Analysis of the rpn11-m1 proteasomal mutant reveals connection between cell cycle and mitochondrial biogenesis

The proteasomal lid subunit Rpn11 is essential for maintaining a correct cell cycle and mitochondrial morphology in Saccharomyces cerevisiae. In this paper, we show that the rpn11-m1 mutant has a peculiar cell cycle defect reminiscent of mutants defective in the FEAR pathway that delay the release of the Cdc14 protein phosphatase from the nucleolus. We analyzed the rpn11-m1 phenotypes and found that overexpression of Cdc14 suppresses all the rpn11-m1 defects, including the mitochondrial ones. Suppression by Cdc14 of the rpn11-m1 mitochondrial morphology defect reveals an uncharacterized connection between mitochondrial and cell cycle events. Interestingly, the overexpression of Cdc14 also partially restores the tubular network in an Δmmm2 strain, which lacks a mitochondrial protein belonging to the complex necessary to anchor the mitochondrion to the actin cytoskeleton. Altogether our findings indicate, for the first time, a cross-talk between the cell cycle and mitochondrial morphology.     

Integrity of the Saccharomyces cerevisiae Rpn11 Protein Is Critical for Formation of Proteasome Storage Granules (PSG) and Survival in Stationary Phase

Decline of proteasome activity has been reported in mammals, flies and yeasts during aging. In the yeast Saccharomyces cerevisiae, the reduction of proteolysis in stationary phase is correlated with disassembly of the 26S proteasomes into their 20S and 19S subcomplexes. However a recent report showed that upon entry into the stationary phase, proteasome subunits massively re-localize from the nucleus into mobile cytoplasmic structures called proteasome storage granules (PSGs). Whether proteasome subunits in PSG are assembled into active complexes remains an open question that we addressed in the present study. We showed that a particular mutant of the RPN11 gene (rpn11-m1), encoding a proteasome lid subunit already known to exhibit proteasome assembly/stability defect in vitro, is unable to form PSGs and displays a reduced viability in stationary phase. Full restoration of long-term survival and PSG formation in rpn11-m1 cells can be achieved by the expression in trans of the last 45 amino acids of the C-terminal domain of Rpn11, which was moreover found to co-localize with PSGs. In addition, another rpn11 mutant leading to seven amino acids change in the Rpn11 C-terminal domain, which exhibits assembled-26S proteasomes, is able to form PSGs but with a delay compared to the wild type situation. Altogether, our findings indicate that PSGs are formed of fully assembled 26S proteasomes and suggest a critical role for the Rpn11 protein in this process.     

A proteasome assembly defect in rpn3 mutants is associated with Rpn11 instability and increased sensitivity to stress

Rpn11 is a proteasome-associated deubiquitinating enzyme that is essential for viability. Recent genetic studies showed that Rpn11 is functionally linked to Rpn10, a major multiubiquitin chain binding receptor in the proteasome. Mutations in Rpn11 and Rpn10 can reduce the level and/or stability of proteasomes, indicating that both proteins influence its structural integrity. To characterize the properties of Rpn11, we examined its interactions with other subunits in the 19S regulatory particle and detected strong binding to Rpn3. Two previously described rpn3 mutants are sensitive to protein translation inhibitors and an amino acid analog. These mutants also display a mitochondrial defect. The abundance of intact proteasomes was significantly reduced in rpn3 mutants, as revealed by strongly reduced binding between 20S catalytic with 19S regulatory particles. Proteasome interaction with the shuttle factor Rad23 was similarly reduced. Consequently, higher levels of multiUb proteins were associated with Rad23, and proteolytic substrates were stabilized. The availability of Rpn11 is important for maintaining adequate levels of intact proteasomes, as its depletion caused growth and proteolytic defects in rpn3. These studies suggest that Rpn11 is stabilized following its incorporation into proteasomes. The instability of Rpn11 and the defects of rpn3 mutants are apparently caused by a failure to recruit Rpn11 into mature proteasomes.     

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.     

