Proteasome dynamics between proliferation and quiescence stages of Saccharomyces cerevisiae

The ubiquitin-proteasome system (UPS) plays a critical role in cellular protein homeostasis and is required for the turnover of short-lived and unwanted proteins, which are targeted by poly-ubiquitination for degradation. Proteasome is the key protease of UPS and consists of multiple subunits, which are organized into a catalytic core particle (CP) and a regulatory particle (RP). In Saccharomyces cerevisiae, proteasome holo-enzymes are engaged in degrading poly-ubiquitinated substrates and are mostly localized in the nucleus during cell proliferation. While in quiescence, the RP and CP are sequestered into motile and reversible storage granules in the cytoplasm, called proteasome storage granules (PSGs). The reversible nature of PSGs allows the proteasomes to be transported back into the nucleus upon exit from quiescence. Nuclear import of RP and CP through nuclear pores occurs via the canonical pathway that includes the importin-αβ heterodimer and takes advantage of the Ran-GTP gradient across the nuclear membrane. Dependent on the growth stage, either inactive precursor complexes or mature holo-enzymes are imported into the nucleus. The present review discusses the dynamics of proteasomes including their assembly, nucleo-cytoplasmic transport during proliferation and the sequestration of proteasomes into PSGs during quiescence.

     

20S proteasome biogenesis

26S proteasomes are multi-subunit protease complexes responsible for the turnover of short-lived proteins. Proteasomal degradation starts with the autocatalytic maturation of the 20S core particle. Here, we summarize different models of proteasome assembly. 20S proteasomes are assembled as precursor complexes containing alpha and unprocessed beta subunits. The propeptides of the beta subunits are thought to prevent premature conversion of the precursor complexes into matured particles and are needed for efficient beta subunit incorporation. The complex biogenesis is tightly regulated which requires additional components such as the maturation factor Ump1/POMP, an ubiquitous protein in eukaryotic cells. Ump1/POMP is associated with precursor intermediates and degraded upon final maturation. Mammalian proteasomes are localized all over the cell, while yeast proteasomes mainly localize to the nuclear envelope/endoplasmic reticulum (ER) membrane network. The major localization of yeast proteasomes may point to the subcellular place of proteasome biogenesis.     

Structural defects in the regulatory particle-core particle interface of the proteasome induce a novel proteasome stress response

Proteasomes consist of a 19-subunit regulatory particle (RP) and 28-subunit core particle (CP), an α(7)β(7)β(7)α(7) structure. The RP recognizes substrates and translocates them into the CP for degradation. At the RP-CP interface, a heterohexameric Rpt ring joins to a heteroheptameric CP α ring. Rpt C termini insert individually into the α ring pockets to form a salt bridge with a pocket lysine residue. We report that substitutions of α pocket lysine residues produce an unexpected block to CP assembly, arising from a late stage defect in β ring assembly. Substitutions α5(K66A) and α6(K62A) resulted in abundant incorporation of immature CP β subunits, associated with a complete β ring, into proteasome holoenzymes. Incorporation of immature CP into the proteasome depended on a proteasome-associated protein, Ecm29. Using ump1 mutants, we identified Ecm29 as a potent negative regulator of RP assembly and confirmed our previous findings that proper RP assembly requires the CP. Ecm29 was enriched on proteasomes of pocket lysine mutants, as well as those of rpt4-Δ1 and rpt6-Δ1 mutants, in which the C-terminal residue, thought to contact the pocket lysine, is deleted. In both rpt6-Δ1 and α6(K62A) proteasomes, Ecm29 suppressed opening of the CP substrate translocation channel, which is gated through interactions between Rpt C termini and the α pockets. The ubiquitin ligase Hul5 was recruited to these proteasomes together with Ecm29. Proteasome remodeling through the addition of Ecm29 and Hul5 suggests a new layer of the proteasome stress response and may be a common response to structurally aberrant proteasomes or deficient proteasome function.     

