Proteasomal ATPase-associated factor 1 negatively regulates proteasome activity by interacting with proteasomal ATPases

The 26S proteasome, composed of the 20S core and the 19S regulatory complex, plays a central role in ubiquitin-dependent proteolysis by catalyzing degradation of polyubiquitinated proteins. In a search for proteins involved in regulation of the proteasome, we affinity purified the 19S regulatory complex from HeLa cells and identified a novel protein of 43 kDa in size as an associated protein. Immunoprecipitation analyses suggested that this protein specifically interacted with the proteasomal ATPases. Hence the protein was named proteasomal ATPase-associated factor 1 (PAAF1). Immunoaffinity purification of PAAF1 confirmed its interaction with the 19S regulatory complex and further showed that the 19S regulatory complex bound with PAAF1 was not stably associated with the 20S core. Overexpression of PAAF1 in HeLa cells decreased the level of the 20S core associated with the 19S complex in a dose-dependent fashion, suggesting that PAAF1 binding to proteasomal ATPases inhibited the assembly of the 26S proteasome. Proteasomal degradation assays using reporters based on green fluorescent protein revealed that overexpression of PAAF1 inhibited the proteasome activity in vivo. Furthermore, the suppression of PAAF1 expression that is mediated by small inhibitory RNA enhanced the proteasome activity. These results suggest that PAAF1 functions as a negative regulator of the proteasome by controlling the assembly/disassembly of the proteasome.     

Subcellular localization, stoichiometry, and protein levels of 26 S proteasome subunits in yeast.

The 26 S proteasome of eukaryotes is responsible for the degradation of proteins targeted for proteolysis by the ubiquitin system. Yeast has been an important model organism for understanding eukaryotic proteasome structure and function. Toward a quantitative characterization of the proteasome, we have determined the localization, cellular levels, and stoichiometry of proteasome subunits. The subcellular localization of two ATPase components of the regulatory complex of the proteasome, Sug2/Rpt4 and Sug1/Rpt6, and a subunit of the 20 S proteasome, Pre1, were determined by immunofluorescence. In contrast to findings in multicellular organisms, these proteins are localized almost exclusively to the nucleus throughout the cell cycle. We have also determined the cellular abundance and stoichiometry of these proteasome subunits. Sug1/Rpt6, Sug2/Rpt4, and Pre1 are present in roughly equal stoichiometry with an abundance of 15,000-30,000 molecules/cell, corresponding to a concentration of 13-26 microM in the nucleus. Also, in contrast to mammalian cells, we find no evidence of a p27-containing "modulator" of the proteasome in yeast. This information will be useful in comparing and contrasting the yeast and mammalian proteasomes and should contribute to a mechanistic understanding of how this complex functions.     

Cdc48-Ufd1-Npl4: stuck in the middle with Ub

The ubiquitin-proteasome pathway has a well-defined beginning and end. Target proteins are initially recognized by upstream components and tagged with polyubiquitin chains. The 26S proteasome then degrades these polyubiquitinated proteins. Until recently, it was not known what, if any, steps occurred between the initial polyubiquitination of target proteins and their final degradation. Several new papers investigating the function of the Cdc48-Ufd1-Npl4 complex indicate that there is indeed a middle to the ubiquitin-proteasome pathway. The Cdc48-Ufd1-Npl4 complex functions in the recognition of several polyubiquitin-tagged proteins and facilitates their presentation to the 26S proteasome for processive degradation or even more specific processing. The elucidation of Cdc48, Ufd1 and Npl4 action not only provides long-sought functions for these specific proteins, but illuminates a poorly understood part of the ubiquitin-proteasome pathway.     

