Localisation of 26S proteasomes with different subunit composition in insect muscles undergoing programmed cell death

The 26S proteasome is a large multisubunit complex involved in degrading both cytoplasmic and nuclear proteins. We have investigated the subcellular distribution of four regulatory ATPase subunits (S6 (TBP7/MS73), S6' (TBP1), S7 (MSS1), and S10b (SUG2)) together with components of 20S proteasomes in the intersegmental muscles (ISM) of Manduca sexta during developmentally programmed cell death (PCD). Immunogold electron microscopy shows that S6 is located in the heterochromatic part of nuclei of ISM fibres. S6' is present in degraded material only outside intact fibres. S7 can be detected in nuclei, cytoplasm and also in degraded material. S10b, on the other hand, is initially found in nuclei and subsequently in degraded cytoplasmic locations during PCD. 20S proteasomes are present in all areas where ATPase subunits are detected, consistent with the presence of intact 26S proteasomes. These results are discussed in terms of heterogeneity of 26S proteasomes, 26S proteasome disassembly and the possible role of ATPases in non-proteasome complexes in the process of PCD. Cell Death and Differentiation (2000) 7, 1210 - 1217.     

Proteasomes and their associated ATPases: a destructive combination

Protein degradation by 20S proteasomes in vivo requires ATP hydrolysis by associated hexameric AAA ATPase complexes such as PAN in archaea and the homologous ATPases in the eukaryotic 26S proteasome. This review discusses recent insights into their multistep mechanisms and the roles of ATP. We have focused on the PAN complex, which offers many advantages for mechanistic and structural studies over the more complex 26S proteasome. By single-particle EM, PAN resembles a "top-hat" capping the ends of the 20S proteasome and resembles densities in the base of the 19S regulatory complex. The binding of ATP promotes formation of the PAN-20S complex, which induces opening of a gate for substrate entry into the 20S. PAN's C-termini, containing a conserved motif, docks into pockets in the 20S's alpha ring and causes gate opening. Surprisingly, once substrates are unfolded, their translocation into the 20S requires ATP-binding but not hydrolysis and can occur by facilitated diffusion through the ATPase in its ATP-bound form. ATP therefore serves multiple functions in proteolysis and the only step that absolutely requires ATP hydrolysis is the unfolding of globular proteins. The 26S proteasome appears to function by similar mechanisms.     

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.     

Docking of the proteasomal ATPases' carboxyl termini in the 20S proteasome's alpha ring opens the gate for substrate entry

The 20S proteasome functions in protein degradation in eukaryotes together with the 19S ATPases or in archaea with the homologous PAN ATPase complex. These ATPases contain a conserved C-terminal hydrophobic-tyrosine-X motif (HbYX). We show that these residues are essential for PAN to associate with the 20S and open its gated channel for substrate entry. Upon ATP binding, these C-terminal residues bind to pockets between the 20S's alpha subunits. Seven-residue or longer peptides from PAN's C terminus containing the HbYX motif also bind to these sites and induce gate opening in the 20S. Gate opening could be induced by C-terminal peptides from the 19S ATPase subunits, Rpt2, and Rpt5, but not by ones from PA28/26, which lack the HbYX motif and cause gate opening by distinct mechanisms. C-terminal residues in the 19S ATPases were also shown to be critical for gating and stability of 26S proteasomes. Thus, the C termini of the proteasomal ATPases function like a "key in a lock" to induce gate opening and allow substrate entry.     

Quaternary structure of the ATPase complex of human 26S proteasomes determined by chemical cross-linking

The 26S proteasome is the major protease responsible for nonlysosomal protein degradation in eukaryotic cells. The enzyme is composed of two subparticles: the 20S proteasome, and a 19S regulatory particle (PA700) which binds to the ends of the 20S proteasome cylinder and accounts for ATP dependence and substrate specificity. Among the approximately 18 subunits of PA700 regulator, six are ATPases. The ATPases presumably recognize, unfold, and translocate substrates into the interior of the 26S proteasome. It is generally believed that the ATPases form a hexameric ring. By means of chemical cross-linking, immunoprecipitation, and blotting, we have determined that the ATPases are organized in the order S6-S6'-S10b-S8-S4-S7. Additionally, we found cross-links between the ATPase S10b and the 20S proteasome subunit alpha6. Together with the previously known interaction between S8 and alpha1 and between S4 and alpha7, these data establish the relative orientations of ATPases with respect to the 20S 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.     

