19S Complex Research Articles

Characterization of the proteasome interaction with the Sec61 channel in the endoplasmic reticulum

Biogenesis of secretory proteins requires their translocation into the endoplasmic reticulum (ER) through the Sec61 channel. Proteins that fail to fold are transported back into the cytosol and are degraded by proteasomes. For many substrates this retrograde transport is affected by mutations in the Sec61 channel, and can be promoted by ATP and the 19S regulatory particle of the proteasome, which binds directly to the Sec61 channel via its base. Here, we identify mutations in SEC61 which reduce proteasome binding to the channel, and demonstrate that proteasomes and ribosomes bind differently to cytosolic domains of the channel. We found that Sec63p and BiP coprecipitate with ER-associated proteasomes, but Sec63p does not contribute to proteasome binding to the ER. The 19S base contains six AAA-ATPase subunits (Rpt proteins) that have non-equivalent functions in proteasome-mediated protein turnover and form a hetero-hexamer. Mutations in the ATP-binding sites of individual Rpt proteins all reduced the affinity of 19S complexes for the ER, suggesting that the 19S base in the ATP-bound conformation docks at the Sec61 channel.

HRpn13/ADRM1/GP110 is a novel proteasome subunit that binds the deubiquitinating enzyme, UCH37

The 26S proteasome catalyzes the degradation of most proteins in mammalian cells. To better define its composition and associated regulatory proteins, we developed affinity methods to rapidly purify 26S proteasomes from mammalian cells. By this approach, we discovered a novel 46-kDa (407 residues) subunit of its 19S regulatory complex (previously termed ADRM1 or GP110). As its N-terminal half can be incorporated into the 26S proteasome and is homologous to Rpn13, a 156-residue subunit of the 19S complex in budding yeast, we renamed it human Rpn13 (hRpn13). The C-terminal half of hRpn13 binds directly to the proteasome-associated deubiquitinating enzyme, UCH37, and enhances its isopeptidase activity. Knockdown of hRpn13 in 293T cells increases the cellular levels of ubiquitin conjugates and decreases the degradation of short-lived proteins. Surprisingly, an overproduction of hRpn13 also reduced their degradation. Furthermore, transfection of the C-terminal half of hRpn13 slows proteolysis and induces cell death, probably by acting as a dominant-negative form. Thus in human 26S proteasomes, hRpn13 appears to be important for the binding of UCH37 to the 19S complex and for efficient proteolysis.

Proteasome recruitment and activation of the Uch37 deubiquitinating enzyme by Adrm1

Uch37 is one of the three principal deubiquitinating enzymes (DUBs), and the only ubiquitin carboxy-terminal hydrolase (UCH)-family protease, that is associated with mammalian proteasomes. We show that Uch37 is responsible for the ubiquitin isopeptidase activity in the PA700 (19S) proteasome regulatory complex. PA700 isopeptidase disassembles Lys 48-linked polyubiquitin specifically from the distal end of the chain, a property that may be used to clear poorly ubiquitinated or unproductively bound substrates from the proteasome. To better understand Uch37 function and the mechanism responsible for its specificity, we investigated how Uch37 is recruited to proteasomes. Uch37 binds through Adrm1, a previously unrecognized orthologue of Saccharomyces cerevisiae Rpn13p, which in turn is bound to the S1 (also known as Rpn2) subunit of the 19S complex. Adrm1 (human Rpn13, hRpn13) binds the carboxy-terminal tail of Uch37, a region that is distinct from the UCH catalytic domain, which we show inhibits Uch37 activity. Following binding, Adrm1 relieves Uch37 autoinhibition, accelerating the hydrolysis of ubiquitin-7-amido-4-methylcoumarin (ubiquitin-AMC). However, neither Uch37 alone nor the Uch37-Adrm1 or Uch37-Adrm1-S1 complexes can hydrolyse di-ubiquitin efficiently; rather, incorporation into the 19S complex is required to enable processing of polyubiquitin chains.

