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.     

Affinity labeling of the proteasome by a belactosin A derived inhibitor

Belactosin A is a potent proteasome inhibitor isolated from Streptomyces metabolites. Here we show that a hydrophobic belactosin A derivative, dansyl-KF33955, can covalently, and specifically, affinity label the catalytic subunits of the 26S proteasome, which consists of the 20S protein degrading core particle and the 19S regulatory particles. The labeling of catalytic subunits proceeds faster in intact proteasomes in vivo than in isolated 20S core particles. These data suggest that the 19S regulatory particle may facilitate entry of the inhibitor into the 20S core particle. This cell-permeable chemical probe is an excellent tool with which to study the interactions of this proteasome inhibitor with proteasomes in intact cells.     

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.     

Comparative proteome analysis of changes in the 26S proteasome during oocyte maturation in goldfish

Proteasomes are large, multi-subunit particles that act as the proteolytic machinery for most of the regulated intracellular protein degradation in eukaryotic cells. An alteration of proteasome function may be important for the regulation of the meiotic cell cycle. To study the change at the subunit level of the 26S proteasome during meiotic maturation, we purified 26S proteasomes from immature and mature oocytes of goldfish. Two-dimensional polyacrylamide gel electrophoresis was used to separate proteins. For differential analysis, whole spots of the 26S proteasome from goldfish oocytes were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and database analysis. Four spots that were different (only detected in mature oocyte 265 proteasomes and not in immature ones) and four protein spots that were up- or down-regulated were identified unambiguously. The mature-specific spots were not 26S proteasome components but rather their interacting proteins, and were identified as chaperonin-containing TCP-1 subunits and myosin light chain. Minor spots of three subunits of the 20S core particle and one of the 19S regulatory particle showed meiotic cell cycle-dependent changes. These results demonstrate that modifications of proteasomal subunits and cell cycle phase-dependent interactions of proteins with proteasomes occur during oocyte maturation in goldfish.     

20 S proteasomes are imported as precursor complexes into the nucleus of yeast

The mechanism by which yeast 20 S proteasomes are imported into the nucleus is still unresolved. Here, we provide the first evidence that 20 S proteasomes are imported as precursor complexes into the nucleus. By using the srp1-49 mutant which is deficient in nuclear import of cargos with classical nuclear localization sequences (cNLS), we show that proteasome precursor complexes associate with importin/karyopherin alphabeta, the cNLS receptor, and that they accumulate inside the cytoplasm. Reconstitution assays revealed that only precursor complexes are targeted to the nuclear envelope (NE) by karyopherin alphabeta. In support, the green fluorescent protein (GFP)-labelled maturation factor Ump1, marking precursor complexes, mainly localizes to the nucleus and around the NE. Our data suggest that nuclear 20 S proteasomes are finally matured inside the nucleus.     

The assembly pathway of the 19S regulatory particle of the yeast 26S proteasome

The 26S proteasome consists of the 20S proteasome (core particle) and the 19S regulatory particle made of the base and lid substructures, and it is mainly localized in the nucleus in yeast. To examine how and where this huge enzyme complex is assembled, we performed biochemical and microscopic characterization of proteasomes produced in two lid mutants, rpn5-1 and rpn7-3, and a base mutant DeltaN rpn2, of the yeast Saccharomyces cerevisiae. We found that, although lid formation was abolished in rpn5-1 mutant cells at the restrictive temperature, an apparently intact base was produced and localized in the nucleus. In contrast, in DeltaN rpn2 cells, a free lid was formed and localized in the nucleus even at the restrictive temperature. These results indicate that the modules of the 26S proteasome, namely, the core particle, base, and lid, can be formed and imported into the nucleus independently of each other. Based on these observations, we propose a model for the assembly process of the yeast 26S proteasome.     

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.     

Reconstitution of hybrid proteasomes from purified PA700-20 S complexes and PA28alphabeta activator: ultrastructure and peptidase activities.

The activity of the proteasome, the major non-lysosomal proteinase in eukaryotes, is stimulated by two activator complexes, PA700 and PA28. PA700-20 S-PA700 proteasome complexes, generally designated as 26 S proteasomes, degrade proteins, whereas complexes of the type PA28-20 S-PA28 degrade only peptides. We report, for the first time, the in vitro reconstitution of previously identified hybrid proteasomes (PA700-20 S-PA28) from purified PA700-20 S proteasome complexes and PA28 activator. In electron micrographs, the hybrid appears as a corkscrew-shaped particle with a PA700 and a PA28 activator each bound to a terminal alpha-disk of the 20 S core proteasome. The multiple peptidase activities of hybrid proteasomes are not different from those of PA28-20 S-PA28 or PA700-20 S-PA700 complexes.     