Structural features of the 26S proteasome complex isolated from rat testis and sperm tail

We have previously cloned a cDNA encoding TBP-1, a protein present in the rat spermatid manchette and outer dense fibers of the developing sperm. TBP-1 contains a heptad repeat of six-leucine zipper fingers at the amino terminus and highly conserved ATPase and DNA/RNA helicase motifs toward the carboxyl terminus. TBP-1 is one of the 20 subunits forming the 19S regulatory complex of the 26S proteasome, an ATP-dependent multisubunit protease found in most eukaryotic cells. We now report the isolation of the 26S proteasome from rat testis and sperm tail and its visualization by whole-mount electron microscopy using negative staining. The 26S proteasome from rat testis was fractionated by Sephacryl S-400/Mono-Q chromatography using homogenates suspended in a 10% glycerol-supplemented buffer. Chromatographic fractions were analyzed by immunoblotting using a specific anti-TBP-1 serum. During the purification of Sak57, a keratin filament present in outer dense fibers from epididymal sperm, we detected a substantial amount of 26S proteasomes. Intact 26S proteasomes from rat testis display a rod-shaped particles about 45 nm in length and 11-17 nm in diameter. Each particle consists of a 20S barrel-shaped component formed by four rings (alphabetabetaalpha), capped by two polar 19S regulatory complexes, each identified by an element known as the "Chinese dragon head motif". TBP-1 is an ATPase-containing subunit of the 19S regulatory cap. Rat sperm preparations displayed both dissociated 26S proteasomes and Sak57 filaments. We hypothesize that 26S proteasomes in the perinuclear-arranged manchette are in a suitable location for recognition, sequestration, and degradation of accumulating ubiquitin-conjugated somatic and transient testis-specific histones during spermiogenesis. In the sperm tail, the 26S proteasome may have a role in the remodeling of the outer dense fibers and other tail components during epididymal transit.     

Demonstration that a human 26S proteolytic complex consists of a proteasome and multiple associated protein components and hydrolyzes ATP and ubiquitin-ligated proteins by closely linked mechanisms.

It is known that two types of high-molecular-mass protease complexes are present in the cytosol of mammalian cells; a 20S latent multicatalytic proteinase named the proteasome, and a large proteolytic complex with an apparent sedimentation coefficient of 26S that catalyzes ATP-dependent breakdown of proteins conjugated with ubiquitin. In this work, we first demonstrated that a low concentration of SDS was required for activation of the latent proteasome, whereas the 26S complex degraded substrates for proteasomes in the absence of SDS. Moreover, the 26S complex was greatly stabilized in the presence of 2 mM ATP and 20% glycerol. Based on these characteristics, we next devised a novel procedure for purification of the 26S proteolytic complexes from human kidney. In this procedure, the proteolytic complexes were precipitated from cytoplasmic extracts by ultracentrifugation for 5 h at 105000 x g, and the large 26S complexes were clearly separated from the 20S proteasomes by molecular-sieve chromatography on a Biogel A-1.5 m column. The 26S enzyme was then purified to apparent homogeneity by successive chromatographies on hydroxyapatite and Q Sepharose, then by glycerol density-gradient centrifugation. Electrophoretic and immunochemical analyses showed that the purified human 26S complex consisted of multiple subunits of proteasomes with molecular masses of 21-31 kDa and 13-15 protein components ranging in molecular mass over 35-110 kDa, which were directly associated with the proteasome. The purified 26S proteolytic complex degraded 125I-labeled lysozyme-ubiquitin conjugates in an ATP-dependent manner. The 26S enzyme also showed high ATPase activity, which was copurified with the complex. Vanadate and hemin strongly inhibited not only ATP cleavage, but also ATP-dependent breakdown of ubiquitinligated proteins, suggesting that the 26S complex hydrolyzes ATP and ubiquitinated proteins by closely linked mechanisms. These findings indicate that the 26S complex consists of a proteasome with proteolytic function and multiple other components including an ATPase that regulates energy-dependent, ubiquitin-mediated protein degradation.     

Proteasome function is regulated by cyclic AMP-dependent protein kinase through phosphorylation of Rpt6

Dysregulation of the proteasome has been documented in a variety of human diseases such as Alzheimer, muscle atrophy, cataracts etc. Proteolytic activity of 26 S proteasome is ATP- and ubiquitin-dependent. O-GlcNAcylation of Rpt2, one of the AAA ATPases in the 19 S regulatory cap, shuts off the proteasome through the inhibition of ATPase activity. Thus, through control of the flux of glucose into O-GlcNAc, the function of the proteasome is coupled to glucose metabolism. In the present study we found another metabolic control of the proteasome via cAMP-dependent protein kinase (PKA). Contrary to O-Glc-NAcylation, PKA activated proteasomes both in vitro and in vivo in association with the phosphorylation at Ser(120) of another AAA ATPase subunit, Rpt6. Mutation of Ser(120) to Ala blocked proteasome function. The stimulatory effect of PKA and the phosphorylation of Rpt6 were reversible by protein phosphatase 1 gamma. Thus, hormones using the PKA system can also regulate proteasomes often in concert with glucose metabolism. This finding might lead to novel strategies for the treatment of proteasome-related diseases.     

