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J. Biol. Chem., Vol. 282, Issue 48, 34735-34747, November 30, 2007
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1
From the
Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid 28040, Spain and the Department of Microbiology, Imperial College London, London SW7 2AZ, United Kingdom
Received for publication, August 13, 2007 , and in revised form, September 27, 2007.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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R) impairs formation of PacC27 and leads to persistence of PacC53. Wild-type PacC53 becomes multiply phosphorylated upon alkaline pH exposure. Processing-impaired 4KR PacC53 becomes excessively phosphorylated. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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B p50 is generated from its 105-kDa precursor by proteasome-mediated proteolytic processing (2). Since then, the list of substrates processed (i.e. partially proteolyzed to yield a functional form) by the proteasome has increased sufficiently to indicate that this mechanism is not uncommon. Additional, relevant examples are the Drosophila melanogaster zinc finger transcription factor Ci (3) and its metazoan homologue Gli3 (4, 5).
The molecular mechanisms mediating the proteolytic processing of NF-B p105 and Ci/Gli3 are remarkably complex. Proteins are usually (but not exclusively) targeted to the 26 S proteasome after polyubiquitination of one or more of their Lys residues (1). NF-B p105 is polyubiquitinated in different regions, which involves at least two ubiquitin ligases. Following signal-dependent phosphorylation of p105, its C-terminal domain recruits a multisubunit E3 through the WD40/F-box protein β-TrCP, promoting p105 ubiquitination and proteasome-mediated processing but additionally leading to its degradation (6, 7). Thus, the final output is determined by a delicate balance between complete degradation and processing (6, 8). Notably, a recent report demonstrates that NF-B p105 can be processed post-translationally by purified proteasomes in the absence of polyubiquitination, reflecting its intrinsic tendency to be targeted to the proteasome. It has also been reported that NF-B p105 is processed co-translationally (9). Finally, a glycine-rich repeat acting as a proteasome stop signal has been shown to play a key role in processing (8, 10–12) but is dispensable for NF-B p105 processing in yeast (13).
Ci, the transducer of the hedgehog signal, is synthesized as a 155-kDa precursor, which can be processed to a 75-kDa repressor. In the presence of hedgehog signaling, processing is prevented, and Ci155 translocates into the nucleus to activate hedgehog-responsive genes (3). The E3 component WD40/F-box protein Slimb (the fly orthologue of β-TrCP) binds phosphorylated target motifs in Ci to promote such processing (14, 15). Hedgehog signaling prevents Ci155 processing to Ci75 but promotes Ci155 conversion to a labile form of the precursor (16), reflecting the delicate balance between processing and degradation inherent to proteasome-mediated processing.
What determines whether a protein is processed rather than destroyed by the proteasome? Substrates need to be actively translocated into the catalytic chamber, and such translocation involves energy-dependent substrate unfolding (1). Matouschek and colleagues (12, 17) elegantly demonstrated that substrates are processively unraveled from degradation signals mediating substrate recognition by multichambered proteases and established the requirements for proteolytic processing by the proteasome. These include, in addition to a degradation signal targeting the substrate to the proteasome, the presence of a tightly folded (thus recalcitrant to unfolding) domain in the direction of proteasome movement that leads, when encountered by the proteasome, to release of a partially degraded substrate molecule (12). Release is favored by the presence of a low complexity sequence preceding the folded domain in the direction of proteasome movement (12). The NF-B p105 glycinerich region mentioned above is one such low complexity sequence (12). The N-terminally located Rel homology domain in NF-B p105 and the zinc finger region in Ci155 play the key role of the tightly folded domain in their respective proteins (12).
Previous work (18, 19) highlighted the similarities between the zinc finger regions of Ci/Gli and PacC, the Aspergillus nidulans transcription factor mediating regulation of gene expression by ambient pH in fungi (20, 21). 674-residue PacC contains an N-terminal DNA binding domain with three canonical C2H2 zinc fingers (22) and undergoes two-step proteolytic activation removing 
425 residues from the C terminus in response to alkaline ambient pH (22–24). The 72-kDa PacC72 translation product is converted to 53-kDa PacC53 by a "signaling" protease. PacC53 is the substrate of a second proteolytic processing reaction, which results in the 27-kDa final product PacC27 (Fig. 1A). The signaling protease is almost certainly the cysteine protease PalB (25, 26). The subject of this work is the identity of the "processing" protease mediating the second step.
PacC72 is held in a "closed" conformation, inaccessible to the processing protease, by intramolecular interactions involving three regions, denoted A, B, and C (schematically depicted in Fig. 1A). pH-responsive signaling protease cleavage removes interactive region C, thus disrupting intramolecular interactions and making the resulting C-terminally truncated product PacC53 accessible to the processing protease (27). Therefore, PacC53 meets one of the requirements for a proteasome substrate, with exposure of unstructured regions that the proteasome needs to initiate polypeptide translocation (28).
