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.     

Genetic analyses of the Arabidopsis 26S proteasome regulatory particle reveal its importance during light stress and a specific role for the N-Terminus of RPT2 in development.

The 26S proteasome subunit RPT2 is a component of the hexameric ring of AAA-ATPases that forms the base of the 19S regulatory particle (RP). This subunit has specific roles in the yeast and mammalian proteasomes by helping promote assembly of the RP with the 20S core protease (CP) and gate the CP to prevent indiscriminate degradation of cytosolic and nuclear proteins. In plants, this subunit plays an important role in diverse processes that include shoot and root apical meristem maintenance, cell size regulation, trichome branching, and stress responses. Recently, we reported that mutants in RPT2 and several other RP subunits have reduced histone levels, suggesting that at least some of the pleiotropic phenotypes observed in these plants result from aberrant nucleosome assembly. Here, we expand our genetic analysis of RPT2 in Arabidopsis to shed additional light on the roles of the N- and C-terminal ends. We also present data showing that plants bearing mutations in RP subunit genes have their seedling phenotypes exacerbated by prolonged light exposure.     

The RPT2 subunit of the 26S proteasome directs complex assembly, histone dynamics, and gametophyte and sporophyte development in Arabidopsis

The regulatory particle (RP) of the 26S proteasome contains a heterohexameric ring of AAA-ATPases (RPT1-6) that unfolds and inserts substrates into the core protease (CP) for degradation. Through genetic analysis of the Arabidopsis thaliana gene pair encoding RPT2, we show that this subunit plays a critical role in 26S proteasome assembly, histone dynamics, and plant development. rpt2a rpt2b double null mutants are blocked in both male and female gamete transmission, demonstrating that the subunit is essential. Whereas rpt2b mutants are phenotypically normal, rpt2a mutants display a range of defects, including impaired leaf, root, trichome, and pollen development, delayed flowering, stem fasciation, hypersensitivity to mitomycin C and amino acid analogs, hyposensitivity to the proteasome inhibitor MG132, and decreased 26S complex stability. The rpt2a phenotype can be rescued by both RPT2a and RPT2b, indicative of functional redundancy, but not by RPT2a mutants altered in ATP binding/hydrolysis or missing the C-terminal hydrophobic sequence that docks the RPT ring onto the CP. Many rpt2a phenotypes are shared with mutants lacking the chromatin assembly factor complex CAF1. Like caf1 mutants, plants missing RPT2a or reduced in other RP subunits contain less histones, thus implicating RPT2 specifically, and the 26S proteasome generally, in plant nucleosome assembly.     

Structure and functional analyses of the 26S proteasome subunits from plants – Plant 26S proteasome

As initial steps to define how the 26S proteasome degrades ubiquitinated proteins in plants, we have characterized many of the subunits that comprise the proteolytic complex from Arabidopsis thaliana. A set of 23 Arabidopsis genes encoding the full complement of core particle (CP) subunits and a collection encoding 12 out of 18 known eukaryotic regulatory particle (RP) subunits, including six AAA-ATPase subunits, were identified. Several of these 26S proteasome genes could complement yeast strains missing the corresponding orthologs. Using this ability of plant subunits to functionally replace yeast counterparts, a parallel structure/function analysis was performed with the RP subunit RPN10/MCB1, a putative receptor for ubiquitin conjugates. RPN10 is not essential for yeast viability but is required for amino acid analog tolerance and degradation of proteins via the ubiquitin-fusion degradation pathway, a subpathway within the ubiquitin system. Surprisingly, we found that the C-terminal motif required for conjugate recognition by RPN10 is not essential for in vivo functions. Instead, a domain near the N-terminus is required. We have begun to exploit the moss Physcomitrella patens as a model to characterize the plant 26S proteasome using reverse genetics. By homologous recombination, we have successfully disrupted the RPN10 gene. Unlike yeast rpn10 strains which grow normally, Physcomitrella rpn10 strains are developmentally arrested, being unable to initiate gametophorogenesis. Further analysis of these mutants revealed that RPN10 is likely required for a developmental program triggered by plant hormones.     