SOCS-1 localizes to the microtubule organizing complex-associated 20S proteasome

The regulation of cytokine signaling is critical for controlling cellular proliferation and activation during an immune response. SOCS-1 is a potent inhibitor of Jak kinase activity and of signaling initiated by several cytokines. SOCS-1 protein levels are tightly regulated, and recent data suggest that SOCS-1 may regulate the protein levels of some signaling proteins by the ubiquitin proteasome pathway; however, the cellular mechanism by which SOCS-1 directs proteins for degradation is unknown. In this report, SOCS-1 is found to colocalize and biochemically copurify with the microtubule organizing complex (MTOC) and its associated 20S proteasome. The SOCS-1 SH2 domain is required for the localization of SOCS-1 to the MTOC. Overexpression of SOCS-1 targets Jak1 in an SH2-dependent manner to a perinuclear distribution resembling the MTOC-associated 20S proteasome. Analysis of MTOCs fractionated from SOCS-1-deficient cells demonstrates that SOCS-1 may function redundantly to regulate the localization of Jak1 to the MTOC. Nocodazole inhibits the protein turnover of SOCS-1, demonstrating that the minus-end transport of SOCS-1 to the MTOC-associated 20S proteasome is required to regulate SOCS-1 protein levels. These data link SOCS-1 directly with the proteasome pathway and suggest another function for the SH2 domain of SOCS-1 in the regulation of Jak/STAT signaling.     

The Ccr4a (CNOT6) and Ccr4b (CNOT6L) deadenylase subunits of the human Ccr4-Not complex contribute to the prevention of cell death and senescence

A key step in cytoplasmic mRNA degradation is the shortening of the poly(A) tail, which involves several deadenylase enzymes. Relatively little is known about the importance of these enzymes for the cellular physiology. Here we focused on the role of the highly similar Ccr4a (CNOT6) and Ccr4b (CNOT6L) deadenylase subunits of the Ccr4-Not complex. In addition to a role in cell proliferation, Ccr4a and Ccr4b play a role in cell survival, in contrast to the Caf1a (CNOT7) and Caf1b (CNOT8) deadenylase subunits or the CNOT1 and CNOT3 noncatalytic subunits of the Ccr4-Not complex. Underscoring the differential contributions of the deadenylase subunits, we found that knockdown of Caf1a/Caf1b or Ccr4a/Ccr4b differentially affects the formation of cytoplasmic foci by processing-body components. Furthermore, we demonstrated that the amino-terminal leucine-rich repeat (LRR) domain of Ccr4b influenced its subcellular localization but was not required for the deadenylase activity of Ccr4b. Moreover, overexpression of Ccr4b lacking the LRR domain interfered with cell cycle progression but not with cell viability. Finally, gene expression profiling indicated that distinct gene sets are regulated by Caf1a/Caf1b and Ccr4a/Ccr4b and identified Ccr4a/Ccr4b as a key regulator of insulin-like growth factor-binding protein 5, which mediates cell cycle arrest and senescence via a p53-dependent pathway.     

Sum1, a component of the fission yeast eIF3 translation initiation complex,is rapidly relocalized during environmental stress and interacts with components of the 26S proteasome

Eukaryotic translation initiation factor 3 (eIF3) is a multisubunit complex that plays a central role in translation initiation. We show that fission yeast Sum1, which is structurally related to known eIF3 subunits in other species, is essential for translation initiation, whereas its overexpression results in reduced global translation. Sum1 is associated with the 40S ribosome and interacts stably with Int6, an eIF3 component, in vivo, suggesting that Sum1 is a component of the eIF3 complex. Sum1 is cytoplasmic under normal growth conditions. Surprisingly, Sum1 is rapidly relocalized to cytoplasmic foci after osmotic and thermal stress. Int6 and p116, another putative eIF3 subunit, behave similarly, suggesting that eIF3 is a dynamic complex. These cytoplasmic foci, which additionally comprise eIF4E and RNA components, may function as translation centers during environmental stress. After heat shock, Sum1 additionally colocalizes stably with the 26S proteasome at the nuclear periphery. The relationship between Sum1 and the 26S proteasome was further investigated, and we find cytoplasmic Sum1 localization to be dependent on the 26S proteasome. Furthermore, Sum1 interacts with the Mts2 and Mts4 components of the 26S proteasome. These data indicate a functional link between components of the structurally related eIF3 translation initiation and 26S proteasome complexes.     