Indeed, several arguments strongly suggested the proteasome as the likely processing protease. (i) Extensive mutational analyses have failed to identify the PacC-processing protease, which would agree with the essential role of the proteasome. (ii) The processing protease is conserved in S. cerevisiae, despite the fact that the yeast PacC orthologue Rim101p undergoes a single step "signaling protease cleavage" proteolytic activation (29, 30). (iii) Resembling Ci155 processing, PacC processing is independent of sequence at the processing cleavage site (12, 31). (iv) The C terminus of PacC27 (32) (this work) is heterogeneous, as expected from the model of processing by the proteasome of Matouschek and co-workers (12). (v) The site(s) of processing is dictated by sequence or structure determinants that are remote from the processing site (32). (vi) A processing efficiency determinant has been mapped to a region located C-terminal to the processing site, which would be exposed after signaling protease cleavage (24). (vii) The zinc finger regions of PacC and Ci share distinctive structural features (18, 20), and indeed the C-terminal fingers (those that the proteasome would first encounter) share, in addition to the zinc finger fold, high amino acid sequence identity (19).
Thus, we explored the possible role of the proteasome in PacC processing by exploiting the amenability of S. cerevisiae and A. nidulans to classical and reverse genetic manipulation. Our data demonstrating that partial loss-of-function mutations affecting the active site of the orthologous proteasome β5 subunits Pre2p/PreB characteristically alter the specificity of the processing reaction provide strong evidence that the proteasome is indeed the PacC-processing protease.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Media for A. nidulans Adjusted to Different Ambient pH Conditions—Cultures for protein extraction and Western analysis were made in Aspergillus fermentation medium as described (26). In pH shift experiments, the pH of the cultures before shifting mycelia to alkaline conditions (pH 8.4) was 4.3. Overnight (14–16-h) cultures in relatively uniform pH conditions were made after adding 100 mM NaH2PO4 plus 100 mM NaCl for acidic conditions (starting and final pH values were 5.5 and 5.1–5.4, respectively) and 100 mM Na2HPO4 for alkaline conditions (starting and final pH values were 8.0 and 7.2–7.4, respectively). Harvesting of mycelia and protein extraction have been described (26).
Yeast Techniques—S. cerevisiae strains with active site missense mutations pre3T20A, pup1T30A, pre2K108A, pre2K108R, and pre2T76S single mutations as well as pre3T20Apup1T30A and pre2K108Rpre3T20A double mutations in genes encoding the proteasome proteolytic subunits (36) were isogenic to the WCG4a strain (MATa leu2–3,112 ura3-
5 his3–11,15 canS GAL2 and were obtained from Prof. Dr. D. Wolf (University of Stuttgart, Germany). pRS416-Myc3-PacC14 was made by inserting a pacC cDNA fragment encoding PacC residues 5–492 (thereby equivalent to the pacCc14 product) with three copies of the Myc epitope fused to its N terminus into pRS416, using HindIII and BamHI restriction sites. Strains were transformed with pRS416-Myc3-PacC14 following a LiAc-based procedure (37) and cultured in minus uracil-selective YM4 minimal medium. Whole cell yeast samples for immunoblotting were prepared from 4 A600 units of yeast in log phase following an alkaline lysis procedure as described by Stimpson et al. (38).
Western Blot Analyses—50 µgof A. nidulans protein per sample (as determined by the Bradford assay) were used unless otherwise indicated. Myc3-PacC, NimE-GFP, and actin were detected with the anti-c-MYC mouse monoclonal antibody 9E10 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 1:1,000, a mouse monoclonal antibody anti-GFP mixture (clones 7.1 and 13.1; Roche Applied Science) at 1:1,000, and a mouse anti-actin monoclonal antibody (clone C4; ICN Biomedicals Inc.) at 1:25,000, respectively. Peroxidase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories) was used as secondary antibody at a 1:4,000 dilution. Peroxidase activity was detected using Amersham Biosciences ECL. If required, chemiluminiscence was quantified using a TDI 06-LAS3000Q Chemiluminiscence Image Analyzer with Fujifilm Multi Gauge software.
pacC Gene Replacements—A. nidulans pacC mutations were all constructed by gene replacement using plasmid pSpacC900 as described (26) with the correction that the triple MYC epitope and linker sequence preceding the major PacC initiator Met is MAEQKLISEEDLNGEQKLISEEDLNGEQKLISEEDLNGT. Missense and deletion mutations were introduced using Stratagene's QuikChange methodology. MAD265 (yA2 pabaA1 biA1 pyrG89 pacC::pyr-4 argB2 was used as recipient strain. A. nidulans transformants were selected in the presence of 12.5–25 mM sodium molybdate. Those carrying the expected gene replacement were identified by Southern analysis and meiotically crossed to remove the pyrG89 mutation as described (26). Myc-tagged pacC alleles segregating in crosses were additionally genotyped by PCR. The gene-replaced pacC alleles used in this work are listed in Table 1.
