Use of RNA Interference and Complementation To Study the Function of the Drosophila and Human 26S Proteasome Subunit S13

Abstract

The S13 subunit (also called Pad1, Rpn11, and MPR1) is a component of the 19S complex, a regulatory complex essential for the ubiquitin-dependent proteolytic activity of the 26S proteasome. To address the functional role of S13, we combined double-stranded RNA interference (RNAi) against the Drosophila proteasome subunit DmS13 with expression of wild-type and mutant forms of the homologous human gene, HS13. These studies show that DmS13 is essential for 26S function. Loss of the S13 subunit in metazoan cells leads to increased levels of ubiquitin conjugates, cell cycle defects, DNA overreplication, and apoptosis. In vivo assays using short-lived proteasome substrates confirmed that the 26S ubiquitin-dependent degradation pathway is compromised in S13-depleted cells. In complementation experiments using Drosophila cell lines expressing HS13, wild-type HS13 was found to fully rescue the knockdown phenotype after DmS13 RNAi treatment, while an HS13 containing mutations (H113A-H115A) in the proposed isopeptidase active site was unable to rescue. A mutation within the conserved MPN/JAMM domain (C120A) abolished the ability of HS13 to rescue the Drosophila cells from apoptosis or DNA overreplication. However, the C120A mutant was found to partially restore normal levels of ubiquitin conjugates. The S13 subunit may possess multiple functions, including a deubiquitinylating activity and distinct activities essential for cell cycle progression that require the conserved C120 residue.The ubiquitin-proteasome system is the major nonlysosomal pathway responsible for the degradation of intracellular proteins in eukaryotic cells. This system participates in the regulation of a vast number of cellular pathways through timely and specific conjugation of target proteins with multiple ubiquitin molecules followed by proteolysis by the 26S proteasome (52). The 26S proteasome is a large protease (2,500 kDa) composed of a 20S catalytic core and a 19S regulatory complex that associates with the ends of the 20S proteasome in an ATPdependent manner. The 20S core (700 kDa) is a compartmentalized multicatalytic complex composed of a total of 28 ␣ and ␤ subunits arranged in four stacked heptameric rings. The subunits generate a hollow cylindrical structure that separates the cytosolic environment from the catalytic sites located at the lumenal face of the ␤-rings (16,30,46,60). In the absence of regulatory factors, the 20S proteasome exists in an autoinhibited (latent) state in which the free N-terminal tails of its ␣ subunits extend into the 20S pores and thus sterically block substrate access to the lumen (17). Activation of the 20S proteasome requires interactions of the ␣ subunits with specific regulators, such as the PA28␣␤ or -␥ heptamer (35), PA200 (47), or the 19S regulatory complex (8).The 19S complex has been directly implicated in nonproteolytical regulatory functions, such as nucleotide excision-repair (10, 38) and transcription elongation (7,14). Its main function however, is to carry out several distinct steps critical for u...

z-Leucinyl-Leucinyl-Norleucinal Induces Apoptosis of Human Glioblastoma Tumor–Initiating Cells by Proteasome Inhibition and Mitotic Arrest Response

“…The upregulation of the expression of some genes coding for proteasomal proteins, as a consequence of the inhibition of proteasome activity by both RNA interference and small molecules such as GM132, is a well-established phenomenon observed in several organisms including mammals (48)(49)(50)(51). Therefore, the data reported here, which show the ability of LLNle to inhibit the proteasome activity and concomitantly to upregulate the expression of genes coding for several proteasome subunits, are in line with these previous studies.…”

The 19 S Proteasomal Subunit POH1 Contributes to the Regulation of c-Jun Ubiquitination, Stability, and Subcellular Localization

“…Embedded within the JAMM sequence is a Cys-box-like motif of unknown function. The relevant cysteine (Cys-120) of this motif is not believed to be essential for the DUB activity of POH1 (12,14) but may be required for deubiquitination of specific subsets of proteins (24). The alignment also identified a short hydrophobic peptide (LALLKML) located near the N terminus that resembles the ubiquitin-binding motif of RPN10 (LALALR(L/V)) (25).…”

“…A recent study has shown that a C120A mutation of POH1 produced specific defects related to DNA replication and apoptosis. The authors proposed the cysteine might confer specificity for particular substrates that were associated with these phenotypes (24). One such substrate is likely to be c-Jun.…”

The JAMM motif of human deubiquitinase Poh1 is essential for cell viability

“…To evaluate the role of the Rpn11 JAMM motif in metal ion -dependent deubiquitination, various laboratories have mutated one or both conserved histidines to alanines and reintroduced the mutant protein into proteasomes for functional evaluation. Such studies have shown that mutations in JAMM motif histidines are lethal in yeast and fly, leading to elevated levels of ubiquitin conjugates (10,11,19,23,24). Additionally, purified yeast proteasomes containing Rpn11 point mutants are incompetent for deubiquitination and degradation of polyubiquitinated substrates (10).…”