Smashing Protein Complex Ions to Bits Reveals Their Structural Organization

Most cell diagrams ably illustrate the cell's major structural elements, but they can't convey the incredibly dynamic nature of cellular life. One cell can contain millions of ribosomes, each churning out a protein a minute. To make way for new proteins, obsolete proteins must be removed. Short-lived and misshapen proteins get tagged with ubiquitin proteins and sent to the proteasome, a massive enzyme complex that degrades proteins into peptides through a process called proteolysis. First isolated from yeast, the “26S” proteasome (named after a measure of its macromolecular size) consists of two major subcomplexes, the 20S proteolytic core and the 19S regulatory particle. The 19S particle forms a cap over the 20S core; it has a base that binds to the core and a lid that sits atop the base. After the 19S particle recognizes ubiquitinated substrates and removes their ubiquitin tag, they're unfolded and sent to the core for degradation. To better understand how this important regulatory complex functions, researchers need precise information on its structure—no small task, given that the lid's many subunits presumably undergo dynamic assembly in the cell. In a new study, Michal Sharon, Carol Robinson, and colleagues present an innovative approach for identifying the structural composition and organization of intact macromolecular complexes. By combining this approach with chemical cross-linking, which provides additional artificial bonds between the 19S components, the researchers were able to determine the lid's structural organization and gain insight into the likely functional interactions between the components. Sharon et al. first isolated the intact 19S complexes from yeast, then “electrosprayed” the macromolecules, converting them into ions for mass spectral analysis. Mass spectrometry reveals the protein composition of a sample based on its molecular mass. (Ions travel through a “time-of-flight” analyzer, which times the speed of ions as they travel through an electric field; the sorted ions reach a detector that shows the relative abundance of different masses in the sample as peaks across a spectrum.) The resulting mass spectrum confirmed previous reports that the lid consists of nine protein components, and indicated the presence of an intact complex (roughly 376 kiloDaltons) as well as a smaller subcomplex (roughly 185 kiloDaltons). To further characterize these components, Sharon et al. used tandem mass spectrometry (MS/MS), a technique that involves multiple steps of sorting ions to isolate and analyze the complex's components. The ionized sample accelerates through a gas-filled “collision chamber.” As multiple collisions yield increasing energy, the ionized sample (precursor ion) dissociates into “product ions” (individual subunits). Using this approach on the intact lid, the researchers could infer how the individual pieces of the lid fit together by identifying which of the components stuck together longer. From one round of MS/MS, they identified the two proteins Rpn9 and Rpn12. Then a second, higher energy round of MS/MS triggered the dissociation of a third protein, Rpn6. The researchers concluded that Rpn6, Rpn9, and Rpn12 interact weakly around the lid's periphery. Yet another round of MS/MS, this time on the lid subcomplex, identified Rpn5, Rpn6, and Rpn9 as subunits that can form different interactions with Rpn8, a subunit that had not been detected by itself. Finally, using a technique called chemical cross-linking (which creates covalent bonds between neighboring proteins so they can be examined), the researchers identified additional interaction partners within the lid. Based on the results of their MS/MS and cross-linking experiments, along with previously published data, the researchers proposed a model of how all these components come together to form the lid complex. The lid consists of two subcomplexes—one comprising Rpn5, Rpn6, Rpn8, and Rpn9; the other of Rpn3, Rpn7, Rpn12, and Sem1—with a link forged between Rpn5 and Rpn3. Rnp11, the enzyme responsible for coupling deubiquitination with degradation, is likely recruited by the first subcomplex. These insights into the configuration and possible role of the lid's subunits will facilitate future investigations into the function and regulation of the 19S lid. And this technique—which can identify the subunit organization of intact complexes based on their mass spectra—should illuminate the architecture of a diverse range of macromolecules still uncharacterized.