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preB Gene Replacements—A genomic DNA fragment containing 0.35 kb of flanking DNA on either side of the preB gene was PCR-amplified (with Pfu polymerase) and cloned in pBS-SK+ using NotI and SpeI ends. A BglII fragment containing the Aspergillus fumigatus pyrG gene was inserted downstream of preB, followed by a 1.5-kb fragment corresponding to preB 3'-untranslated region. Mutant versions encoding PreBK101A and PreBK101R were constructed using mutagenic PCR with Stratagene's QuikChange and appropriate oligonucleotides. 5,525-bp NotI-ClaI preB-pyrGfum-3'-untranslated regionpreB wild-type or mutant DNA fragments were used to transform the nonhomologous end-joining pathway-deficient A. nidulans pyroA4 pyrG89 nkuA::argB+ argB2 strain by selecting transformants on pyrimidine-deficient Aspergillus complete medium (39). Primary transformants were purified on the same medium by mycelial transfer (to ensure heterokaryon recovery) before proceeding to homokaryon purification from conidiospores as described (40) (also see "Results"). preB2 and preB0 homokaryosis were confirmed by Southern blot analysis and by direct sequencing of the preB2 mutation following PCR amplification.
Assessment of Proteasome Proteolytic Activities—Mycelia were harvested by filtration, washed, frozen in liquid nitrogen, and lyophilized. Lyophilized cells were broken using a Fast Prep cell disruptor FP120 and a 0.5-mm ceramic bead with a 10-s pulse at a setting of 4. The resulting powder was resuspended in 0.5 ml of Tris-HCl, pH 8.2, 2 mM ATP, 5 mM MgCl2, 1 mM dithiothreitol, and 20% glycerol (v/v). 0.7 ml of 0.45-mm glass beads was added to the suspension, which was shaken for 20 s at a setting of 6 in the Fast Prep. Crude extracts were clarified by centrifugation for 30 min at 16,000 x g and 4 °C. Proteasome proteolytic activities were determined using these crude extracts by monitoring cleavage of fluorogenic peptide substrates essentially as described (41, 42), using N-succinyl-LLVY-7-amido-4-methylcoumarin (N-succinyl-LLVY-AMC), t-butoxylcarbonyl-LRR-AMC (Boc-LRR-AMC), and benzyloxycarbonyl-LLE-AMC (Z-LLE-AMC) as substrates for chymotrypsin, trypsin, and PGPH activities, respectively. Fluorescence readings of released AMC were made using 380 and 460 nm excitation and emission wavelengths, respectively, and were converted to pmol of AMC by using a standard curve.
Bacteriophage 
-Phosphatase Treatment—Treatment with -phosphatase was carried out using 100 µg of protein with incubation for 30 min at 37 °C in 100-µl reaction mixtures in -phosphatase buffer (New England Biolabs) with 2 mM MnCl2 and 400 milliunits of -phosphatase (Biolabs, Northbrook, IL). Where indicated, 10 mM Na3VO4 was used as phosphatase inhibitor. Mock incubations were carried out in the absence of enzyme and inhibitor.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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252–254, is heterogeneous. Using pacC900 in Western blots (Fig. 1B), we reproducibly detected four different C termini for PacC27, corresponding to polypeptides that we denoted a–d (Fig. 1B). Predominating forms a and b seemingly correspond to the major form(s) of PacC27 detected in electrophoretic mobility shift assays and thus differ in 5 residues (see Fig. 3B in Ref. 32). PacC72 and PacC53 also showed some heterogeneity (Fig. 1; see below). Processing Specificity of PacC Is Altered in a Class of S. cerevisiae Proteasome Active Site Mutants—Data from S. cerevisiae showed that proteasome inhibitors such as MG132 are inefficiently taken up by fungi (43), a problem that would be exacerbated in A. nidulans, because its greater metabolic versatility compared with yeast would increase the likelihood that these compounds are catabolized, further enhancing their ineffectiveness in vivo.
To address the possible role of the proteasome in generating PacC27, we initially followed a transgenic approach using S. cerevisiae, taking advantage of a mutant PacC with a nonsense mutation in codon 493 (the product of the pacCc14 allele). This C-terminally truncated version of PacC resembles PacC53 (whose C terminus lies within residues 493–500) and is therefore processed to PacC27 in an ambient pH-independent manner (23, 24). The pacCc14 product is processed ex vivo in S. cerevisiae (32), allowing us to use extant viable mutants impairing the yeast proteasome, following the approach reported by the Maniatis group for their analysis of proteolytic processing of NF-B p105 (2, 13).