Arabidopsis thaliana AtHAL3: a flavoprotein related to salt and osmotic tolerance and plant growth

The 26S proteasome is a multi-subunit ATP-dependent protease responsible for degrading most short-lived intracellular proteins targeted for breakdown by ubiquitin conjugation. The complex is composed of two relatively stable subparticles, the 20S proteasome, a hollow cylindrical structure which contains the proteolytic active sites in its lumen, and the 19S regulatory particle (RP) which binds to either end of the cylinder and provides the ATP-dependence and the specificity for ubiquitinated proteins. Among the approximately 18 subunits of the RP from yeast and animals are a set of six proteins, designated RPT1-6 for regulatory particle triple-A ATPase, that form a distinct family within the AAA superfamily. Presumably, these subunits use ATP hydrolysis to help assemble the 26S holocomplex, recognize and unfold appropriate substrates, and/or translocate the substrates to the 20S complex for degradation. Here, we describe the RPT gene family from Arabidopsis thaliana. From a collection of cDNAs and genomic sequences, a family of genes encoding all six of the RPT subunits was identified with significant amino acid sequence similarity to their yeast and animal counterparts. Five of the six RPT sub- units are encoded by two genes; the exception being RPT3 which is encoded by a single gene. mRNA for each of the six proteins is present in all tissue types examined. Five of the subunits (RPT1 and 3-6) complemented yeast mutants missing their respective orthologs, indicating that the yeast and Arabidopsis proteins are functionally equivalent. Taken together, these results demonstrate that the RP, like the 20S proteasome, is functionally and structurally conserved among eukaryotes and indicate that the plant RPT subunits, like their yeast counterparts, have non-redundant functions.     

Order of the Proteasomal ATPases and Eukaryotic Proteasome Assembly

The 26S proteasome is responsible for a large fraction of the regulated protein degradation in eukaryotic cells. The enzyme complex is composed of a 20S proteolytic core particle (CP) capped on one or both ends with a 19S regulatory particle (RP). The RP recognizes and unfolds substrates and translocates them into the CP. The RP can be further divided into lid and base subcomplexes. The base contains a ring of six AAA+ ATPases (Rpts) that directly abuts the CP and is responsible for unfolding substrates and driving them into the CP for proteolysis. Although 120 arrangements of the six different ATPases within the ring are possible in principle, they array themselves in one specific order. The high sequence and structural similarity between the Rpt subunits presents special challenges for their ordered association and incorporation into the assembling proteasome. In this review, we discuss recent advances in our understanding of proteasomal RP base biogenesis, with emphasis on potential specificity determinants in ring arrangement, and the implications of the ATPase ring arrangement for proteasome assembly.     

Substrate access and processing by the 20S proteasome core particle

Intracellular proteolysis is an essential process. In eukaryotes, most proteins in the cytosol and nucleus are degraded by the ubiquitin (Ub)-proteasome pathway. A major component within this system is the 26S proteasome, a 2.5MDa molecular machine, built from more than 31 different subunits. This complex is formed by a cylinder-shaped multimeric complex referred to as the proteolytic 20S proteasome (core particle, CP) capped at each end by another multimeric component called the 19S complex (regulatory particle, RP) or PA700. Structure, assembly and enzymatic mechanism have been elucidated only for the CP, whereas the organization of the RP is less well understood. The CP is composed of 28 subunits, which are arranged as an alpha7beta7beta7alpha7-complex in four stacked rings. The interior of the free core particle, which harbors the active sites, is inaccessible for folded and unfolded substrates and represents a latent state. This inhibition is relieved upon binding of the RP to the CP by formation of the 26S proteasome holoenzyme. This review summarizes the current knowledge of the structural features of 20S proteasomes.     

Identification of the 19S regulatory particle subunits from the rice 26S proteasome.

The 26S proteasome, a protein complex consisting of a 20S proteasome and a pair of 19S regulatory particles (RP), is involved in ATP-dependent proteolysis in eukaryotes. In yeast, the RP contains six different ATPase subunits and, at least, 11 non-ATPase subunits. In this study, we identified the rice homologs of yeast RP subunit genes from the rice expressed sequence tag (EST) library. The complete nucleotide sequences of the homologs for five ATPase subunits, OsRpt1, OsRpt2, OsRpt4, OsRpt5 and OsRpt6, and five non-ATPase subunits, OsRpn7, OsRpn8, OsRpn10, OsRpn11 and OsRpn12, and the partial sequences of one ATPase subunit, OsRpt3, and six non-ATPase subunits, OsRpn1, OsRpn2, OsRpn3, OsRpn5, OsRpn6 and OsRpn9, were determined. Gene homologs of four ATPase subunits, OsRpt1, OsRpt2, OsRpt4 and OsRpt5, and three non- ATPase subunits, OsRpn1, OsRpn2 and OsRpn9, were found to be encoded by duplicated genes. The rice RP was purified by immunoaffinity chromatography with a Protein A column immobilized antibody against rice 20S proteasome, and the subunit composition was determined. The homologs obtained from the rice EST library were identified as genes encoding subunits of RP purified from rice, including the both products of duplicated genes by using electrospray ionization quadrupole time-of-flight mass spectrometry. Post-translational modifications and processing in rice RP subunits were also identified. Various types of RP complex with different subunit compositions are present in rice cells, suggesting the multiple functions of rice proteasome.     