Regulation of Murine Cardiac 20S Proteasomes

Our recent studies have provided a proteomic blueprint of the 26S proteasome complexes in the heart, among which 20S proteasomes were found to contain cylinder-shaped structures consisting of both alpha and beta subunits. These proteasomes exhibit a number of features unique to the myocardium, including striking differences in post-translational modifications (PTMs) of individual subunits and novel PTMs that have not been previously reported. To date, mechanisms contributing to the regulation of this myocardial proteolytic core system remain largely undefined; in particular, little is known regarding PTM-dependent regulation of cardiac proteasomes. In this investigation, we seek to elucidate the function and regulation of 20S proteasome complexes in the heart. Functionally viable murine cardiac 20S proteasomes were purified. Tandem mass spectrometry analyses, combined with native gel electrophoresis, immunoprecipitation, and immunoblotting, revealed the identification of 2 previously unrecognized functional partners in the endogenous intact cardiac 20S complexes: protein phosphatase 2A (PP2A), and protein kinase A (PKA). Furthermore, our results demonstrated that PP2A and PKA profoundly impact the proteolytic function of 20S proteasomes: phosphorylation of 20S complexes enhances the peptidase activity of individual subunits in a substrate-specific fashion. Moreover, inhibition of PP2A or the addition of PKA significantly modified both the serine- and threonine-phosphorylation profile of proteasomes; multiple individual subunits of 20S (eg, alpha1 and beta2) were targets of PP2A and PKA. Taken together, these studies provide the first demonstration that the function of cardiac 20S proteasomes is modulated by associating partners and that phosphorylation may serve as a key mechanism for regulation.

Mapping the Murine Cardiac 26S Proteasome Complexes

The importance of proteasomes in governing the intracellular protein degradation process has been increasingly recognized. Recent investigations indicate that proteasome complexes may exist in a species- and cell-type-specific fashion. To date, despite evidence linking impaired protein degradation to cardiac disease phenotypes, virtually nothing is known regarding the molecular composition, function, or regulation of cardiac proteasomes. We have taken a functional proteomic approach to characterize 26S proteasomes in the murine heart. Multidimensional chromatography was used to obtain highly purified and functionally viable cardiac 20S and 19S proteasome complexes, which were subjected to electrophoresis and tandem mass spectrometry analyses. Our data revealed complex molecular organization of cardiac 26S proteasomes, some of which are similar to what were reported in yeast, whereas others exhibit contrasting features that have not been previously identified in other species or cell types. At least 36 distinct subunits (17 of 20S and 19 of 19S) are coexpressed and assembled as 26S proteasomes in this vital cardiac organelle, whereas the expression of PA200 and 11S subunits were detected with limited participation in the 26S complexes. The 19S subunits included a new alternatively spliced isoform of Rpn10 (Rpn10b) along with its primary isoform (Rpn10a). Immunoblotting and immunocytochemistry verified the expression of key alpha and beta subunits in cardiomyocytes. The expression of 14 constitutive alpha and beta subunits in parallel with their three inducible subunits (beta1i, beta2i, and beta5i) in the normal heart was not expected; these findings represent a distinct level of structural complexity of cardiac proteasomes, significantly different from that of yeast and human erythrocytes. Furthermore, liquid chromatography/tandem mass spectroscopy characterized 3 distinct types of post-translational modifications including (1) N-terminal acetylation of 19S subunits (Rpn1, Rpn5, Rpn6, Rpt3, and Rpt6) and 20S subunits (alpha2, alpha5, alpha7, beta3, and beta4); (2) N-terminal myristoylation of a 19S subunit (Rpt2); and (3) phosphorylation of 20S subunits (eg, alpha7)). Taken together, this report presents the first comprehensive characterization of cardiac 26S proteasomes, providing critical structural and proteomic information fundamental to our future understanding of this essential protein degradation system in the normal and diseased myocardium.