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-subunits (44). Since and β subunits contribute to the overall architecture of the CP, deletion of any single subunit in the 20 S CP, with the single exception of Pre9p, is lethal. Three of the seven S. cerevisiae β-subunits, denoted β1/Pre3p (peptidylglutamyl-peptide hydrolytic), β2/Pup1p (trypsin-like), and β5/Pre2p (chymotrypsin-like), have endoproteolytic active site Thr residues, sequestered within the catalytic chamber formed by β-rings. Catalytic Thr residues are N-terminally exposed after proteolytic processing of proprotein precursors that takes place after proteasome assembly (44, 45). Proteolytic subunits can be mutationally inactivated by substituting Ala for the catalytic Thr residue (Thr30 in β2/Pup1p, Thr20 in β1/Pre3p, and Thr76 in β5/Pre2p proteolytic subunit precursors). Although missense mutations leading to Pup1p T30A and Pre3p T20A substitutions, either singly or in combination, are viable, T76A substitution in Pre2p is lethal (36). However, Pre2p activity can be markedly impaired by introducing substitutions involving Lys108 (corresponding to Lys33 in the mature protein), which critically contributes to the active site (36, 45), with K108A causing a more extreme phenotype than K108R (36).
We constructed a plasmid driving expression of Myc3-PacC-(5–492) in S. cerevisiae and used a panel of yeast proteasome mutants to analyze PacC53 processing (Fig. 2). In the wild type, we detected a prominent band corresponding to Myc3-PacC-(5–492) and a processed band with the mobility of PacC27 (Fig. 2, lanes 2 and 10; an A. nidulans extract containing PacC53 and PacC27 was loaded as control in lane 1). Pre3p T20A and Pup1p T30A substitutions inactivating the PGPH- and trypsin-like proteolytic centers, respectively, had no significant effect on Myc3-PacC (5–492) processing (Fig. 2, compare lanes 3 and 4 with lanes 2 and 10). The double mutant (Fig. 2, lane 5) had no effect either. In marked contrast, substitutions involving Pre2p Lys108 had a marked effect. In single mutants, both Pre2p K108A and K108R led to a distinctive pattern of misprocessing characterized by the appearance of two abnormally high mobility forms of PacC27 (denoted β and 
in Fig. 2), apparently at the expense of the major wild-type form (indicated by ). These abnormal β and forms were absent in the Pre2p T76S mutant (Fig. 2, lane 8) (Thr76 is the N-terminal, catalytic residue in the mature subunit). Although Pre2p substitutions involving Lys108 lead to markedly deficient chymotrypsin-like activity, Ser functionally substitutes for Thr in the N terminus of mature Pre2p (36). Thus, these data strongly suggest that PacC27 originates from proteasome-mediated processing of Myc3-PacC-(5–492) and that Pre2p plays a key role in determining the actual size (i.e. the C terminus) of the processed product, since impairing the Pre2p catalytic activity shifts the position(s) of the scissile peptide bond(s). This would be consistent with previous data (and our working model) according to which the position of the scissile bond(s) would be determined by their relative distance from an N-terminal sequence or structure determinant. In agreement, the double pre3T20A pre2K108R mutation led to the characteristic misprocessing resulting from Pre2pK108R but additionally led to a marked reduction of form , indicating that processing to this polypeptide involves the proteolytic activity of Pre3p (Fig. 2, lanes 6 and 9, representing two independent clones).
A preB Mutation Resulting in K101A Substitution Is Lethal—In view of the above results, we set out to obtain loss-of-function mutations in the A. nidulans gene encoding the proteasome β5 subunit. We identified chromosome II-located AN3932.3 (available on the World Wide Web) as the PRE2 orthologue, which we denoted preB, and confirmed its predicted exon-intron structure by cDNA sequencing (supplementary Fig. S1). preB encodes a 296-residue polypeptide showing high amino acid sequence identity with Pre2p (supplementary Fig. S2; similarity is lost in the region of the propeptide, which is released upon proteolytic activation). As expected, key residues involved in proteolysis are invariant. The equivalents in the PreB proenzyme of yeast Pre2p Thr76 (the N-terminal catalytic Thr after proenzyme proteolytic activation) and Lys108 (Lys33 in the mature protein) are Thr69 and Lys101, respectively.
In S. cerevisiae Pre2p, K108A substitution has a more extreme phenotype than K108R (36, 45). We constructed preB alleles encoding mutant proteins carrying the corresponding single residue substitutions by gene replacement (supplementary Fig. S1), using an nkuA recipient strain, deficient in the nonhomologous end-joining recombination pathway (46). Our preB gene replacement protocol uses transforming linear fragments carrying A. fumigatus pyrG as a selective marker (pyrG encodes orotidine 5'-phosphate decarboxylase) and involves a double recombination event. One cross-over must occur at the preB 3'-untranslated region to ensure pyrG integration into the genome. In contrast, the second cross-over can take place either upstream (leading to a replacement by the mutant allele) or downstream (leading to a wild-type allele) of the mutated preB Lys101 codon (supplementary Fig. S1). We performed three separate transformation experiments with preB1K101A, preB2K101R, and wild-type preB0 alleles, respectively.