Effect of high hydrostatic pressures on 20S proteasome activity.

The 20S proteasome is the catalytic core of the ubiquitin proteolytic pathway, which is implicated in many cellular processes. The cylindrical structure of this complex consists of four stacked rings of seven subunits each. The central cavity is formed by two beta catalytic subunit rings in which protein substrates are progressively degraded. The 20S proteasome is isolated in a latent form which can be activated in vitro by various chemical and physical treatments. In this study, the effects of high hydrostatic pressures on 20S proteasome enzymatic activity were investigated. When proteasomes were subjected to increasing hydrostatic pressures, a progressive loss of peptidase activities was observed between 75 and 150 MPa. The inactivation also occurred when proteasomes were pressurized in the presence of synthetic peptide substrates; this may be the result of the dissociation of the 20S particle into its subunits under pressure, as was shown by PAGE. Pressurized proteasomes also lost their caseinolytic activity. In contrast, in the presence of casein, the pressure-induced inactivation and the dissociation of the 20S particles were prevented. In addition, in comparison to that observed at atmospheric pressure, their caseinolytic activity was increased under pressure. Following depressurization, the caseinolytic activity returned to basal levels but was further enhanced following an additional pressurization treatment. Thus, the structure of the 20S particle exhibits a certain degree of plasticity. This pressure-induced activation of the 20S proteasome is discussed in relation to its hollow structure, its currently accepted proteolytic mechanism and the general effect of high pressures on the biochemical reactions and structures of biopolymers.     

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.     

Affinity Purification of the Arabidopsis 26 S Proteasome Reveals a Diverse Array of Plant Proteolytic Complexes

Selective proteolysis in plants is largely mediated by the ubiquitin (Ub)/proteasome system in which substrates, marked by the covalent attachment of Ub, are degraded by the 26 S proteasome. The 26 S proteasome is composed of two subparticles, the 20 S core protease (CP) that compartmentalizes the protease active sites and the 19 S regulatory particle that recognizes and translocates appropriate substrates into the CP lumen for breakdown. Here, we describe an affinity method to rapidly purify epitope-tagged 26 S proteasomes intact from Arabidopsis thaliana. In-depth mass spectrometric analyses of preparations generated from young seedlings confirmed that the 2.5-MDa CP-regulatory particle complex is actually a heterogeneous set of particles assembled with paralogous pairs for most subunits. A number of these subunits are modified post-translationally by proteolytic processing, acetylation, and/or ubiquitylation. Several proteasome-associated proteins were also identified that likely assist in complex assembly and regulation. In addition, we detected a particle consisting of the CP capped by the single subunit PA200 activator that may be involved in Ub-independent protein breakdown. Taken together, it appears that a diverse and highly dynamic population of proteasomes is assembled in plants, which may expand the target specificity and functions of intracellular proteolysis.     

GFP-labelling of 26S proteasomes in living yeast: insight into proteasomal functions at the nuclear envelope/rough ER

26S proteasomes are multisubunit protease complexes that play the central role in the ubiquitin-dependent protein degradation pathway. The proteolytically active core is formed by the 20S proteasome. Regulatory subunits, principally the 19S cap complex, confer the specificity towards ubiquitinated substrates and an ATP-dependence on proteolysis. Green fluorescence protein (GFP)-tagged versions of either an -subunit of the 20S core or an ATPase subunit of the 19S cap complex were functionally incorporated into the protease complex, thus allowing to monitor the subcellular distribution of 26S proteasomes in living yeast. Our localization studies suggest that proteasomal proteolysis mainly occurs at the nuclear envelope (NE)/rough ER. Implications of proteasomal functions at the NE/rough ER are discussed in the context of published work on ER degradation and with regard to possible targeting mechanisms.     