ATP Binding to PAN or the 26S ATPases Causes Association with the 20S Proteasome, Gate Opening, and Translocation of Unfolded Proteins

The archaeal ATPase complex PAN, the homolog of the eukaryotic 26S proteasome-regulatory ATPases, was shown to associate transiently with the 20S proteasome upon binding of ATP or ATPgammaS, but not ADP. By electron microscopy (EM), PAN appears as a two-ring structure, capping the 20S, and resembles two densities in the 19S complex. The N termini of the archaeal 20S alpha subunits were found to function as a gate that prevents entry of seven-residue peptides but allows entry of tetrapeptides. Upon association with the 20S particle, PAN stimulates gate opening. Although degradation of globular proteins requires ATP hydrolysis, the PAN-20S complex with ATPgammaS translocates and degrades unfolded and denatured proteins. Rabbit 26S proteasomes also degrade these unfolded proteins upon ATP binding, without hydrolysis. Thus, although unfolding requires energy from ATP hydrolysis, ATP binding alone supports ATPase-20S association, gate opening, and translocation of unfolded substrates into the proteasome, which can occur by facilitated diffusion through the ATPase.

Ubiquitin-proteasome-dependent proteolytic activity remains elevated after zymosan-induced sepsis in rats while muscle mass recovers

We studied the role of the ubiquitin-proteasome system in rat skeletal muscle during sepsis and subsequent recovery. Sepsis was induced with intraperitoneal zymosan injections. This model allows one to study a sustained and reversible catabolic phase and mimics the events that prevail in septic and subsequently recovering patients. In addition, the role of the ubiquitin-proteasome system during muscle recovery is poorly documented. There was a trend for increased ubiquitin-conjugate formation in the muscle wasting phase, which was abolished during the recovery phase. The trypsin- and chymotrypsin-like peptidase activities of the 20S proteasome peaked at day 6 following zymosan injection (i.e. when both muscle mass and muscle fiber cross-sectional area were reduced the most), but remained elevated when muscle mass and muscle fiber cross-sectional area were recovering (11 days). This clearly suggests a role for the ubiquitin-proteasome pathway in the muscle remodeling and/or recovery process. Protein levels of 19S complex and 20S proteasome subunits did not increase throughout the study, pointing to alternative mechanisms regulating proteasome activities. Overall these data support a role for ubiquitin-proteasome dependent proteolysis in the zymosan septic model, in both the catabolic and muscle recovery phases.

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.

RETRACTED: ATP Hydrolysis-Dependent Disassembly of the 26S Proteasome Is Part of the Catalytic Cycle

ATP hydrolysis is required for degradation of polyubiquitinated proteins by the 26S proteasome but is thought to play no role in proteasomal stability during the catalytic cycle. In contrast to this view, we report that ATP hydrolysis triggers rapid dissociation of the 19S regulatory particles from immunopurified 26S complexes in a manner coincident with release of the bulk of proteasome-interacting proteins. Strikingly, this mechanism leads to quantitative disassembly of the 19S into subcomplexes and free Rpn10, the polyubiquitin binding subunit. Biochemical reconstitution with purified Sic1, a prototype substrate of the Cdc34/SCF ubiquitin ligase, suggests that substrate degradation is essential for triggering the ATP hydrolysis-dependent dissociation and disassembly of the 19S and that this mechanism leads to release of degradation products. This is the first demonstration that a controlled dissociation of the 19S regulatory particles from the 26S proteasome is part of the mechanism of protein degradation.

Subcellular Redistribution of Components of the Ubiquitin–Proteasome Pathway during Lens Differentiation and Maturation

To determine the subcellular distribution of components of the ubiquitin-proteasome pathway (UPP) in lens epithelium and differentiating fibers and to evaluate potential roles of the UPP in eliminating nuclei and other organelles during maturation of lens fibers. Adult bovine lens cryosections were stained for immunofluorescence and analyzed by confocal microscopy. The specificities of the antibodies used in this study were determined by Western blot. results Cryosections of bovine lenses show that E1 and Ubc1 were present in both the cytoplasm and the nucleus in epithelial cells, whereas Ubc3 and ubiquitin conjugates were mostly confined to the nucleus, and Ubc4/5 was preferentially localized in clusters in the vicinity of the nuclear membrane. The 19S and 20S proteasome complexes were preferentially localized in the cytoplasm. When the epithelial cells differentiated into fiber cells at the transition zone, all components of the UPP were primarily present in the nucleus, with the exception of Ubc4/5, which was associated with the nuclear membrane. conclusions The results show that during lens fiber differentiation and maturation, components of the UPP are redistributed at subcellular levels. Subcellular localization of an enzyme indicates where the reaction takes place. The primary nuclear localization of the UPP components in the differentiating fibers supports the hypothesis that the UPP may play a role in elimination of nuclei and other organelles during differentiation and maturation of lens fibers.