Proteasome Assembly Influences Interaction with Ubiquitinated Proteins and Shuttle Factors

A major fraction of intracellular protein degradation is mediated by the proteasome. Successful degradation of these substrates requires ubiquitination and delivery to the proteasome followed by protein unfolding and disassembly of the multiubiquitin chain. Enzymes, such as Rpn11, dismantle multiubiquitin chains, and mutations can affect proteasome assembly and activity. We report that different rpn11 mutations can affect proteasome interaction with ubiquitinated proteins. Moreover, proteasomes are unstable in rpn11-1 and do not form productive interactions with multiubiquitinated proteins despite high levels in cell extracts. However, increased levels of ubiquitinated proteins were found associated with shuttle factors. In contrast to rpn11-1, proteasomes expressing a catalytically inactive mutant (rpn11^AXA) were more stable and bound very high amounts of ubiquitinated substrates. Expression of the carboxyl-terminal domain of Rpn11 partially suppressed the growth and proteasome stability defects of rpn11-1. These results indicate that ubiquitinated substrates are preferentially delivered to intact proteasome.     

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.     

Genetic evidence for interaction between components of the yeast 26S proteasome: combination of a mutation in RPN12 (a lid component gene) with mutations in RPT1 (an ATPase gene) causes synthetic lethality

The 19S regulatory particle of the yeast 26S proteasome consists of six related ATPases (Rpt proteins) and at least 11 non-ATPase proteins (Rpn proteins). RPN12 (formerly NIN1) encodes an Rpn component of the 19S regulatory particle and is essential for growth. To determine which subunit(s) of the 26S proteasome interact(s) with Rpn12, we attempted to screen for mutations that cause synthetic lethality in the presence of the rpn12-1 (formerly nin1-1) mutation. Among the candidates recovered was a new allele of RPT1 (formerly CIM5). This mutant allele was designated rpt1-2; on its own this mutation caused no phenotypic change, whereas the rpn12-1 rpt1-2 double mutant was lethal, suggesting a strong interaction between Rpn12 and Rpt1. The site of the rpt1-2 mutation was determined by DNA sequencing of the RPT1 locus retrieved from the mutant, and a single nucleotide alteration was found. This changes amino acid 446 of the RPT1 product from alanine to valine. The alanine residue is conserved in all Rpt proteins, except Rpt5, but no function has yet been assigned to the region that contains it. We propose that this region is necessary for Rpt1 to interact with Rpn12. The terminal phenotype of the rpn12-1 rpt1-2 double mutant was not cell cycle specific, suggesting that in the double mutant cells the function of the 26S proteasome is completely eliminated, thereby inducing multiple defects in cellular functions.     

Genetic evidence for interaction between components of the yeast 26S proteasome: combination of a mutation in RPN12 (a lid component gene) with mutations in RPT1 (an ATPase gene) causes synthetic lethality

The 19S regulatory particle of the yeast 26S proteasome consists of six related ATPases (Rpt proteins) and at least 11 non-ATPase proteins (Rpn proteins). RPN12 (formerly NIN1) encodes an Rpn component of the 19S regulatory particle and is essential for growth. To determine which subunit(s) of the 26S proteasome interact(s) with Rpn12, we attempted to screen for mutations that cause synthetic lethality in the presence of the rpn12-1 (formerly nin1-1) mutation. Among the candidates recovered was a new allele of RPT1 (formerly CIM5). This mutant allele was designated rpt1-2; on its own this mutation caused no phenotypic change, whereas the rpn12-1 rpt1-2 double mutant was lethal, suggesting a strong interaction between Rpn12 and Rpt1. The site of the rpt1-2 mutation was determined by DNA sequencing of the RPT1 locus retrieved from the mutant, and a single nucleotide alteration was found. This changes amino acid 446 of the RPT1 product from alanine to valine. The alanine residue is conserved in all Rpt proteins, except Rpt5, but no function has yet been assigned to the region that contains it. We propose that this region is necessary for Rpt1 to interact with Rpn12. The terminal phenotype of the rpn12-1 rpt1-2 double mutant was not cell cycle specific, suggesting that in the double mutant cells the function of the 26S proteasome is completely eliminated, thereby inducing multiple defects in cellular functions. Received: 1 February 1999 / Accepted: 5 May 1999     

Rpn5 is a conserved proteasome subunit and required for proper proteasome localization and assembly

