We found a third pair ofproteasome subunits expressed reciprocally in response to IFN-y. Molecular cloning of a cDNA encoding one subunit designated as Z, downregulated by IFN-% showed that it is a novel proteasomal subunit with high homology to MECL1, which is markedly induced by IFN-% Thus, IFN-~/induces subunit replacements of not only X and Y by LMP7 and LMP2, respectively, but also of Z by MECL1, producing proteasomes responsible for immunological processing of endogenous antigens. When processed from their precursors, three pairs of the 10 homologous, but distinct, [3-type subunits of eukaryotic proteasomes, that is, X/LMP7, Y/LMP2, and Z/MECL1, have an NH2-terminal threonine residue, assumed to be part of a catalytic center. These findings suggest that the altered molecular organization of the proteasome induced by IFN-y may be responsible for acquisition of its functional change.
The primary structures of two proteins that comprise PA28, an activator of the 20S proteasome, have been determined by cDNA cloning and sequencing. These protein subunits, termed PA28a and PA28JB, are about 50% identical to one another and are highly conserved between rat and human. PA28a and PA28~ are homologous to a previously described protein, Ki antigen, whose function is unknown. PA28c~, but neither PA28~ nor Ki antigen, contains a 'KEKE motif', which has been postulated to promote the binding of proteins having this structural feature. PA28a and PA2818 were coordinately regulated by y-interferon, which greatly induced mRNA levels of both proteins in cultured cells. The mRNA level of the Ki antigen also increased in response to "y-interferon treatment, but the magnitude of the increase was less than that for the PA28s, and the effect was transient. These results demonstrate the existence of a new protein family, at least two of whose members are involved in proteasome activation. They also provide the basis for future structure/function studies of PA28 subunits and the determination of their relative physiological roles in the regulation of proteasome activity.
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