The UBA2 Domain Functions as an Intrinsic Stabilization Signal that Protects Rad23 from Proteasomal Degradation

The proteasome-interacting protein Rad23 is a long-lived protein. Interaction between Rad23 and the proteasome is required for Rad23's functions in nucleotide excision repair and ubiquitin-dependent degradation. Here, we show that the ubiquitin-associated (UBA)-2 domain of yeast Rad23 is a cis-acting, transferable stabilization signal that protects Rad23 from proteasomal degradation. Disruption of the UBA2 domain converts Rad23 into a short-lived protein that is targeted for degradation through its N-terminal ubiquitin-like domain. UBA2-dependent stabilization is required for Rad23 function because a yeast strain expressing a mutant Rad23 that lacks a functional UBA2 domain shows increased sensitivity to UV light and, in the absence of Rpn10, severe growth defects. The C-terminal UBA domains of Dsk2, Ddi1, Ede1, and the human Rad23 homolog hHR23A have similar protective activities. Thus, the UBA2 domain of Rad23 is an evolutionarily conserved stabilization signal that allows Rad23 to interact with the proteasome without facing destruction.

Overexpression of Proteasome β5 Assembled Subunit Increases the Amount of Proteasome and Confers Ameliorated Response to Oxidative Stress and Higher Survival Rates

The proteasome is the major cellular proteolytic machinery responsible for the degradation of both normal and damaged proteins. Proteasomes play a fundamental role in retaining cellular homeostasis. Alterations of proteasome function have been recorded in various biological phenomena including aging. We have recently shown that the decrease in proteasome activity in senescent human fibroblasts relates to the down-regulation of β-type subunits. In this study we have followed our preliminary observation by developing and further characterizing a number of different human cell lines overexpressing the β5 subunit. Stable overexpression of the β5 subunit in WI38/T and HL60 cells resulted in elevated levels of other β-type subunits and increased levels of all three proteasome activities. Immunoprecipitation experiments have shown increased levels of assembled proteasomes in stable clones. Analysis by gel filtration has revealed that the recorded higher level of proteasome assembly is directly linked to the efficient integration of “free” (not integrated) α-type subunits identified to accumulate in vector-transfected cells. In support we have also found low proteasome maturation protein levels in β5 transfectants, thus revealing an increased rate/level of proteasome assembly in these cells as opposed to vector-transfected cells. Functional studies have shown that β5-overexpressing cell lines confer enhanced survival following treatment with various oxidants. Moreover, we demonstrate that this increased rate of survival is due to higher degradation rates following oxidative stress. Finally, because oxidation is considered to be a major factor that contributes to aging and senescence, we have overexpressed the β5 subunit in primary IMR90 human fibroblasts and observed a delay of senescence by 4-5 population doublings. In summary, these data demonstrate the phenotypic effects following genetic up-regulation of the proteasome and provide insights toward a better understanding of proteasome regulation.

A voyage to the inner space of cells.

The title I have chosen for my personal recollections describes, in a nutshell, the direction my scientific endeavors from the time of my Ph.D. thesis, which I began in 1970, to the present day. Over the years, I have changed fields a number of times. There were periods when I was preoccupied with the development of methods; at other times, the focus was on biological problems. Science can be advanced by new hypotheses about how things work, which can be tested and proven right or wrong, and by new methods which enable us to tackle questions that we were unable to address with the existing methods. Or, as Richard Feynman put it, “Science means, sometimes, a special method of finding things out. Sometimes it means the body of knowledge arising from the things found out. It may also mean the new things you can do when you have found something out, or the actual doing of new things. This last field is usually called technology. . . .” (R.P. Feynman in the John Danz Lectures, 1963 [Feynman 1998]).