Localization of the 26S proteasome during mitosis and meiosis in fission yeast.

The 26S proteasome is a large multisubunit complex involved in degrading both cytoplasmic and nuclear proteins. We have investigated the localization of this complex in the fission yeast, Schizosaccharomyces pombe. Immunofluorescence microscopy shows a striking localization pattern whereby the proteasome is found predominantly at the nuclear periphery, both in interphase and throughout mitosis. Electron microscopic analysis revealed a concentration of label near the inner side of the nuclear envelope. The localization of green fluorescent protein (GFP)-tagged 26S proteasomes was analyzed in live cells during mitosis and meiosis. Throughout mitosis the proteasome remained predominantly at the nuclear periphery. During meiosis the proteasome was found to undergo dramatic changes in its localization. Throughout the first meiotic division, the signal is more dispersed over the nucleus. During meiosis II, there was a dramatic re-localization, and the signal became restricted to the area between the separating DNA until the end of meiosis when the signal dispersed before returning to the nuclear periphery during spore formation. These findings strongly imply that the nuclear periphery is a major site of protein degradation in fission yeast both in interphase and throughout mitosis. Furthermore they raise interesting questions as to the spatial organization of protein degradation during meiosis.     

Mass spectrometric characterization of the affinity-purified human 26S proteasome complex

The 26S proteasome is a multisubunit complex responsible for degradation of ubiquitinated substrates, which plays a critical role in regulating various biological processes. To fully understand the function and regulation of the proteasome complex, an important step is to elucidate its subunit composition and posttranslational modifications. Toward this goal, a new affinity purification strategy has been developed using a derivative of the HB tag for rapid isolation of the human 26S proteasome complex for subsequent proteomic analysis. The purification of the complex is achieved from stable 293 cell lines expressing a HB-tagged proteasome subunit and by high-affinity streptavidin binding with TEV cleavage elution. The complete composition of the 26S proteasome complex, including recently assigned new subunits, is identified by LC-MS/MS. In addition, all known proteasome activator proteins and components involved in the ubiquitin-proteasome degradation pathway are identified. Aside from the subunit composition, the N-terminal modification and phosphorylation of the proteasome subunits have been characterized. Twelve novel phosphorylation sites from eight subunits have been identified, and N-terminal modifications are determined for 25 subunits, 12 of which have not been previously reported in mammals. We also observe different N-terminal processing of subunit Rpn2, which results in identification of two different N-termini of the protein. This work presents the first comprehensive characterization of the human 26S proteasome complex by affinity purification and tandem mass spectrometry. The detailed proteomic profiling obtained here is significant to future studies aiming at a complete understanding of the structure-function relationship of the human 26S proteasome complex.     

The multiubiquitin-chain-binding protein Mcb1 is a component of the 26S proteasome in Saccharomyces cerevisiae and plays a nonessential, substrate-specific role in protein turnover.

The 26S proteasome is an essential proteolytic complex that is responsible for degrading proteins conjugated with ubiquitin. It has been proposed that the recognition of substrates by the 26S proteasome is mediated by a multiubiquitin-chain-binding protein that has previously been characterized in both plants and animals. In this study, we identified a Saccharomyces cerevisiae homolog of this protein, designated Mcb1. Mcb1 copurified with the 26S proteasome in both conventional and nickel chelate chromatography. In addition, a significant fraction of Mcb1 in cell extracts was present in a low-molecular-mass form free of the 26S complex. Recombinant Mcb1 protein bound multiubiquitin chains in vitro and, like its plant and animal counterparts, exhibited a binding preference for longer chains. Surprisingly, (delta)mcb1 deletion mutants were viable, grew at near-wild-type rates, degraded the bulk of short-lived proteins normally, and were not sensitive to UV radiation or heat stress. These data indicate that Mcb1 is not an essential component of the ubiquitin-proteasome pathway in S.cerevisiae. However, the (delta)mcb1 mutant exhibited a modest sensitivity to amino acid analogs and had increased steady-state levels of ubiquitin-protein conjugates. Whereas the N-end rule substrate, Arg-beta-galactosidase, was degraded at the wild-type rate in the (delta)mcb1 strain, the ubiquitin fusion degradation pathway substrate, ubiquitin-Pro-beta-galactosidase, was markedly stabilized. Collectively, these data suggest that Mcb1 is not the sole factor involved in ubiquitin recognition by the 26S proteasome and that Mcb1 may interact with only a subset of ubiquitinated substrates.     