A Highlights from MBoC Selection: Proteasome Nuclear Import Mediated by Arc3 Can Influence Efficient DNA Damage Repair and Mitosis in Schizosaccharomyces Pombe

Proteasomes must remove regulatory molecules and abnormal proteins throughout the cell, but how proteasomes can do so efficiently remains unclear. We have isolated a subunit of the Arp2/3 complex, Arc3, which binds proteasomes. When overexpressed, Arc3 rescues phenotypes associated with proteasome deficiencies; when its expression is repressed, proteasome deficiencies intensify. Arp2/3 is best known for regulating membrane dynamics and vesicular transport; thus, we performed photobleaching experiments and showed that proteasomes are readily imported into the nucleus but exit the nucleus slowly. Proteasome nuclear import is reduced when Arc3 is inactivated, leading to hypersensitivity to DNA damage and inefficient cyclin-B degradation, two events occurring in the nucleus. These data suggest that proteasomes display Arc3-dependent mobility in the cell, and mobile proteasomes can efficiently access substrates throughout the cell, allowing them to effectively regulate cell-compartment–specific activities.     

The HEAT repeat protein Blm10 regulates the yeast proteasome by capping the core particle

Proteasome activity is fine-tuned by associating the proteolytic core particle (CP) with stimulatory and inhibitory complexes. Although several mammalian regulatory complexes are known, knowledge of yeast proteasome regulators is limited to the 19-subunit regulatory particle (RP), which confers ubiquitin-dependence on proteasomes. Here we describe an alternative proteasome activator from Saccharomyces cerevisiae, Blm10. Synthetic interactions between blm10Delta and other mutations that impair proteasome function show that Blm10 functions together with proteasomes in vivo. This large, internally repetitive protein is found predominantly within hybrid Blm10-CP-RP complexes, representing a distinct pool of mature proteasomes. EM studies show that Blm10 has a highly elongated, curved structure. The near-circular profile of Blm10 adapts it to the end of the CP cylinder, where it is properly positioned to activate the CP by opening the axial channel into its proteolytic chamber.     

Intracellular localization of the proteasome in response to stress conditions

The ubiquitin–proteasome system fulfills an essential role in regulating protein homeostasis by spatially and temporally controlling proteolysis in an ATP- and ubiquitin-dependent manner. However, the localization of proteasomes is highly variable under diverse cellular conditions. In yeast, newly synthesized proteasomes are primarily localized to the nucleus during cell proliferation. Yeast proteasomes are transported into the nucleus through the nuclear pore either as immature subcomplexes or as mature enzymes via adapter proteins Sts1 and Blm10, while in mammalian cells, postmitotic uptake of proteasomes into the nucleus is mediated by AKIRIN2, an adapter protein essentially required for nuclear protein degradation. Stressful growth conditions and the reversible halt of proliferation, that is quiescence, are associated with a decline in ATP and the reorganization of proteasome localization. Cellular stress leads to proteasome accumulation in membraneless granules either in the nucleus or in the cytoplasm. In quiescence, yeast proteasomes are sequestered in an ubiquitin-dependent manner into motile and reversible proteasome storage granules in the cytoplasm. In cancer cells, upon amino acid deprivation, heat shock, osmotic stress, oxidative stress, or the inhibition of either proteasome activity or nuclear export, reversible proteasome foci containing polyubiquitinated substrates are formed by liquid–liquid phase separation in the nucleus. In this review, we summarize recent literature revealing new links between nuclear transport, ubiquitin signaling, and the intracellular organization of proteasomes during cellular stress conditions.     

Proteasomes: protein and gene structures.

Proteasomes are ring- or cylinder-shaped particles that have a sedimentation coefficient of 20S and are composed of a characteristic set of small polypeptides. These particles have a latent multicatalytic proteinase activity. Recently, proteasomes were found to combine reversibly with multiple protein components to form 26S proteolytic complexes that catalyze ATP-dependent, selective breakdown of proteins ligated with ubiquitin. This suggests that the 26S complexes are a new type of ATP-requiring protease in eukaryotic cells. We have studied the structures of various eukaryotic proteasomes at the molecular level by physicochemical and recombinant DNA techniques and have proposed that the gross structures of proteasomes, such as their size and shape, have been highly conserved during evolution. Proteasome subunits appear to be encoded by a family of homologous genes named the "proteasome gene family," which may have evolved from a common ancestral gene. Evidence obtained by genetic analyses in yeast and studies on the levels of proteasome expression in various eukaryotic cells indicates that proteasomes have essential roles in the cell. In this review, we summarize available information on the protein and gene structures of proteasomes and discuss the biological functions of proteasomes.     