Recombinant ATPases of the yeast 26S proteasome activate protein degradation by the 20S proteasome

The 26S proteasome contains a proteolytic core, 20S proteasome, and its regulatory particle, 19S complex. That regulatory particle contains six ATPases that are involved in unfolding and translocation of substrates to the 20S proteasome's catalytic chamber. We expressed ATPase-encoding genes of the regulatory particle of Saccharomyces cerevisiae and found that some recombinant ATPases can self-assemble into a high-molecular-weight protein complex in Escherichia coli. Purification of the Rpt1Rpt2 hetero-complex and the Rpt4 homo-complex for functional characterization demonstrated their contribution to energy-dependent protein degradation. Our finding, production of a functional subunit of the 19S regulatory particle in bacteria, is a simpler and technically advanced system to functionally characterize individual subunits.

Linkage between the proteasome pathway and neurodegenerative diseases and aging.

During aging, the production of free radicals increases. This can result in damage to protein, the accumulation of which is characteristic of the aging process. This questions the efficacy of proteolytic systems. Among these systems, the proteasome and the adenosine triphosphate-ubiquitin-dependent pathway have been shown to play an important role in the elimination of abnormal proteins. There are two major steps in the ubiquitin-proteasome pathway: the conjugation of a polyubiquitin degradation signal to the substrate and the subsequent degradation of the tagged protein by the 26S proteasome. The 26S proteasome is build-up from the 20S proteasome, which is a cylinder-shaped multimeric complex, and two additional 19S complexes. The 20S proteasome can also bind to 11S regulator and is then implicated in antigen presentation. These regulators confer a high adaptability on proteasome. With advancing age, predisposition to neurodegenerative diseases increases. These diseases are also characterized by protein aggregation. Several findings such as the presence of ubiquinated proteins, usually broken down by proteasomes, and genetic anomalies involving the ubiquitin-proteasome system (parkin, UCH-L1) suggest a link between the ubiquitin-proteasome pathway and the genesis of these diseases.

Electron cryomicroscopy structure of N-ethyl maleimide sensitive factor at 11 A resolution.

N-ethyl maleimide sensitive factor (NSF) belongs to the AAA family of ATPases and is involved in a number of cellular functions, including vesicle fusion and trafficking of membrane proteins. We present the three-dimensional structure of the hydrolysis mutant E329Q of NSF complexed with an ATP-ADP mixture at 11 A resolution by electron cryomicroscopy and single-particle averaging of NSF.alpha-SNAP.SNARE complexes. The NSF domains D1 and D2 form hexameric rings that are arranged in a double-layered barrel. Our structure is more consistent with an antiparallel orientation of the two rings rather than a parallel one. The crystal structure of the D2 domain of NSF was docked into the EM density map and shows good agreement, including details at the secondary structural level. Six protrusions corresponding to the N domain of NSF (NSF-N) emerge from the sides of the D1 domain ring. The density corresponding to alpha-SNAP and SNAREs is located on the 6-fold axis of the structure, near the NSF-N domains. The density of the N domain is weak, suggesting conformational variability in this part of NSF.

Use of RNA interference and complementation to study the function of the Drosophila and human 26S proteasome subunit S13.

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.

Ras promotes p21(Waf1/Cip1) protein stability via a cyclin D1-imposed block in proteasome-mediated degradation.

Ras promotes the accumulation of the cyclin-dependent kinase inhibitor p21(Waf1/Cip1) (p21). Previous studies reported that acute Raf/MEK/ERK activation elevates p21 protein levels by increased transcription. However, we have found that p21 induction in Ras-transformed murine fibroblasts occurs principally by a post-translational mechanism. Chronic activation of the Raf/MEK/ERK pathway blocked proteasome-mediated p21 degradation, resulting in accumulation of p21 protein with an elevated half-life. The stabilization of p21 by Ras was accompanied by high levels of p21-associated cyclin D1 and, similarly to Ras, cyclin D1 was sufficient to inhibit the proteasome-mediated p21 degradation. Knock-down of cyclin D1 by RNA interference confirmed that Ras-induced p21 stabilization was dependent upon cyclin D1 expression. We show that p21 directly binds to the C8alpha subunit of the 20S proteasome complex and that by competing for binding, cyclin D1 inhibits p21 degradation by purified 20S complexes in vitro. Therefore, we propose that Ras stabilizes p21 by promoting the formation of p21-cyclin D1 complexes that prevent p21 association with, and subsequent degradation by, the 20S proteasome.