Transformants obtained with the preB0 (i.e. preB+) control grew normally on minus-pyrimidine medium and were phenotypically stable upon subsequent subculturing. They carried the expected recombination event, as determined by Southern blot hybridization and diagnostic PCR (data not shown). Although the replaced wild-type allele was denoted preB0 to underscore that, unlike the "true" wild type, it contains A. fumigatus pyrG downstream of the preB open reading frame, the preB0 and preB+ strains were shown to be phenotypically indistinguishable (see below). In contrast, two classes were noticeable among preBK101A and preBK101R transformants (supplemental Fig. S1). Transformants in one class resembled those in the single preB0 class and were shown to contain the wild-type preB gene. Transformants in the second class grew slowly (preB2K101R) or very slowly (preB1K101A) compared with the wild type. Slowly growing colonies were shown to be heterokaryons whose cells contain recipient (preB+, pyrimidine-requiring) and transformed (preB1K101A or preB2K101R, pyrimidine-nonrequiring) nuclei.
A. nidulans conidiospores contain a single nucleus and thus the heterokaryotic state cannot propagate through conidia. In contrast to preB0 or preB2K101R transformants, conidiospores corresponding to slowly growing preBK101A transformants were able to form colonies only when plated on medium containing pyrimidines (i.e. under conditions supporting growth of the recipient strain) but not on selective medium lacking pyrimidines (supplemental Fig. S1), suggesting that preBK101A::pyrGfum conidia were not viable (40).
To characterize the terminal phenotype of the preBK101A:: pyrGfum allele, we examined the ability of conidiospores developed from heterokaryotic colonies to germinate. Control preB0 conidia were fully germinated after 24 h irrespective of the presence or absence of exogenous pyrimidines. In contrast, conidia from a strain carrying preBK101A::pyrGfum in heterokaryosis were indistinguishable from the pyrG89 control in that they formed hyphae in synthetic medium supplemented with pyrimidines but did not give rise to germ tubes after 24 or 72 h at 25 °C in the absence of pyrimidine supplementation (supplemental Fig. S3). The absence of the expected class of mutant preB conidiospores that would be unable to germinate irrespective of the presence or absence of pyrimidines strongly suggests that a strain carrying preBK101A::pyrGfum in heterokaryosis does not give rise to viable preBK101A::pyrGfum conidia, possibly because this mutation abrogates conidiospore development. (In A. nidulans, conidiospore development involves singly nucleated cells from its very early stages (47).) In any case, these data demonstrate that the preBK101A::pyrGfum allele is lethal and can only be maintained in heterokaryosis.
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Enzyme assays using fluorogenic peptide substrates specific for each of the activities of the proteasome demonstrated that preB2 mutant extracts were markedly deficient in proteasomal chymotryptic activity, which was determined to be 20% of that in the isogenic preB0 wild type (Fig. 3A). In contrast, their trypsin and PGPH activities are possibly somewhat increased (Fig. 3A), resembling the situation reported for pre2K108R mutants in S. cerevisiae (36).
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preB2 Results in a Marked Decrease in the Steady State Levels of PacC and Affects the Fidelity of Processing to PacC27—To address the involvement of the proteasome in PacC processing, we crossed pacC900 into the preB2 mutant background. Unexpectedly, PacC was barely detectable in preB2 extracts when equal amounts of wild-type and preB2 protein extracts were analyzed by Western blotting (Fig. 4A). Detection of PacC processing at equal sensitivity required a considerable increase in the amount of mutant preB2 protein extracts loaded on Western blots (Fig. 4B). Digital and quantitative chemiluminiscence analyses confirmed that the steady state of PacC in the mutant is 3–5 times lower than in the wild type (not shown). Since Northern blot analyses demonstrated that preB2 does not result in a significant reduction in pacC transcription (supplemental Fig. S6), these experiments suggest that preB2 leads to marked destabilization of PacC.