Catalytic components of proteasomes and the regulation of proteinase activity

The proteasome (multicatalytic proteinase complex) is a large multimeric complex which is found in the nucleus and cytoplasm of eukaryotic cells. It plays a major role in both ubiquitin-dependent and ubiquitin-independent nonlysosomal pathways of protein degradation. Proteasome subunits are encoded by members of the same gene family and can be divided into two groups based on their similarity to the and subunits of the simpler proteasome isolated fromThermoplasma acidophilum. Proteasomes have a cylindrical structure composed of four rings of seven subunits. The 26S form of the proteasome, which is responsible for ubiquitin-dependent proteolysis, contains additional regulatory complexes. Eukaryotic proteasomes have multiple catalytic activities which are catalysed at distinct sites. Since proteasomes are unrelated to other known proteases, there are no clues as to which are the catalytic components from sequence alignments. It has been assumed from studies with yeast mutants that -type subunits play a catalytic role. Using a radiolabelled peptidyl chloromethane inhibitor of rat liver proteasomes we have directly identified RC7 as a catalytic component. Interestingly, mutants in Prel, the yeast homologue of RC7, have already been reported to have defective chymotrypsin-like activity. These results taken together confirm a direct catalytic role for these -type subunits. Proteasome activities are sensitive to conformational changes and there are several ways in which proteasome function may be modulatedin vivo. Our recent studies have shown that in animal cells at least two proteasome subunits can undergo phosphorylation, the level of which is likely to be important for determining proteasome localization, activity or ability to form larger complexes. In addition, we have isolated two isoforms of the 26S proteinase.     

Dissection of the assembly pathway of the proteasome lid in Saccharomyces cerevisiae

The 26S proteasome is a highly conserved multisubunit protease that degrades ubiquitinated proteins in eukaryotic cells. It comprises a 20S core particle and two 19S regulatory particles that are further divided into the lid and base complexes. The lid is a nine subunits complex that is structurally related to the COP9 signalosome and the eukaryotic initiation factor 3. Although the assembly pathway of the 20S and the base are well described, that of the lid is still unclear. In this study, we dissected the lid assembly using yeast lid mutant cells, rpn7-3, Δrpn9, and rpn12-1. Using mass spectrometry, we identified a number of lid subassemblies, such as Rpn3-Rpn7 pair and a lid-like complex lacking Rpn12, in the mutants. Our analysis suggests that the assembly of the lid is a highly ordered and multi-step process; first, Rpn5, 6, 8, 9, and 11 are assembled to form a core module, then a second module, consisting of Rpn3, 7, and Sem1, is attached, followed by the incorporation of Rpn12 to form the lid complex.     

Regulation of proteasome complexes by gamma-interferon and phosphorylation

Proteasomes play a major role in non-lysosomal proteolysis and also in the processing of proteins for presentation by the MHC class I pathway. In animal cells they exist in several distinct molecular forms which contribute to the different functions. 26S proteasomes contain the core 20S proteasome together with two 19S regulatory complexes. Alternatively, PA28 complexes can bind to the ends of the 20S proteasome to form PA28-proteasome complexes and PA28-proteasome-19S hybrid complexes have also been described. Immunoproteasome subunits occur in 26S proteasomes as well as in PA28-proteasome complexes. We have found differences in the subcellular distribution of the different forms of proteasomes. The gamma-interferon inducible PA28 alpha and beta subunits are predominantly located in the cytoplasm, while 19S regulatory complexes (present at significant levels only in 26S complexes) are present in the nucleus as well as in the cytoplasm. Immunoproteasomes are greatly enriched at the endoplasmic reticulum (ER) where they may facilitate the generation of peptides for transport into the lumen of the ER. We have also investigated the effects of gamma-interferon on the levels and subcellular distribution of inducible subunits and regulator subunits. In each case gamma-interferon was found to increase the level but not to alter the distribution. Several subunits of proteasomes are phosphorylated including alpha subunits C8 (alpha7) and C9 (alpha3), and ATPase subunit S4 (rpt2). Our studies have shown that gamma-interferon treatment decreases the level of phosphorylation of proteasomes. We have investigated the role of phosphorylation of C8 by casein kinase II by site directed mutagenesis. The results demonstrate that phosphorylation at either one of the two sites is essential for the association of 19S regulatory complexes and that the ability to undergo phosphorylation at both sites gives the most efficient incorporation of C8 into the 26S proteasome.     