Proper function of the 26 S proteasome requires assembly of the regulatory complex, which is composed of the lid and base subcomplexes. We characterized Rpn5, a lid subunit, in fission yeast. We show that Rpn5 associates with the proteasome rpn5. Deletion (rpn5Delta) exacerbates the growth defects in proteasome mutants, leading to mitotic abnormalities, which correlate with accumulation of polyubiquitinated proteins, such as Cut2/securin. Rpn5 expression is tightly controlled; both overexpression and deletion of rpn5 impair proteasome functions. The proteasome is assembled around the inner nuclear membrane in wild-type cells; however, in rpn5Delta cells, proteasome subunits are improperly assembled and/or localized. In the lid mutants, Rpn5 is mislocalized in the cytosol, while in the base mutants, Rpn5 can enter the nucleus, but is left in the nucleoplasm, and not assembled into the nuclear membrane. These results suggest that Rpn5 is a dosage-dependent proteasome regulator and plays a role in mediating proper proteasome assembly. Moreover, the Rpn5 assembly may be a cooperative process that involves at least two steps: 1) nuclear import and 2) subsequent assembly into the nuclear membrane. The former step requires other components of the lid, while the latter requires the base. Human Rpn5 rescues the phenotypes associated with rpn5Delta and is incorporated into the yeast proteasome, suggesting that Rpn5 functions are highly conserved.     

Mitochondrial effects of the pleiotropic proteasomal mutation mpr1/rpn11: uncoupling from cell cycle defects in extragenic revertants

We have previously characterized a Saccharomyces cerevisiae mutant which contains a mutation in the essential rpn11/mpr1 gene coding for the proteasomal regulatory subunit Rpn11. The mpr1-1 mutation shows the phenotypic characteristics generally associated with proteasomal mutations, such as cell cycle defects and accumulation of polyubiquitinated proteins. However, for the first time, mitochondrial defects have also been found to be a consequence of a mutation in a proteasomal gene (Mol. Biol. Cell 9 (1998) 2917-2931). Since the mutant strain is thermosensitive both on glucose and on glycerol, we searched for revertants in order to shed light on the Rpn11/Mpr1 functions. Spontaneous revertants able to grow on glucose but not on glycerol at 36 degrees C were isolated, and, only from them, revertants able to grow at 36 degrees C on glycerol were selected. Revertants of the two classes were found to be extragenic. The detailed characterization of these extragenic suppressors demonstrates that the phenotypes related to cell cycle defects can be dissociated from those concerned with mitochondrial organization.     

Functional analysis of Rpn6p, a lid component of the 26 S proteasome, using temperature-sensitive rpn6 mutants of the yeast Saccharomyces cerevisiae

Rpn6p is a component of the lid of the 26 S proteasome. We isolated and analyzed two temperature-sensitive rpn6 mutants in the yeast, Saccharomyces cerevisiae. Both mutants showed defects in protein degradation in vivo. However, the affinity-purified 26 S proteasome of the rpn6 mutants grown at the permissive temperature degraded polyubiquitinated Sic1p efficiently, even at a higher temperature. Interestingly, their enzyme activity was even higher at a higher temperature, indicating that once made mutant proteasomes are stable and have little defect in the proteolytic function. These results suggest that the deficiency in protein degradation observed in vivo is rather due to a defect in the assembly of a holoenzyme at the restrictive temperature. Indeed, both rpn6 mutants grown at the restrictive temperature were defective in assembling the 26 S proteasome. A striking feature of the rpn6 mutants at the restrictive temperature was that there appeared a protein complex composed of only four of the nine lid components, Rpn5p, Rpn8p, Rpn9p, and Rpn11p. Altogether, we conclude that Rpn6p is essential for the integrity/assembly of the lid in the sense that it is necessary for the incorporation of Rpn3p, Rpn7p, Rpn12p, and Sem1p (Rpn15p) into the lid, thereby playing an essential role in the proper function of the 26 S proteasome.     

Rpn7 Is required for the structural integrity of the 26 S proteasome of Saccharomyces cerevisiae

Rpn7 is one of the lid subunits of the 26 S proteasome regulatory particle. The RPN7 gene is known to be essential, but its function remains to be elucidated. To explore the function of Rpn7, we isolated and characterized temperature-sensitive rpn7 mutants. All of the rpn7 mutants obtained accumulated poly-ubiquitinated proteins when grown at the restrictive temperature. The N-end rule substrate (Ub-Arg-beta-galactosidase), the UFD pathway substrate (Ub-Pro-beta-galactosidase), and cell cycle regulators (Pds1 and Clb2) were found to be stabilized in experiments using one of the rpn7 mutants termed rpn7-3 at the restrictive temperature, indicating its defect in the ubiquitin-proteasome pathway. Subsequent analysis of the structure of the 26 S proteasome in rpn7-3 cells suggested that the defect was in the assembly of the 26 S holoenzyme. The most striking characteristic of the proteasome of the rpn7-3 mutant was that a lid subcomplex affinity-purified from the rpn7-3 cells grown at the restrictive temperature contained only 5 of the 8 lid components, a phenomenon that has not been reported in the previously isolated lid mutants. From these results, we concluded that Rpn7 is required for the integrity of the 26 S complex by establishing a correct lid structure.     