Western analyses of PacC in mutant preB2 cells revealed that the kinetics of the two-step processing of PacC72 to PacC53 and PacC27 were not greatly affected (Fig. 4B). However, a closer inspection of the PacC processing pattern revealed two striking effects of preB2 on the processing protease reaction. One was the abnormally high levels of PacC27 relative to PacC72 seen under acidic pH conditions, under which processing is barely detectable in preB+ cells (Fig. 4, A and B). However, the most conspicuous effect of preB2 is on the fidelity of the processing protease reaction. As noted above, PacC27 is heterogeneous and involves four major polypeptides, denoted a, b, c, and d in decreasing size order (Fig. 1B). In the wild type, PacC27b largely predominates, PacC27a follows in abundance, and forms c and d are very minor (Fig. 4C). In marked contrast, this highly reproducible pattern is altered by preB2, such that c and d become very prominent under persistent alkaline ambient pH conditions, apparently at the expense of PacC27a and b (Fig. 4C). The effects on the fidelity of processing that we observed in the A. nidulans preB2 mutant are markedly similar to those seen in the corresponding S. cerevisiae pre2K108R mutant, although the actual electrophoretic mobilities of the minor bands increased in prominence are clearly different in yeast and Aspergillus (Fig. 4D). The fact that the fidelity of PacC processing is altered by a single residue change affecting the active site of the A. nidulans proteasome β5 subunit constitutes evidence that PacC is a direct substrate of the proteasome and that this is indeed the processing protease.
pacC910 Rescues the Low Levels of PacC Resulting from the preB2 Mutation by Favoring Its Proteolytic Processing—Previous work established that residues 266–407 contain a processing efficiency determinant (PED). We constructed by gene replacement pacC910, a Myc-tagged version of pacC10 encoding a PacC mutant deleted for the PED (24) for comparison with pacC900. In agreement with our published data using electrophoretic mobility shift assays, a major proportion of PacC910 from acidic ambient pH conditions is detected as the (mutant) primary translation product, with a minor proportion seen as PacC27 (Fig. 5A, lane 1). Since the deleted region 266–407 includes the nearly entire interactive region B (including critical Leu340; see also below) and part of region A, PacC910 must be in the open conformation and thus accessible to the processing protease under any ambient pH condition (24, 27). However, since deletion of the PED markedly reduces processing, the primary PacC910 translation product predominates under acidic conditions. Upon shifting cells to alkaline conditions, PacC910 is converted to mutant PacC53, which, unlike the situation in the wild type, remains stable over time and is not converted to PacC27 (24) (Fig. 5A). Notably, PacC910 protein levels were 4–6 times higher than in the wild type (data not shown) (Fig. 5A, compare lanes 1–7 with lane 8). This is attributable, at least in part, to elevated levels of pacC transcription in the mutant, as determined by Northern analyses (supplemental Fig. S6).
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3-fold increase in overall PacC levels in the preB2 background (Fig. 5B) (data not shown). However, unlike the situation in the preB+ background, mutant (lacking the PED) PacC91053 accumulates 30 min after the shift but becomes barely detectable at later time points, apparently without conversion to PacC27 (Fig. 5B). Most notably, in stark contrast with the pacC910 single mutant, where the full-length PacC910 largely predominates under acidic conditions (Fig. 5A, lane 1), PacC27 is the almost exclusive PacC form in pacC910 preB2 cells and the one that accounts for the elevated levels relative to the pacC900 preB2 single mutant (Fig. 5B). pacC910 preB2 cells show the typical preB2 PacC27 pattern characterized by conspicuous high mobility bands PacC27c and PacC27d (Fig. 5B). From experiments in Figs. 4 and 5 we conclude that deletion of the PED largely precludes PacC processing in the context of the wild-type proteasome, whereas in the context of the preB2 proteasome active site mutant, deletion of the PED favors a processing pathway apparently at the expense of a degradative pathway. We therefore conclude that the proteasome is the PacC-processing protease.
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Alkalinity-mimicking pacCc gain-of-function mutations result in pH-independent processing to PacC27, because their mutant products are synthesized in the processing protease-accessible conformation (23, 24, 32) (Fig. 6B, preB+ lanes). Notably, preB2 did not prevent processing of the pacCc14900 and pacCc69900 products to PacC27 but led to the characteristically altered PacC27 pattern typical of preB2 mutants and to a 4-fold reduction in the levels of PacC, resembling the situation with pacC910 (Fig. 6B, note the different loadings in preB+ and preB2 lanes). (We used here cells continuously cultured under moderately acidic (where a small but detectable amount of signaling occurs) or alkaline conditions rather than the pH shift protocol, since pacCc14900 and pacCc69900 result in PacC processing at any ambient pH.) The increase in PacC27 levels that takes place in preB2 pacC910, preB2 pacCc14900, and preB2 pacCc69900 double mutants relative to the single preB2 mutant, which is most noticeable under pH 5.5 conditions (Fig. 6B, preB2 lanes), possibly underlies their increased tolerance to molybdate.