The 20S Proteasome as an Assembly Platform for the 19S Regulatory Complex

26S proteasomes consist of cylindrical 20S proteasomes with 19S regulatory complexes attached to the ends. Treatment with high concentrations of salt causes the regulatory complexes to separate into two sub-complexes, the base, which is in contact with the 20S proteasome, and the lid, which is the distal part of the 19S complex. Here, we describe two assembly intermediates of the human regulatory complex. One is a dimer of the two ATPase subunits, Rpt3 and Rpt6. The other is a complex of nascent Rpn2, Rpn10, Rpn11, Rpn13, and Txnl1, attached to preexisting 20S proteasomes. This early assembly complex does not yet contain Rpn1 or any of the ATPase subunits of the base. Thus, assembly of 19S regulatory complexes takes place on preexisting 20S proteasomes, and part of the lid is assembled before the base.     

Assembly of the regulatory complex of the 26S proteasome

The 19S regulatory complex (RC) of 26S proteasomes is a 900–1000 kDa particle composed of 18 distinct subunits (S1–S15) ranging in molecular mass from 25 to 110 kDa. This particle confers ATP-dependence and polyubiquitin (polyUb) recognition to the 26S proteasome. The symmetry and homogenous structure of the proteasome contrasts sharply with the remarkable complexity of the RC. Despite the fact that the primary sequences of all the subunits are now known, insight has been gained into the function of only eight subunits. The six ATPases within the RC constitute a subfamily (S4-like ATPases) within the AAA superfamily and we have shown that they form specific pairs in vitro[1]. We have now determined that putative coiled-coils within the variable N-terminal regions of these proteins are likely to function as recognition elements that direct the proper placement of the ATPases within the RC. We have also begun mapping putative interactions between non-ATPase subunits and S4-like ATPases. These studies have allowed us to build a model for the specific arrangement of 9 subunits within the human regulatory complex. This model agrees with recent findings by Glickman et al. [2] who have reported that two subcomplexes, termed the base and the lid, form the RC of budding yeast 26S proteasomes.     

Regulation of the proteasome by AMPK in endothelial cells: the role of O-GlcNAc transferase (OGT)

26S proteasome is a macromolecular multi-subunit complex responsible for recognizing, unfolding, and ultimately destroying proteins. It remains poorly understood how 26S proteasome activity is regulated. The present study was to investigate if AMP-activated protein kinase (AMPK) functions as a physiological suppressor of the 26S proteasome in endothelial cells. 26S proteasome assembly, activity, and O-GlcNAcylation of P700 were assayed in cultured human umbilical vein endothelial cells (HUVEC) and mouse aortas isolated from C57BL6 wild type and AMPKα2 knockout mice with or without being exposed to selective AMPK activators or inhibitors. Pharmacological and genetic activation of AMPK effectively suppresses 26S proteasomes in endothelial cells. Conversely, inactivation of AMPK either pharmacologically or genetically increases 26S proteasome activity; furthermore, the inactivation decreases the O-GlcNAcylation of PA700/S10B (the regulatory complex in 26S proteasomes) and increases the assembly of 26S proteasomes. In contrast, AMPK activation increases levels of O-GlcNAcylated PA700/S10B, likely through enhanced association of PA700 with O-GlcNAc transferase (OGT), the enzyme that catalyzes protein O-GlcNAcylation. Finally, aortas from AMPK-KO vs wild type mice exhibit elevated 26S proteasome activity in parallel with decreased PA700/S10B O-GlcNAcylation and PA700/S10B-OGT association. Taken together, we conclude that AMPK functions as a physiological suppressor of 26S proteasomes.     

The ATP Costs and Time Required to Degrade Ubiquitinated Proteins by the 26 S Proteasome

The degradation of ubiquitinated proteins by 26 S proteasomes requires ATP hydrolysis. To investigate if the six proteasomal ATPases function independently or in a cyclic manner, as proposed recently, we used yeast mutants that prevent ATP binding to Rpt3, Rpt5, or Rpt6. Although proteasomes contain six ATPase subunits, each of these single mutations caused a 66% reduction in basal ATP hydrolysis, and each blocked completely the 2–3-fold stimulation of ATPase activity induced by ubiquitinated substrates. Therefore, the ATPase subunits must function in a ordered manner, in which each is required for the stimulation of ATPase activity by substrates. Although ATP is essential for multiple steps in proteasome function, when the rate of ATP hydrolysis was reduced incrementally, the degradation of Ub₅-DHFR (where Ub is ubiquitin and DHFR is dihydrofolate reductase) decreased exactly in parallel. This direct proportionality implies that a specific number of ATPs is consumed in degrading a ubiquitinated protein. When the ubiquitinated DHFR was more tightly folded (upon addition of the ligand folate), the rate of ATP hydrolysis was unchanged, but the time to degrade a Ub₅-DHFR molecule (∼13 s) and the energy expenditure (50–80 ATPs/Ub₅-DHFR) both increased by 2-fold. With a mutation in the ATPase C terminus that reduced gate opening into the 20 S proteasome, the energy costs and time required for conjugate degradation also increased. Thus, different ubiquitin conjugates activate similarly the ATPase subunit cycle that drives proteolysis, but polypeptide structure determines the time required for degradation and thus the energy cost.     

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.