Bidirectional remodeling of β1-integrin adhesions during chemotropic regulation of nerve growth

Background

The proteasome is a multi-subunit protein machine that is the final destination for cellular proteins that have been marked for degradation via an ubiquitin (Ub) chain appendage. These ubiquitylated proteins either bind directly to the intrinsic proteasome ubiqutin chain receptors Rpn10, Rpn13, or Rpt5, or are shuttled to the proteasome by Rad23, Dsk2, or Ddi1. The latter proteins share an Ub association domain (UBA) for binding poly-Ub chains and an Ub-like-domain (UBL) for binding to the proteasome. It has been proposed that shuttling receptors dock on the proteasome via Rpn1, but the precise nature of the docking site remains poorly defined.

Results

To shed light on the recruitment of shuttling receptors to the proteasome, we performed both site-directed mutagenesis and genetic screening to identify mutations in Rpn1 that disrupt its binding to UBA-UBL proteins. Here we demonstrate that delivery of Ub conjugates and docking of Ddi1 (and to a lesser extent Dsk2) to the proteasome are strongly impaired by an aspartic acid to alanine point mutation in the highly-conserved D517 residue of Rpn1. Moreover, degradation of the Ddi1-dependent proteasome substrate, Ufo1, is blocked in rpn1-D517A yeast cells. By contrast, Rad23 recruitment to the proteasome is not affected by rpn1-D517A.

Conclusions

These studies provide insight into the mechanism by which the UBA-UBL protein Ddi1 is recruited to the proteasome to enable Ub-dependent degradation of its ligands. Our studies suggest that different UBA-UBL proteins are recruited to the proteasome by distinct mechanisms.     

Conformational Dynamics of the Rpt6 ATPase in Proteasome Assembly and Rpn14 Binding

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Highlights? Proteasome ATPase Rpt6 undergoes helix-coil exchange in its C-terminal domain ? Rpt6 G^360,387A has a stabilized four-helix bundle and raised melting temperature ? Assembly chaperone Rpn14 binds selectively to the four-helix bundle Rpt6 conformer ? Rpt6 G^360,387A (rpt6AA) is synthetically defective with an rpn14 null mutation     

Complementary roles for Rpn11 and Ubp6 in deubiquitination and proteolysis by the proteasome

Substrates destined for degradation by the 26 S proteasome are labeled with polyubiquitin chains. These chains can be dismantled by deubiquitinating enzymes (DUBs). A number of reports have identified different DUBs that can hydrolyze ubiquitin from substrates bound to the proteasome. We measured deubiquitination by both isolated lid and base-core particle subcomplexes, suggesting that at least two different DUBs are intrinsic components of 26 S proteasome holoenzymes. In agreement, we find that highly purified proteasomes contain both Rpn11 and Ubp6, situated within the lid and base subcomplexes, respectively. To study their relative contributions, we purified proteasomes from a mutant in the putative metalloprotease domain of Rpn11 and from a ubp6 null. Interestingly, in both preparations we observed slower deubiquitination rates, suggesting that Rpn11 and Ubp6 serve complementary roles. In accord, the double mutant is synthetically lethal. In contrast to WT proteasomes, proteasomes lacking the lid subcomplex or those purified from the rpn11 mutant are less sensitive to metal chelators, supporting the prediction that Rpn11 may be a metalloprotein. Treatment of proteasomes with ubiquitin-aldehyde or with cysteine modifiers also inhibited deubiquitination but simultaneously promoted degradation of a monoubiquitinated substrate along with the ubiquitin tag. Degradation is unique to 26 S proteasome holoenzymes; we could not detect degradation of a ubiquitinated protein by "lidless" proteasomes, although they were competent for deubiquitination. The fascinating observation that a single ubiquitin moiety is sufficient for targeting an otherwise stable substrate to proteasomes exposes how rapid deubiquitination of poorly ubiquitinated substrates may counteract degradation.     