Base-CP proteasome can serve as a platform for stepwise lid formation

26S proteasome, a major regulatory protease in eukaryotes, consists of a 20S proteolytic core particle (CP) capped by a 19S regulatory particle (RP). The 19S RP is divisible into base and lid sub-complexes. Even within the lid, subunits have been demarcated into two modules: module 1 (Rpn5, Rpn6, Rpn8, Rpn9 and Rpn11), which interacts with both CP and base sub-complexes and module 2 (Rpn3, Rpn7, Rpn12 and Rpn15) that is attached mainly to module 1. We now show that suppression of RPN11 expression halted lid assembly yet enabled the base and 20S CP to pre-assemble and form a base-CP. A key role for Regulatory particle non-ATPase 11 (Rpn11) in bridging lid module 1 and module 2 subunits together is inferred from observing defective proteasomes in rpn11–m1, a mutant expressing a truncated form of Rpn11 and displaying mitochondrial phenotypes. An incomplete lid made up of five module 1 subunits attached to base-CP was identified in proteasomes isolated from this mutant. Re-introducing the C-terminal portion of Rpn11 enabled recruitment of missing module 2 subunits. In vitro, module 1 was reconstituted stepwise, initiated by Rpn11–Rpn8 heterodimerization. Upon recruitment of Rpn6, the module 1 intermediate was competent to lock into base-CP and reconstitute an incomplete 26S proteasome. Thus, base-CP can serve as a platform for gradual incorporation of lid, along a proteasome assembly pathway. Identification of proteasome intermediates and reconstitution of minimal functional units should clarify aspects of the inner workings of this machine and how multiple catalytic processes are synchronized within the 26S proteasome holoenzymes.     

Evidence that the Arabidopsis Ubiquitin C-terminal Hydrolases 1 and 2 associate with the 26S proteasome and the TREX-2 complex

The 26S proteasome interacts with a number of different proteins, while the TREX-2 complex is an important component of the mRNA export machinery. In animals and yeast, members of the Ubiquitin C-terminal Hydrolase 37 (UCH37) family are found to associate with the 26S proteasome, but this has not been demonstrated in plants. The Arabidopsis UCH1 and UCH2 are orthologous to UCH37. Here, we show that UCH1 and UCH2 interact with the 26S proteasome lid subunits. In addition, the two UCHs also interact with TREX-2 components. Our data suggest that Arabidopsis UCHs may serve as a link between the 26S proteasome lid complex and the TREX-2 complex.     

Assembly of the 20S proteasome

The proteasome is a cellular protease responsible for the selective degradation of the majority of the intracellular proteome. It recognizes, unfolds, and cleaves proteins that are destined for removal, usually by prior attachment to polymers of ubiquitin. This macromolecular machine is composed of two subcomplexes, the 19S regulatory particle (RP) and the 20S core particle (CP), which together contain at least 33 different and precisely positioned subunits. How these subunits assemble into functional complexes is an area of active exploration. Here we describe the current status of studies on the assembly of the 20S proteasome (CP). The 28-subunit CP is found in all three domains of life and its cylindrical stack of four heptameric rings is well conserved. Though several CP subunits possess self-assembly properties, a consistent theme in recent years has been the need for dedicated assembly chaperones that promote on-pathway assembly. To date, a minimum of three accessory factors have been implicated in aiding the construction of the 20S proteasome. These chaperones interact with different assembling proteasomal precursors and usher subunits into specific slots in the growing structure. This review will focus largely on chaperone-dependent CP assembly and its regulation.     

Subunit interaction maps for the regulatory particle of the 26S proteasome and the COP9 signalosome.

The 26S proteasome plays a major role in eukaryotic protein breakdown, especially for ubiquitin-tagged proteins. Substrate specificity is conferred by the regulatory particle (RP), which can dissociate into stable lid and base subcomplexes. To help define the molecular organization of the RP, we tested all possible paired interactions among subunits from Saccharomyces cerevisiae by yeast two-hybrid analysis. Within the base, a Rpt4/5/3/6 interaction cluster was evident. Within the lid, a structural cluster formed around Rpn5/11/9/8. Interactions were detected among synonymous subunits (Csn4/5/7/6) from the evolutionarily related COP9 signalosome (CSN) from Arabidopsis, implying a similar quaternary arrangement. No paired interactions were detected between lid, base or core particle subcomplexes, suggesting that stable contacts between them require prior assembly. Mutational analysis defined the ATPase, coiled-coil, PCI and MPN domains as important for RP assembly. A single residue in the vWA domain of Rpn10 is essential for amino acid analog resistance, for degrading a ubiquitin fusion degradation substrate and for stabilizing lid-base association. Comprehensive subunit interaction maps for the 26S proteasome and CSN support the ancestral relationship of these two complexes.     