Blm10 facilitates nuclear import of proteasome core particles

Short‐lived proteins are degraded by proteasome complexes, which contain a proteolytic core particle (CP) but differ in the number of regulatory particles (RPs) and activators. A recently described member of conserved proteasome activators is Blm10. Blm10 contains 32 HEAT‐like modules and is structurally related to the nuclear import receptor importin/karyopherin β. In proliferating yeast, RP‐CP assemblies are primarily nuclear and promote cell division. During quiescence, RP‐CP assemblies dissociate and CP and RP are sequestered into motile cytosolic proteasome storage granuli (PSG). Here, we show that CP sequestration into PSG depends on Blm10, whereas RP sequestration into PSG is independent of Blm10. PSG rapidly clear upon the resumption of cell proliferation and proteasomes are relocated into the nucleus. Thereby, Blm10 facilitates nuclear import of CP. Blm10‐bound CP serves as an import receptor–cargo complex, as Blm10 mediates the interaction with FG‐rich nucleoporins and is dissociated from the CP by Ran‐GTP. Thus, Blm10 represents the first CP‐dedicated nuclear import receptor in yeast.     

Tissue- and developmental stage-specific changes in the subcellular localization of the 26S proteasome in the ovary of Drosophila melanogaster

The intracellular localization of the 26S proteasome in the different ovarian cell types of Drosophila melanogaster was studied by means of immunofluorescence staining and laser scanning microscopy, with the use of antibodies specific for regulatory complex subunits or the catalytic core of the 26S proteasome. During the previtellogenic phase of oogenesis (stages 1-6), strong cytoplasmic staining was observed in the nurse cells and follicular epithelial cells, but the proteasome was not detected in the nuclei of these cell types. The subcellular distribution of the 26S proteasome was completely different in the oocyte. Besides a constant, very faint cytoplasmic staining, there was a gradual nuclear accumulation of proteasomes during the previtellogenic phase of oogenesis. A characteristic subcellular redistribution of the 26S proteasome occurred in the ovarian cells during the vitellogenic phase of oogenesis. There was a gradual decline in the concentration of the 26S proteasome in the nucleus of the oocyte, and in the stage 10 oocyte the proteasome could barely be detected in the nucleus. This was accompanied by a massive nuclear accumulation of proteasomes in the follicular epithelial cells. These results demonstrate that the subcellular distribution of the 26S proteasome in higher eukaryotes is strictly tissue- and developmental stage-specific.     

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.     

The unfolding of substrates and ubiquitin-independent protein degradation by proteasomes

26S proteasomes are composed of a 20S proteolytic core and two ATPase-containing 19S regulatory particles. To clarify the role of these ATPases in proteolysis, we studied the PAN complex, the archaeal homolog of the 19S ATPases. When ATP is present, PAN stimulates protein degradation by archaeal 20S proteasomes. PAN is a molecular chaperone that catalyzes the ATP-dependent unfolding of globular proteins. If 20S proteasomes are present, this unfoldase activity is linked to degradation. Thus PAN, and presumably the 26S ATPases, unfold substrates and facilitate their entry into the 20S particle. 26S proteasomes preferentially degrade ubiquitinated proteins. However, we found that calmodulin (CaM) and troponin C are degraded by 26S proteasomes without ubiquitination. Ca(2+)-free native CaM and in vitro 'aged' CaM are degraded faster than the Ca(2+)-bound form. Ubiquitination of CaM does not enhance its degradation. Degradation of ovalbumin normally requires ubiquitination, but can occur without ubiquitination if ovalbumin is denatured. The degradation of these proteins still requires ATP and the 19S particle. Thus, ubiquitin-independent degradation by 26S proteasomes may be more important than generally assumed.     

Sts1 plays a key role in targeting proteasomes to the nucleus

The evidence that nuclear proteins can be degraded by cytosolic proteasomes has received considerable experimental support. However, the presence of proteasome subunits in the nucleus also suggests that protein degradation could occur within this organelle. We determined that Sts1 can target proteasomes to the nucleus and facilitate the degradation of a nuclear protein. Specific sts1 mutants showed reduced nuclear proteasomes at the nonpermissive temperature. In contrast, high expression of Sts1 increased the levels of nuclear proteasomes. Sts1 targets proteasomes to the nucleus by interacting with Srp1, a nuclear import factor that binds nuclear localization signals. Deletion of the NLS in Sts1 prevented its interaction with Srp1 and caused proteasome mislocalization. In agreement with this observation, a mutation in Srp1 that weakened its interaction with Sts1 also reduced nuclear targeting of proteasomes. We reported that Sts1 could suppress growth and proteolytic defects of rad23Δ rpn10Δ. We show here that Sts1 suppresses a previously undetected proteasome localization defect in this mutant. Taken together, these findings explain the suppression of rad23Δ rpn10Δ by Sts1 and suggest that the degradation of nuclear substrates requires efficient proteasome localization.     