The bipartite nuclear localization sequence of Rpn2 is required for nuclear import of proteasomal base complexes via karyopherin alphabeta and proteasome functions

26 S proteasomes fulfill final steps in the ubiquitin-dependent degradation pathway by recognizing and hydrolyzing ubiquitylated proteins. As the 26 S proteasome mainly localizes to the nucleus in yeast, we addressed the question how this 2-MDa multisubunit complex is imported into the nucleus. 26 S proteasomes consist of a 20 S proteolytically active core and 19 S regulatory particles, the latter composed of two subcomplexes, namely the base and lid complexes. We have shown that 20 S core particles are translocated into the nucleus as inactive precursor complexes via the classic karyopherin alphabeta import pathway. Here, we provide evidence that nuclear import of base and lid complexes also depends on karyopherin alphabeta. Potential classic nuclear localization sequences (NLSs) of base subunits were analyzed. Rpn2 and Rpt2, a non-ATPase subunit and an ATPase subunit of the base complex, harbor functional NLSs. The Rpt2 NLS deletion yielded wild type localization. However, the deletion of the Rpn2 NLS resulted in improper nuclear proteasome localization and impaired proteasome function. Our data support the model by which nuclear 26 S proteasomes are assembled from subcomplexes imported by karyopherin alphabeta.     

The proteasome: a macromolecular assembly designed for controlled proteolysis

In eukaryotic cells, the vast majority of proteins in the cytosol and nucleus are degraded via the proteasome-ubiquitin pathway. The 26S proteasome is a huge protein degradation machine of 2.5 MDa, built of approximately 35 different subunits. It contains a proteolytic core complex, the 20S proteasome and one or two 19S regulatory complexes which associate with the termini of the barrel-shaped 20S core. The 19S regulatory complex serves to recognize ubiquitylated target proteins and is implicated to have a role in their unfolding and translocation into the interior of the 20S complex where they are degraded into oligopeptides. While much progress has been made in recent years in elucidating the structure, assembly and enzymatic mechanism of the 20S complex, our knowledge of the functional organization of the 19S regulator is rather limited. Most of its subunits have been identified, but specific functions can be assigned to only a few of them.     

Treatment of COS-7 cells with proteasome inhibitors or gamma-interferon reduces the increase in caspase 3 activity associated with staurosporine-induced apoptosis.

Proteasomes play a major role in intracellular protein degradation and have been implicated in apoptosis. In this study we have investigated proteasome activity and the effects of inhibition of proteasomes or modulation of proteasome complexes on staurosporine-induced apoptosis in COS-7 cells. Staurosporine treatment of COS-7 cells had little direct effect on proteasome activity and did not cause dissociation of 26S proteasomes. There was also no major redistribution of proteasomes accompanying apoptosis in COS-7 cells. However, when the cells were pretreated with proteasome inhibitors, both the caspase 3 activity of the cells and the percentage of apoptotic cells measured by the TUNEL assay were reduced compared to staurosporine-treated cells, which had no inhibitor added. Proteasome inhibitors were also found to reduce the activation of caspase 3 in living cells which was assayed using a FRET-based method. However, proteasome inhibitors did not prevent some of the morphological changes associated with staurosporine-induced apoptosis. Pretreatment of cells with gamma-interferon, which increases immunoproteasomes and PA28 complexes and reduces 26S proteasome levels, had an antiapoptotic effect. These results are consistent with a role for 26S proteasomes in regulating the activation of caspase 3 through the degradation of key regulatory proteins.     