Fub1p, a novel protein isolated by boundary screening, binds the proteasome complex

Silenced chromatin domains are restricted to specific regions. Eukaryotic chromosomes are organized into discrete domains delimited by domain boundaries. From approximately 6,000 genes in Saccharomyces cerevisiae, we previously isolated 55 boundary genes. In this study, we focus on the molecular function of one of boundary genes, YCR076C/FUB1 (function of boundary), whose function has not been clearly defined in vivo. Biochemical analysis of Fub1p revealed that it interacted with multiple subunits of the 20S proteasome core particle (20S CP). To further clarify the functional link between Fub1p and proteasome, several proteasome mutants were analyzed. Although only 20S CP subunits were isolated as Fub1p interactors, a genetic interaction was also observed for component of 19S regulatory particle (19S RP) suggesting involvement of Fub1p with the whole proteasome. We also analyzed the mechanism of boundary establishment by using proteasome composition factor-deficient strains. Deletion of pre9 and ump1, whose products have effects on the 20S CP, resulted in a decrease in boundary function. Domain analyses of Fub1p identified a minimum functional domain in the C terminus that was essential for boundary establishment and showed a limited sequence homology to the human PSMF1, which is known to inhibit proteasome activity. Finally, boundary assay showed that human PSMF1 also exhibited boundary establishment activity in yeast. Our results defined the functional correlation between Fub1p and PSMF1.     

The substrate translocation channel of the proteasome

The core particle (CP) of the yeast proteasome is composed of four heptameric rings of subunits arranged in a hollow, barrel-like structure. We have found that the CP is autoinhibited by the N-terminal tails of the outer (alpha) ring subunits. Crystallographic analysis showed that deletion of the tail of the alpha3 subunit opens a channel into the proteolytically active interior chamber of the CP, thus derepressing peptide hydrolysis. In the latent state of the particle, the tails prevent substrate entry by imposing topological closure on the CP. Inhibition by the alpha subunit tails is relieved upon binding of the regulatory particle to the CP to form the proteasome holoenzyme. Opening of the CP channel by assembly of the holoenzyme is regulated by the ATPase domain of Rpt2, one of 17 subunits in the RP. Thus, open-channel mutations in CP subunits suppress the closed-channel phenotype of an rpt2 mutant. These results identify a specific mechanism for allosteric regulation of the CP by the RP.     

An atomic model AAA-ATPase/20S core particle sub-complex of the 26S proteasome

The 26S proteasome is the most downstream element of the ubiquitin-proteasome pathway of protein degradation. It is composed of the 20S core particle (CP) and the 19S regulatory particle (RP). The RP consists of 6 AAA-ATPases and at least 13 non-ATPase subunits. Based on a cryo-EM map of the 26S proteasome, structures of homologs, and physical protein-protein interactions we derive an atomic model of the AAA-ATPase-CP sub-complex. The ATPase order in our model (Rpt1/Rpt2/Rpt6/Rpt3/Rpt4/Rpt5) is in excellent agreement with the recently identified base-precursor complexes formed during the assembly of the RP. Furthermore, the atomic CP-AAA-ATPase model suggests that the assembly chaperone Nas6 facilitates CP-RP association by enhancing the shape complementarity between Rpt3 and its binding CP alpha subunits partners.     

Proteasome disassembly and downregulation is correlated with viability during stationary phase

During prolonged starvation, yeast cells enter a stationary phase (SP) during which the synthesis of many proteins is dramatically decreased. We show that a parallel decrease in proteasome-dependent proteolysis also occurs. The reduction in proteolysis is correlated with disassembly of 26S proteasome holoenzymes into their 20S core particle (CP) and 19S regulatory particle (RP) components. Proteasomes are reassembled, and proteolysis resumes prior to cell cycle reentry. Free 20S CPs are found in an autoinhibited state in which the N-terminal tails from neighboring alpha subunits are anchored by an intricate lattice of interactions blocking the channel that leads into the 20S CPs. By deleting channel gating residues of CP alpha subunits, we generated an "open channel" proteasome that exhibits faster rates of protein degradation both in vivo and in vitro, indicating that gating contributes to regulation of proteasome activity. This open channel mutant is delayed in outgrowth from SP and cannot survive following prolonged starvation. In summary, we have found that the ubiquitin-proteasome pathway can be subjected to global downregulation, that the proteasome is a target of this regulation, and that proteasome downregulation is linked to survival of SP cells. Maintaining high viability during SP is essential for evolutionary fitness, which may explain the extreme conservation of channel gating residues in eukaryotic proteasomes.     