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Deletion Analysis of a Gly-Ala-Prorich Sequence within the PED—Our attempts to delimit further the PED by deletion analysis were largely unsuccessful, possibly indicating that it includes different regions, each contributing partially to the efficiency of the processing protease reaction in addition to their role in the intramolecular PacC interactions (data not shown). However, one interesting conclusion came from deletion of a region within the A. nidulans PacC PED, including residues 296–325, which is very rich in Pro, Ala, and Gly (Fig. 7A). In other fungal homologues, this region is markedly rich in Gly residues (27). A Gly-rich region has been proposed as a stop signal preventing the complete degradation by the proteasome of the NF-B p105 processed products p50 and p52 (8, 10–12). It has also been reported that Gly-Ala repeats impair proper substrate unfolding by the proteasome (49). To address the possible role of this region, we constructed pacC913, encoding a Myc-tagged PacC deleted for residues 296–325. In pH shift experiments, PacC913296–325 appears unstable upon shifting cells to alkaline conditions (Fig. 7B). In agreement, pacC913 cells cultured under moderately acidic or alkaline conditions showed markedly reduced levels of PacC compared with wild type (Fig. 7C). Thus, deletion of this Gly-Ala-Pro-rich sequence appears to be required for normal stability of PacC under conditions activating the pH signaling pathway.
Substitution of Lys Residues in the PED and an Adjacent Region by Arg Largely Prevent Processing Proteolysis—One feature that the PED could potentially provide to promote efficient PacC processing would be one or more Lys residues whose ubiquitination targets the PacC substrate to the proteasome. The 266–407 region contains potentially ubiquitinable Lys267 and Lys337 residues. Lys267 belongs to a putative bipartite nuclear localization sequence composed of two triplets of basic residues, which additionally include Lys252 and Lys253 (Fig. 8A). Simultaneous substitution of all six basic residues in this bipartite sequence by Ala (pacC906; Table 1) demonstrated that none of them is involved in PacC processing (data not shown). Thus, we constructed the gene-replaced allele pacC901 encoding a mutant PacC carrying the conservative yet nonubiquitinable K337R substitution and found that this mutant protein was converted to PacC27 slightly less efficiently than the wild type (data not shown). We thus considered the possibility that Lys267 could potentially compensate for the absence of Lys337 and constructed pacC902 encoding a Myc-tagged PacC carrying the double K267R/K337R substitution. Indeed, this double substitution significantly impairs PacC processing (Fig. 8B). This prompted us to test the effect in processing of K252R/K253R substitution alone (pacC903) and in combination with the double K267R/K337R substitution (pacC904). In agreement with the above Ala-scanning mutagenesis data, K252R/K253R had no significant effect on processing (Fig. 8B). In marked contrast, the quadruple Lys to Arg substitution largely prevented processing in pH shift experiments (Fig. 8C) and resulted in detectable mutant PacC53 accumulation and a marked reduction in the steady state level of PacC27 in cells from overnight cultures under alkaline conditions (Fig. 8D).
It is notable that no increase in either PacC72 or PacC53 commensurate with the marked decrease in PacC27 resulting from pacC904 was detected. Since pacC900 and pacC904 show no evident difference in their transcriptional response to alkaline pH (as determined by Northern blots),3 this suggests that the mutant forms might be unstable, although this possibility has not been addressed here. In summary, the negative, additive effect on processing of Arg substitutions involving four Lys residues located within or adjacent to the PED would agree with (although it does not unequivocally demonstrate) a requirement for their ubiquitination in PacC processing, with Lys337 playing the most prominent role.
PacC72 can be phosphorylated (50). The aggregate electrophoretic mobilities of wild-type pacC900 and mutant pacC904 PacC53 decrease over time with exposure to alkaline conditions (Fig. 8C). They consist of several closely spaced bands, with the lower mobility PacC53 bands predominating at late time points, apparently at the expense of faster mobility bands prominent at 15 min (Fig. 8E, untreated lanes). -Phosphatase treatment shifted the mobility of all relatively slow bands toward the fastest mobility band, demonstrating that they all result from phosphorylation (Fig. 8, E and F). (Note that acquisition of negative phosphate group charges results in reduced electrophoretic mobility). The multiple -phosphatase-sensitive PacC53 bands predominating at late time points (Fig. 8E, untreated lanes) therefore result from multiple phosphorylation events on the same molecule.
An even lower mobility PacC53 band, very prominent at late time points in the pacC904 mutant but not in the wild type (Fig. 8F, corresponding to 60 min time points; this band is indicated by arrows), clearly contributes to the relatively lower PacC53 aggregate electrophoretic mobility of the mutant (Fig. 8C), suggesting that the mutant PacC53 is excessively phosphorylated. Sensitivity of this band to -phosphatase treatment (Fig. 8F) shows that it indeed results from PacC53 phosphorylation. We conclude that wild-type PacC53 is phosphorylated and that markedly reduced PacC53 processing resulting from pacC904 correlates with excessive phosphorylation of the quadruple Lys-to-Arg-substituted mutant PacC53.