A novel proteasome interacting protein recruits the deubiquitinating enzyme UCH37 to 26S proteasomes

The 26S proteasome is a multisubunit protease responsible for regulated proteolysis in eukaryotic cells. It is composed of one catalytic 20S proteasome and two 19S regulatory particles attached on both ends of 20S proteasomes. Here, we describe the identification of Adrm1 as a novel proteasome interacting protein in mammalian cells. Although the overall sequence of Adrm1 has weak homology with the yeast Rpn13, the amino- and carboxyl-terminal regions exhibit significant homology. Therefore, we designated it as hRpn13. hRpn13 interacts with a base subunit Rpn2 via its amino-terminus. The majority of 26S proteasomes contain hRpn13, but a portion of them does not, indicating that hRpn13 is not an integral subunit. Intriguingly, we found that hRpn13 recruits UCH37, a deubiquitinating enzyme known to associate with 26 proteasomes. The carboxyl-terminal regions containing KEKE motifs of both hRpn13 and UCH37 are involved in their physical interaction. Knockdown of hRpn13 caused no obvious proteolytic defect but loss of UCH37 proteins and decrease in deubiquitinating activity of 26S proteasomes. Our results indicate that hRpn13 is essential for the activity of UCH37.     

Mammalian 26S proteasomes remain intact during protein degradation

It has been suggested that degradation of polyubiquitylated proteins is coupled to dissociation of 26S proteasomes. In contrast, using several independent types of experiments, we find that mammalian proteasomes can degrade polyubiquitylated proteins without disassembling. Thus, immobilized, (35)S-labeled 26S proteasomes degraded polyubiquitylated Sic1 and c-IAP1 without releasing any subunits. In addition, it is predicted that if 26S proteasomes dissociate into 20S proteasomes and regulatory complexes during a degradation cycle, the reassembly rate would be limiting at low proteasome concentrations. However, the rate with which each proteasome degraded polyubiquitylated Sic1 was independent of the proteasome concentration. Likewise, substrate-dependent dissociation of 26S proteasomes could not be detected by nondenaturing electrophoresis. Lastly, epoxomicin-inhibited 20S proteasomes can trap released regulatory complexes, forming inactive 26S proteasomes, but addition of epoxomicin-inhibited 20S proteasomes had no effect on the degradation of either polyubiquitylated Sic1 or UbcH10 by 26S proteasomes or of endogenous substrates in cell extracts.     

PiZ Mouse Liver Accumulates Polyubiquitin Conjugates That Associate with Catalytically Active 26S Proteasomes

Accumulation of aggregation-prone human alpha 1 antitrypsin mutant Z (AT-Z) protein in PiZ mouse liver stimulates features of liver injury typical of human alpha 1 antitrypsin type ZZ deficiency, an autosomal recessive genetic disorder. Ubiquitin-mediated proteolysis by the 26S proteasome counteracts AT-Z accumulation and plays other roles that, when inhibited, could exacerbate the injury. However, it is unknown how the conditions of AT-Z mediated liver injury affect the 26S proteasome. To address this question, we developed a rapid extraction strategy that preserves polyubiquitin conjugates in the presence of catalytically active 26S proteasomes and allows their separation from deposits of insoluble AT-Z. Compared to WT, PiZ extracts had about 4-fold more polyubiquitin conjugates with no apparent change in the levels of the 26S and 20S proteasomes, and unassembled subunits. The polyubiquitin conjugates had similar affinities to ubiquitin-binding domain of Psmd4 and co-purified with similar amounts of catalytically active 26S complexes. These data show that polyubiquitin conjugates were accumulating despite normal recruitment to catalytically active 26S proteasomes that were available in excess, and suggest that a defect at the 26S proteasome other than compromised binding to polyubiquitin chain or peptidase activity played a role in the accumulation. In support of this idea, PiZ extracts were characterized by high molecular weight, reduction-sensitive forms of selected subunits, including ATPase subunits that unfold substrates and regulate access to proteolytic core. Older WT mice acquired similar alterations, implying that they result from common aspects of oxidative stress. The changes were most pronounced on unassembled subunits, but some subunits were altered even in the 26S proteasomes co-purified with polyubiquitin conjugates. Thus, AT-Z protein aggregates indirectly impair degradation of polyubiquitinated proteins at the level of the 26S proteasome, possibly by inducing oxidative stress-mediated modifications that compromise substrate delivery to proteolytic core.