C Termini of Proteasomal ATPases Play Nonequivalent Roles in Cellular Assembly of Mammalian 26 S Proteasome

Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9040, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 05/2011; 286(30):26652-66. DOI: 10.1074/jbc.M111.246793
Source: PubMed

ABSTRACT The 26 S proteasome comprises two multisubunit subcomplexes as follows: 20 S proteasome and PA700/19 S regulatory particle.
The cellular mechanisms by which these subcomplexes assemble into 26 S proteasome and the molecular determinants that govern
the assembly process are poorly defined. Here, we demonstrate the nonequivalent roles of the C termini of six AAA subunits
(Rpt1–Rpt6) of PA700 in 26 S proteasome assembly in mammalian cells. The C-terminal HbYX motif (where Hb is a hydrophobic residue, Y is tyrosine, and X is any amino acid) of each of two subunits, Rpt3 and Rpt5, but not that of a third subunit Rpt2, was essential for assembly
of 26 S proteasome. The C termini of none of the three non-HbYX motif Rpt subunits were essential for cellular 26 S proteasome assembly, although deletion of the last three residues of
Rpt6 destabilized the 20 S-PA700 interaction. Rpt subunits defective for assembly into 26 S proteasome due to C-terminal truncations
were incorporated into intact PA700. Moreover, intact PA700 accumulated as an isolated subcomplex when cellular 20 S proteasome
content was reduced by RNAi. These results indicate that 20 S proteasome is not an obligatory template for assembly of PA700.
Collectively, these results identify specific structural elements of two Rpt subunits required for 26 S proteasome assembly,
demonstrate that PA700 can be assembled independently of the 20 S proteasome, and suggest that intact PA700 is a direct intermediate
in the cellular pathway of 26 S proteasome assembly.

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    • "Consistent with decreased proteasomal function, the surrogate UPS substrate GFPdgn, when overexpressed in the HS-Csn8KO, was also significantly accumulated (Figure 3A ~ 3C). The marked reduction of the protein levels of Rpt5 (a critical subunit of the 19S base) in HS-Csn8KO livers (Figures 2A, 3D, 3E) may have contributed to the proteasomal impairment because Rpt5 has been elegantly shown to play an indispensable role in 26S proteasome assembly [48,49]. The mechanism for Rpt5 down-regulation is unclear and does not appear to be attributable to an increased caspase activity in HS-Csn8KO livers. "
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    ABSTRACT: The COP9 signalosome (CSN), an evolutionally highly conserved protein complex composed of 8 unique subunits (CSN1 through CSN8) in higher eukaryotes, is purported to modulate protein degradation mediated by the ubiquitin-proteasome system (UPS) but this has not been demonstrated in a critical mitotic parenchymal organ of vertebrates. Hepatocyte-specific knockout of the Cops8 gene (HS-Csn8KO) was shown to cause massive hepatocyte apoptosis and liver malfunction but the underlying mechanism remains unclear. Here, we report that Csn8/CSN exerts profound impacts on hepatic UPS function and is critical to the stability of the pro-apoptotic protein Bim. Significant decreases in CIS (cytokine-inducible Src homology 2 domain-containing protein), a Bim receptor of a cullin2-based ubiquitin ligase, were found to co-exist with a marked increase of Bim proteins. Csn8 deficiency also significantly decreased 19S proteasome subunit Rpt5 and markedly increased high molecular weight neddylated and ubiquitinated proteins. The use of a surrogate UPS substrate further reveals severe impairment of UPS-mediated proteolysis in HS-Csn8KO livers. Inclusion body-like materials were accumulated in Csn8 deficient hepatocytes. In addition to Bim, massive hepatocyte apoptosis in HS-Csn8KO livers is also associated with elevated expression of other members of the Bcl2 family, including pro-apoptotic Bax as well as anti-apoptotic Bcl2 and Bcl-XL. Increased interaction between Bcl2 and Bim, but not between Bcl2 and Bax, was detected. Hence, it is concluded that hepatic CSN8 deficiency impairs the UPS in the liver and the resultant Bim upregulation likely plays an important role in triggering hepatocyte apoptosis via sequestering Bcl2 away from Bax.
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    ABSTRACT: The proteasome is a large and complex protease formed by 66 polypeptides. The assembly of the proteasome is assisted by at least nine chaperones. One of these chaperones, Nas2/p27, binds to the C-terminal region of the AAA-ATPase Rpt5. We report here that the tail of Rpt5 provides two functions. First, it facilitates the previously reported interaction with the proteasome core particle (CP). Second, it is essential for the interaction with Nas2. Deletion of the C-terminal amino acid of Rpt5 disrupts the CP interaction, but not the binding to Nas2. The latter is surprising considering Nas2 contains a PDZ domain, which is often involved in binding to C termini. Interestingly, deletion of the last three amino acids interferes with both functions. The disruption of the Rpt5-CP interactions gave distinct phenotypes different from disruption of the Nas2-Rpt5 interaction. Additionally, proteasomes purified from a Saccharomyces cerevisiae rpt5-Δ3 strain show a strong enrichment of Ecm29. The function of Ecm29, a proteasome-associated protein, is not well understood. Our data show that Ecm29 can inhibit proteasomes, because our Ecm29-containing proteasomes have reduced suc-LLVY-AMC hydrolytic activity. Consistent with this apparent role as negative regulator, the deletion of ECM29 rescues the phenotypes of rpt5-Δ3 and nas2Δ in an hsm3Δ background. In sum, the interactions facilitated by the tail of Rpt5 act synergistically to minimize the formation of faulty proteasomes, thereby preventing recognition and inhibition by Ecm29.
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    ABSTRACT: The 26 S proteasome is a large multi-subunit protein complex that degrades ubiquitinated proteins in eukaryotic cells. Proteasome assembly is a complex process that involves formation of six- and seven-membered ring structures from homologous subunits. Here we report that the assembly of hexameric Rpt ring of the 19 S regulatory particle (RP) requires nucleotide binding but not ATP hydrolysis. Disruption of nucleotide binding to an Rpt subunit by mutation in the Walker A motif inhibits the assembly of the Rpt ring without affecting heterodimer formation with its partner Rpt subunit. Coexpression of the base assembly chaperones S5b and PAAF1 with mutant Rpt1 and Rpt6, respectively, relieves assembly inhibition of mutant Rpts by facilitating their interaction with adjacent Rpt dimers. The mutation in the Walker B motif which impairs ATP hydrolysis does not affect Rpt ring formation. Incorporation of a Walker B mutant Rpt subunit abrogates the ATPase activity of the 19 S RP, suggesting that failure of the mutant Rpt to undergo the conformational transition from an ATP-bound to an ADP-bound state impairs conformational changes in the other five wild-type Rpts in the Rpt ring. In addition, we demonstrate that the C-terminal tails of Rpt subunits possessing core particle (CP)-binding affinities facilitate the cellular assembly of the 19 S RP, implying that the 20 S CP may function as a template for base assembly in human cells. Taken together, these results suggest that the ATP-bound conformational state of an Rpt subunit with the exposed C-terminal tail is competent for cellular proteasome assembly.
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