Proteasomes of the yeastS. cerevisiae: genes,structure and functions

Proteasomes are large multicatalytic protease complexes which fulfil central functions in major intracellular proteolytic pathways of the eukaryotic cell. 20S proteasomes are 700 kDa cylindrically shaped particles, found in the cytoplasm and the nucleus of all eukaryotes. They are composed of a pool of 14 different subunits (MW 22–25 kDa) arranged in a stack of 4 rings with 7-fold symmetry. In the yeastSaccharomyces cerevisiae a complete set of 14 genes coding for 20S proteasome subunits have been cloned and sequenced. 26S proteasomes are even larger proteinase complexes (about 1700 kDa) which degrade ubiquitinylated proteins in an ATP-dependent fashionin vitro. The 26S proteasome is build up from the 20S proteasome as core particle and two additional 19S complexes at both ends of the 20S cylinder. Recently existence of a 26S proteasome in yeast has been demonstrated. Several 26S proteasome specific genes have been cloned and sequenced. They share similarity with a novel defined family of ATPases. 20S and 26S proteasomes are essential for functioning of the eukaryotic cell. Chromosomal deletion of 20S and 26S proteasomal genes in the yeastS. cerevisiae caused lethality of the cell. Thein vivo functions of proteasomes in major proteolytic pathways have been demonstrated by the use of 20S and 26S proteasomal mutants. Proteasomes are needed for stress dependent and ubiquitin mediated proteolysis. They are involved in the degradation of short-lived and regulatory proteins. Proteasomes are important for cell differentiation and adaptation to environmental changes. Proteasomes have also been shown to function in the control of the cell cycle.     

The proteasome-dependent proteolytic system

The 20S proteasome is an intriguingly large complex that acts as a proteolytic catalytic machine. Accumulating evidence indicates the existence of multiple factors capable of regulating the proteasome function. They are classified into two different categories, one type of regulator is PA700 or PA28 that is reversibly associated with the 20S proteasome to form enzymatically active proteasomes and the other type including a 300-kDa modulator and PI31 indirectly influences proteasome activity perhaps by promoting or suppressing the assembly of the 20S proteasome with PA700 or PA28. Thus, there have been documented two types of proteasomes composed of a core catalytic proteasome and a pair of symmetrically disposed PA700 or PA28 regulatory particle. Moreover, the recently-identified proteasome containing both PA28 and PA700 appears to play a significant role in the ATP-dependent proteolytic pathway in cells, as can the 26S proteasome which is known as a eukaryotic ATP-dependent protease.     

Characterization of the 20S proteasome of the lepidopteran,Spodoptera frugiperda

Multiple complexes of 20S proteasomes with accessory factors play an essential role in proteolysis in eukaryotic cells. In this report, several forms of 20S proteasomes from extracts of Spodoptera frugiperda (Sf9) cells were separated using electrophoresis in a native polyacrylamide gel and examined for proteolytic activity in the gel and by Western blotting. Distinct proteasome bands isolated from the gel were subjected to liquid chromatography-tandem mass spectrometry and identified as free core particles (CP) and complexes of CP with one or two dimers of assembly chaperones PAC1-PAC2 and activators PA28γ or PA200. In contrast to the activators PA28γ and PA200 that regulate the access of protein substrates to the internal proteolytic chamber of CP in an ATP-independent manner, the 19S regulatory particle (RP) in 26S proteasomes performs stepwise substrate unfolding and opens the chamber gate in an ATP-dependent manner. Electron microscopic analysis suggested that spontaneous dissociation of RP in isolated 26S proteasomes leaves CPs with different gate sizes related presumably to different stages in the gate opening. The primary structure of 20S proteasome subunits in Sf9 cells was determined by a search of databases and by sequencing. The protein sequences were confirmed by mass spectrometry and verified by 2D gel electrophoresis. The relative rates of sequence divergence in the evolution of 20S proteasome subunits, the assembly chaperones and activators were determined by using bioinformatics. The data confirmed the conservation of regular CP subunits and PA28γ, a more accelerated evolution of PAC2 and PA200, and especially high divergence rates of PAC1.     