Sem1p is a novel subunit of the 26 S proteasome from Saccharomyces cerevisiae

The 26 S proteasome, which catalyzes degradation of polyubiquitinated proteins, is composed of the 20 S proteasome and the 19 S regulatory particle (RP). The RP is composed of the lid and base subcomplexes and regulates the catalytic activity of the 20 S proteasome. In this study, we carried out affinity purification of the lid and base subcomplexes from the tagged strains of Saccharomyces cerevisiae, and we found that the lid contains a small molecular mass protein, Sem1. The Sem1 protein binds with the 26 S proteasome isolated from a mutant with deletion of SEM1 but not with the 26 S proteasome from the wild type. The lid lacking Sem1 is unstable at a high salt concentration. The 19 S RP was immunoprecipitated together with Sem1 by immunoprecipitation using hemagglutinin epitope-tagged Sem1 as bait. Degradation of polyubiquitinated proteins in vivo or in vitro is impaired in the Sem1-deficient 26 S proteasome. In addition, genetic interaction between SEM1 and RPN10 was detected. The human Sem1 homologue hDSS1 was found to be a functional homologue of Sem1 and capable of interacting with the human 26 S proteasome. The results suggest that Sem1, possibly hDSS1, is a novel subunit of the 26 S proteasome and plays a role in ubiquitin-dependent proteolysis.     

The DUG gene of Drosophila melanogaster encodes a structural and functional homolog of the S. cerevisiae SUG1 predicted ATPase associated with the 26S proteasome

The DUG gene of Drosophila encodes a putative ATPase that is a structural and functional homolog of the yeast SUG1 product. When introduced into S. cerevisiae, the Drosophila DUG gene rescued the lethality associated with a SUG1 mutant. Anti-DUG antibodies recognized a protein that migrated in high molecular weight complexes, along with components of the 26S proteasome, and also immunoprecipitated components of the 26S proteasome from embryonic extracts. Proteins recognized by the affinity-purified antibody raised against DUG were localized in either a punctate cytoplasmic distribution or in the nucleus, depending on the cell type, consistent with the subcellular localization of the 26S proteasome in various cell types.     

26S proteasome exhibits endoribonuclease activity controlled by extra-cellular stimuli

26S proteasome is a large multi-subunit protein complex involved in proteolytic degradation of proteins. In addition to its canonical proteolytic activity, the proteasome is also associated with recently characterized endoribonuclease (endo-RNAse) activity. However, neither functional significance, nor the mechanisms of its regulation are currently known. In this report, we show that 26S proteasome is able to hydrolyze various cellular RNAs, including AU-rich mRNA of c-myc and c-fos. The endonucleolytic degradation of these mRNAs is exerted by one of the 26S proteasome subunits, PSMA5 (α5). The RNAse activity of 26S proteasome is differentially affected by various extra-cellular signals. Moreover, this activity contributes to the process of degradation of c-myc mRNA during induced differentiation of K562 cells, and may be controlled by phosphorylation of the adjacent subunits, PSMA1 (α6) and PSMA3 (α7). Collectively, the data presented in this report suggest a causal link between cell signalling pathways, endo-RNAse activity of the 26S proteasome complex and metabolism of cellular RNAs.     