A Mutation in a Novel Yeast Proteasomal Gene, RPN11/MPR1, Produces a Cell Cycle Arrest, Overreplication of Nuclear and Mitochondrial DNA, and an Altered Mitochondrial Morphology

We report here the functional characterization of an essential Saccharomyces cerevisiae gene, MPR1, coding for a regulatory proteasomal subunit for which the name Rpn11p has been proposed. For this study we made use of the mpr1-1 mutation that causes the following pleiotropic defects. At 24°C growth is delayed on glucose and impaired on glycerol, whereas no growth is seen at 36°C on either carbon source. Microscopic observation of cells growing on glucose at 24°C shows that most of them bear a large bud, whereas mitochondrial morphology is profoundly altered. A shift to the nonpermissive temperature produces aberrant elongated cell morphologies, whereas the nucleus fails to divide. Flow cytometry profiles after the shift to the nonpermissive temperature indicate overreplication of both nuclear and mitochondrial DNA. Consistently with the identification of Mpr1p with a proteasomal subunit, the mutation is complemented by the human POH1 proteasomal gene. Moreover, the mpr1-1 mutant grown to stationary phase accumulates ubiquitinated proteins. Localization of the Rpn11p/Mpr1p protein has been studied by green fluorescent protein fusion, and the fusion protein has been found to be mainly associated to cytoplasmic structures. For the first time, a proteasomal mutation has also revealed an associated mitochondrial phenotype. We actually showed, by the use of [rho°] cells derived from the mutant, that the increase in DNA content per cell is due in part to an increase in the amount of mitochondrial DNA. Moreover, microscopy of mpr1-1 cells grown on glucose showed that multiple punctate mitochondrial structures were present in place of the tubular network found in the wild-type strain. These data strongly suggest that mpr1-1 is a valuable tool with which to study the possible roles of proteasomal function in mitochondrial biogenesis.     

Pleiotropic defects caused by loss of the proteasome-interacting factors Rad23 and Rpn10 of Saccharomyces cerevisiae.

Rad23 is a member of a novel class of proteins that contain unprocessed ubiquitin-like (UbL) domains. We showed recently that a small fraction of Rad23 can form an interaction with the 26S proteasome. Similarly, a small fraction of Rpn10 is a component of the proteasome. Rpn10 can bind multiubiquitin chains in vitro, but genetic studies have not clarified its role in vivo. We report here that the loss of both Rad23 and Rpn10 results in pleiotropic defects that are not observed in either single mutant. rad23Delta rpn10Delta displays slow growth, cold sensitivity, and a pronounced G2/M phase delay, implicating overlapping roles for Rad23 and Rpn10. Although rad23Delta rpn10Delta displays similar sensitivity to DNA damage as a rad23Delta single mutant, deletion of RAD23 in rpn10Delta significantly increased sensitivity to canavanine, a phenotype associated with an rpn10Delta single mutant. A mutant Rad23 that is unable to bind the proteasome ((DeltaUbL)rad23) does not suppress the canavanine or cold-sensitive defects of rad23Delta rpn10Delta, demonstrating that Rad23/proteasome interaction is related to these effects. Finally, the accumulation of multiubiquitinated proteins and the stabilization of a specific proteolytic substrate in rad23Delta rpn10Delta suggest that proteasome function is altered.     

Evidence for Distinct Functions of MRE11 in Arabidopsis Meiosis

The evolutionary conserved Mre11/Rad50/Nbs1 complex functions as one of the guardians of genome integrity in eukaryotes; it is required for the double-strand break repair, meiosis, DNA checkpoint, and telomere maintenance. To better understand the role of the MRE11 gene in Arabidopsis, we performed comparative analysis of several mre11 alleles with respect to genome stability and meiosis. The mre11-4 and mre11-2 alleles presumably produce truncated MRE11 proteins composed of the first 499 and 529 amino acids, respectively. Although the putative MRE11 truncated proteins differ only by 30 amino acids, the mutants exhibited strikingly different phenotypes in regards to growth morphology, genome stability and meiosis. While the mre11-2 mutants are fully fertile and undergo normal meiosis, the mre11-4 plants are sterile due to aberrant repair of meiotic DNA breaks. Structural homology analysis suggests that the T-DNA insertion in the mre11-4 allele probably disrupted the putative RAD50 interaction and/or homodimerization domain, which is assumed to be preserved in mre11-2 allele. Intriguingly, introgression of the atm-2 mutant plant into the mre11-2 background renders the double mutant infertile, a phenotype not observed in either parent line. This data indicate that MRE11 partially compensates for ATM deficiency in meiosis of Arabidopsis.