Molecular characterization and intracellular distribution of the alpha 5 subunit of Trypanosoma cruzi 20S proteasome

Three different monoclonal antibodies were produced against Trypanosona cruzi proteasomes. These antibodies were shown to react with a single 27-kDa band on immunoblots of purified proteasomes. Using a 7E5 monoclonal antibody (IgG1) that recognized the α5 subunit of protozoan protease we have studied the intracellular distribution of the T. cruzi 20S proteasome. Contrary to all cell types described to date, T. cruzi 20S proteasome was found not only in the cytoplasm and nucleus but also in the kinetoplast. As revealed by confocal microscopy, the reactivity of monoclonal antibody 7E5 was highly specific for protozoan proteasome because the antibody recognized only the proteasomes from parasites and not those from the mammalian host in T. cruzi infected cells. These findings were confirmed by immunoblots or immunoprecipitations, followed by chymotrypsin-like activity detection in kinetoplasts isolated by differential centrifugation and sucrose density gradients. Proteasome 20S was present in all T. cruzi stages and only slight differences in terms of relative abundance were found. The potential role of the proteasome in kinetoplast remodeling remains to be determined.     

Multiple chaperone-assisted formation of mammalian 20S proteasomes

Protein degradation is essential for maintenance of cellular homeostasis. The majority of proteins are selectively degraded in eukaryotic cells by the ubiquitin-proteasome system. The 26S proteasome selects target proteins that are covalently modified with polyubiquitin chains. The 26S proteasome is a multisubunit protease responsible for regulated proteolysis in eukaryotic cells. The catalytic activities are carried out by the core 20S proteasome. The eukaryotic 20S proteasome is composed of 28 subunits arranged in a cylindrical particle as four heteroheptameric rings, alpha1-7beta1-7beta1-7alpha1-7. Recent studies have revealed the mechanism responsible for the assembly of such a complex structure. This article recounts the observations that disclosed the biogenesis of 20S proteasomes and discusses the difference in the mechanism of assembly between archael, yeast, and mammalian 20S proteasomes.     

Intermediates in the formation of mouse 20S proteasomes: implications for the assembly of precursor beta subunits.

The assembly of individual proteasome subunits into catalytically active mammalian 20S proteasomes is not well understood. Using subunit-specific antibodies, we characterized both precursor and mature proteasome complexes. Antibodies to PSMA4 (C9) immunoprecipitated complexes composed of alpha, precursor beta and processed beta subunits. However, antibodies to PSMA3 (C8) and PSMB9 (LMP2) immunoprecipitated complexes made up of alpha and precursor beta but no processed beta subunits. These complexes possess short half-lives, are enzymatically inactive and their molecular weight is approximately 300 kDa. Radioactivity chases from these complexes into mature, long-lived approximately 700 kDa proteasomes. Therefore, these structures represent precursor proteasomes and are probably made up of two rings: one containing alpha subunits and the other, precursor beta subunits. The assembly of precursor proteasomes occurs in at least two stages, with precursor beta subunits PSMB2 (C7-I), PSMB3 (C10-II), PSMB7 (Z), PSMB9 (LMP2) and PSMB10 (LMP10) being incorporated before others [PSMB1 (C5), PSMB6 (delta), and PSMB8 (LMP7)]. Proteasome maturation (processing of the beta subunits and juxtaposition of the two beta rings) is accompanied by conformational changes in the (outer) alpha rings, and may be inefficient. Finally, interferon-gamma had no significant effect on the half-lives or total amounts of precursor or mature proteasomes.     

Regulation of proteasome structure and function

Proteasomes are cylindrical particles made up of a stack of four heptameric rings. In animal cells the outer rings are made up of 7 different types of alpha subunits and the inner rings are composed of 7 out of 10 possible different beta subunits. Regulatory complexes can bind to the ends of the cylinder. We have investigated aspects of the assembly, activity and subunit composition of core proteasome particles and 26S proteasomes, the localization of proteasome subpopulations, and the possible role of phosphorylation in determining proteasome localization, activities and association with regulatory components.