Changing the DNA landscape: putting a SPN on chromatin.

In eukaryotic cells, transcription and replication each occur on DNA templates that are incorporated into nucleosomes. Formation of chromatin generally limits accessibility of specific DNA sequences and inhibits progression of polymerases as they copy information from the DNA. The processes that select sites for initiating either transcription or replication are therefore strongly influenced by factors that modulate the properties of chromatin proteins. Further, in order to elongate their products, both DNA and RNA polymerases must be able to overcome the inhibition presented by chromatin (Lipford and Bell 2001; Workman and Kingston 1998). One way to adjust the properties of chromatin proteins is to covalently modify them by adding or removing chemical moieties. Both histone and non-histone chromatin proteins are altered by acetylation, methylation, and other changes, and the 'nucleosome modifying' complexes that perform these reactions are important components of pathways of transcriptional regulation (Cote 2002; Orphanides and Reinberg 2000; Roth et al. 2001; Strahl and Allis 2000; Workman and Kingston 1998). Another way to alter the effects of nucleosomes is to change the position of the histone octamers relative to specific DNA sequences (Orphanides and Reinberg 2000; Verrijzer 2002; Wang 2002; Workman and Kingston 1998). Since the ability of a sequence to be bound by specific proteins can vary significantly whether the sequence is in the linkers between nucleosomes or at various positions within a nucleosome, 'nucleosome remodeling' complexes that rearrange nucleosome positioning are also important regulators of transcription. Since the DNA replication machinery has to encounter many of the same challenges posed by chromatin, it seems likely that modifying and remodeling complexes also act during duplication of the genome, but most of the current information on these factors relates to regulation of transcription. This chapter describes the factor known variously as FACT in humans, where it promotes elongation of RNA polymerase II on nucleosomal templates in vitro (Orphanides et al. 1998, 1999), DUF in frogs, where it is needed for DNA replication in oocyte extracts (Okuhara et al. 1999), and CP or SPN in yeast, where it is linked in vivo to both transcription and replication (Brewster et al. 2001; Formosa et al. 2001). Like the nucleosome modifying and remodeling complexes, it is broadly conserved among eukaryotes, affects a wide range of processes that utilize chromatin, and directly alters the properties of nucleosomes. However, it does not have nucleosome modifying or standard ATP-dependent remodeling activity, and therefore represents a third class of chromatin modulating factors. It is also presently unique in the extensive connections it displays with both transcription and replication: FACT/DUF/CP/SPN appears to modify nucleosomes in a way that is directly important for the efficient functioning of both RNA polymerases and DNA polymerases. While less is known about the mechanisms it uses to promote its functions than for other factors that affect chromatin, it is clearly an essential part of the complex mixture of activities that modulate access to DNA within chromatin. Physical and genetic interactions suggest that FACT/DUF/CP/SPN affects multiple pathways within replication and transcription as a member of several distinct complexes. Some of the interactions are easy to assimilate into models for replication or transcription, such as direct binding to DNA polymerase alpha (Wittmeyer and Formosa 1997; Wittmeyer et al. 1999), association with nucleosome modifying complexes (John et al. 2000), and interaction with factors that participate in elongation of RNA Polymerase II (Gavin et al. 2002; Squazzo et al. 2002). Others are more surprising such as an association with the 19S complex that regulates the function of the 20S proteasome (Ferdous et al. 2001; Xu et al. 1995), and the indication that FACT/DUF/CP/SPN can act as a specificity factor for casein kinase II (Keller et al. 2001). This chapter reviews the varied approaches that have each revealed different aspects of the function of FACT/DUF/CP/SPN, and presents a picture of a factor that can both alter nucleosomes and orchestrate the assembly or activity of a broad range of complexes that act upon chromatin.