Tripeptidyl peptidase II. An oligomeric protease complex from Arabidopsis

The breakdown of most nuclear and cytoplasmic proteins involves their partial cleavage by the 26S proteasome followed by further disassembly to free amino acids by the combined action of endo- and exopeptidases. In animals, one important intermediate exopeptidase is tripeptidyl peptidase (TPP)II, which digests peptide products of the 26S proteasome and other endopeptidases into tripeptides. Here, we describe the purification and characterization of TPPII from Arabidopsis (Arabidopsis thaliana). Like its animal counterparts, Arabidopsis TPPII exists as a soluble, approximately 5- to 9-MD complex. Two related species of 153 and 142 kD are present in the purified preparations that are derived from a single TPP2 gene. Sequencing by Edman degradation of the intact polypeptides and mass spectrometry of proteolytic fragments demonstrated that the 142-kD form mainly differs from the 153-kD form by a truncation at the C-terminal end. This serine protease is a member of the subtilisin superfamily and is sensitive to the inhibitors alanine-alanine-phenylalanine-chloromethylketone and butabindide, which are diagnostic for the TPPII subfamily. The Arabidopsis TPP2 gene is widely expressed in many tissue types with related genes evident in other plant genomes. Whereas the 26S proteasome is essential, TPPII appears not as important for plant physiology. An Arabidopsis T-DNA mutant defective in TPP2 expression displays no phenotypic abnormalities and is not hypersensitive to either amino acid analogs or the 26S proteasome inhibitor MG132. As a consequence, plants likely contain other intermediate exopeptidases that assist in amino acid recycling.     

The RPN1 subunit of the 26S proteasome in Arabidopsis is essential for embryogenesis

The 26S proteasome plays a central role in the degradation of regulatory proteins involved in a variety of developmental processes. It consists of two multisubunit protein complexes: the proteolytic core protease and the regulatory particle (RP). The function of most RP subunits is poorly understood. Here, we describe mutants in the Arabidopsis thaliana RPN1 subunit, which is encoded by two paralogous genes, RPN1a and RPN1b. Disruption of RPN1a caused embryo lethality, while RPN1b mutants showed no obvious abnormal phenotype. Embryos homozygous for rpn1a arrested at the globular stage with defects in the formation of the embryonic root, the protoderm, and procambium. Cyclin B1 protein was not degraded in these embryos, consistent with cell division defects. Double mutant plants (rpn1a/RPN1a rpn1b/rpn1b) produced embryos with a phenotype indistinguishable from that of the rpn1a single mutant. Thus, despite their largely overlapping expression patterns in flowers and developing seeds, the two isoforms do not share redundant functions during gametogenesis and embryogenesis. However, complementation of the rpn1a mutation with the coding region of RPN1b expressed under the control of the RPN1a promoter indicates that the two RPN1 isoforms are functionally equivalent. Overall, our data indicate that RPN1 activity is essential during embryogenesis, where it might participate in the destruction of a specific set of protein substrates.     

Incorporation of the Rpn12 subunit couples completion of proteasome regulatory particle lid assembly to lid-base joining

The 26S proteasome, the central eukaryotic protease, comprises a core particle capped by a 19S regulatory particle (RP). The RP is divisible into base and lid subcomplexes. Lid biogenesis and incorporation into the RP remain poorly understood. We report several lid intermediates, including the free Rpn12 subunit and a lid particle (LP) containing the remaining eight subunits, LP2. Rpn12 binds LP2 in vitro, and each requires the other for assembly into 26S proteasomes. Stable Rpn12 incorporation depends on all other lid subunits, indicating that Rpn12 distinguishes LP2 from smaller lid subcomplexes. The highly conserved C terminus of Rpn12 bridges the lid and base, mediating both stable binding to LP2 and lid-base joining. Our data suggest a hierarchical assembly mechanism where Rpn12 binds LP2 only upon correct assembly of all other lid subunits, and the Rpn12 tail then helps drive lid-base joining. Rpn12 incorporation thus links proper lid assembly to subsequent assembly steps.