Adrm1, a putative cell adhesion regulating protein, is a novel proteasome-associated factor

We have identified Adrm1 as a novel component of the regulatory ATPase complex of the 26 S proteasome: Adrm1 was precipitated with an antibody to proteasomes and vice versa. Adrm1 co-migrated with proteasomes on gel-filtration chromatography and non-denaturing polyacrylamide gel electrophoresis. Adrm1 has been described as an interferon-gamma-inducible, heavily glycosylated membrane protein of 110 kDa. However, we found Adrm1 in mouse tissues only as a 42 kDa peptide, corresponding to the mass of the non-glycosylated peptide chain, and it could not be induced in HeLa cells with interferon. Adrm1 was present almost exclusively in soluble 26 S proteasomes, albeit a small fraction was membrane-associated, like proteasomes. Adrm1 was found in cells in amounts equimolar with S6a, a 26 S proteasome subunit. HeLa cells contain no pool of free Adrm1 but recombinant Adrm1 could bind to pre-existing 26 S proteasomes in cell extracts. Adrm1 may be distantly related to the yeast proteasome subunit Rpn13, mutants of which are reported to display no obvious phenotype. Accordingly, knock-down of Adrm1 in HeLa cells had no effect on the amount of proteasomes, or on degradation of bulk cell protein, or accumulation of polyubiquitinylated proteins. This indicates that Adrm1 has a specialised role in proteasome function.     

Evidence for separable functions of Srp1p, the yeast homolog of importin alpha (Karyopherin alpha): role for Srp1p and Sts1p in protein degradation

Srp1p (importin alpha) functions as the nuclear localization signal (NLS) receptor in Saccharomyces cerevisiae. The srp1-31 mutant is defective in this nuclear localization function, whereas an srp1-49 mutant exhibits defects that are unrelated to this localization function, as was confirmed by intragenic complementation between the two mutants. RPN11 and STS1 (DBF8) were identified as high-dosage suppressors of the srp1-49 mutation but not of the srp1-31 mutation. We found that Sts1p interacts directly with Srp1p in vitro and also in vivo, as judged by coimmunoprecipitation and two-hybrid analyses. Mutants of Sts1p that cannot interact with Srp1p are incapable of suppressing srp1-49 defects, strongly suggesting that Sts1p functions in a complex with Srp1p. STS1 also interacted with the second suppressor, RPN11, a subunit of the 26S proteasome, in the two-hybrid system. Further, degradation of Ub-Pro-beta-galactosidase, a test substrate for the ubiquitin-proteasome system, was defective in srp1-49 but not in srp1-31. This defect in protein degradation was alleviated by overexpression of either RPN11 or STS1 in srp1-49. These results suggest a role for Srp1p in regulation of protein degradation separate from its well-established role as the NLS receptor.     

Nucleolar Stress Induces Ubiquitination-independent Proteasomal Degradation of PICT1 Protein

The nucleolar protein PICT1 regulates tumor suppressor p53 by tethering ribosomal protein L11 within the nucleolus to repress the binding of L11 to the E3 ligase MDM2. PICT1 depletion results in the release of L11 to the nucleoplasm to inhibit MDM2, leading to p53 activation. Here, we demonstrate that nucleolar stress induces proteasome-mediated degradation of PICT1 in a ubiquitin-independent manner. Treatment of H1299 cells with nucleolar stress inducers, such as actinomycin D, 5-fluorouridine, or doxorubicin, induced the degradation of PICT1 protein. The proteasome inhibitors MG132, lactacystin, and epoxomicin blocked PICT1 degradation, whereas the inhibition of E1 ubiquitin-activating enzyme by a specific inhibitor and genetic inactivation fail to repress PICT1 degradation. In addition, the 20 S proteasome was able to degrade purified PICT1 protein in vitro. We also found a PICT1 mutant showing nucleoplasmic localization did not undergo nucleolar stress-induced degradation, although the same mutant underwent in vitro degradation by the 20 S proteasome, suggesting that nucleolar localization is indispensable for the stress-induced PICT1 degradation. These results suggest that PICT1 employs atypical proteasome-mediated degradation machinery to sense nucleolar stress within the nucleolus.