The Quadruple Lys to Arg Substitution Impairs Conversion of PacC53 into PacC27—pacCc14900 (see above) encodes a Myc-tagged gene-replaced version of pacCc14 (see above). As reported previously, PacC14900 is efficiently processed to PacC27 irrespective of ambient pH (26), and indeed no primary translation product was detectable in protein extracts from cells cultured under either acidic or alkaline conditions (Figs. 6 and 8D). In contrast, the quadruple Lys to Arg substitution led to detection of the truncated PacC14904 primary translation product (Fig. 8D), strongly suggesting that it impairs PacC53 conversion into PacC27.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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We used a genetic approach to obtain evidence that PacC27 results from proteasome-mediated processing. In S. cerevisiae, substitutions of Lys108 (Lys33 in the mature Pre2p subunit) by Ala or Arg shift about two-thirds of the major processed form toward alternative PacC27 products shortened at their C termini. This effect is specific for Pre2p, since inactivation of the catalytic Thr residues in Pre3p and Pup1p (accounting for the PGPH and trypsin activities of the proteasome, respectively) have no effect and because substitution of Pre2p N-terminal Thr by Ser, which reduces the ability of the proteasome to degrade proteins (51), also reduces the efficiency of processing to PacC27. Involvement of other proteasome subunits in Pre2pK108R shifting of the site of proteolysis to other scissile bonds is likely, because one such abnormal cleavage is impaired by additional inactivation of Pre3p. A. nidulans PreB Lys101 is the equivalent of yeast Pre2p Lys108. We demonstrate that preB1 (K101A) is lethal, but preB2 (K101R) resembles its yeast counterpart in that it shifts the specificity of PacC processing toward otherwise minor cleavage sites, leading to a processing pattern that is remarkably reminiscent of that resulting from the corresponding yeast pre2K108R allele. Thus, equivalent active site mutations in S. cerevisiae and A. nidulans PRE2/preB orthologues leading to partial loss of proteasome chymotryptic activity do not prevent processing but shift specificity from the major processing site toward normally minor (in A. nidulans) or barely detectable (in S. cerevisiae) sites, which implies that PacC is a direct substrate of the proteasome.
Substrates are recruited to the proteasome by polyubiquitin-dependent or -independent signals. Productive engagement additionally requires the presence of an unstructured region in the substrate where the protease begins unfolding (28, 52). Unfolding is required for transfer into the catalytic chamber and precedes degradation, which proceeds sequentially from this initiation site (12, 17, 28, 49, 53). Processing, rather than complete degradation, results from release of partially proteolyzed substrates when the proteasome encounters a stop transfer signal involving a tightly folded domain, generally preceded by a low complexity sequence (12, 49, 53). Our results can be rationalized in the context of this model and the lack of sequence specificity of the proteolytic activities of the proteasome (44). Since the processed form PacC27 retains the N-terminal zinc-finger domain, proteolysis must proceed toward the N terminus, and the actual peptide bonds accessible to the active sites of the proteolytic β subunits will be dictated by their relative distance from the zinc finger domain. In the context of the wild-type proteasome, at least four bonds are scissile, although cleavage of peptide bonds involving the C-terminal residue in PacC27b and, to a lesser extent, that in PacC27ais strongly favored. Impairment of PreB markedly shifts preference from major to minor cleavage sites PacC27c and PacC27d, in striking similarity to the situation in yeast, suggesting that PacC27a and -b are attributable to the chymotryptic activity. In conjunction with yeast data, this additionally suggests that PreB impairment increases accessibility to the active sites of other proteolytic β subunits.
Proteasome Active Site Mutations in A. nidulans—preB1 (K101A) and preB2 (K101R) are the first reported active site proteasome mutations in A. nidulans (although a ts mutation in the gene encoding an outer ring -subunit has been described (54)). preB1K101A results in inviability, which demonstrates that, as expected, the proteasome is essential. preB2K101R allows viability and results in deficient proteasome chymotryptic activity. In S. cerevisiae, substitution by Ala of the corresponding Lys108 is viable but impairs growth to a greater extent than substitution by Arg, possibly as a result of impaired proteasome maturation. Since propeptide autoprocessing is coupled to proteasome assembly (36, 45, 55), this difference appears to reflect that β5K108R is fully autoprocessed, albeit less efficiently, whereas β5K108A is only partially autoprocessed (45). Hence, that preB1K101A results in lethality, whereas preB2K101R allows viability is unsurprising.
The preB2 Proteasome Results in a Marked Reduction in PacC Levels—Steady-state levels of the wild type and the three tested mutant PacC proteins were markedly reduced in preB2 strains (Figs. 4, 5, 6). This was unexpected, because preB2 results in elevated levels of a B-type mitotic cyclin, as expected from partially deficient proteasome-mediated proteolysis.
Since preB2 does not significantly reduce pacC transcription in wild type (nor, incidentally, in pacC910 cells) (supplementary Fig. S6), the reduced PacC levels due to the proteasome mutation in the wild-type (pacC+) and in three different pacC mutant strains are unlikely to be due to reduced synthesis. Hence, assuming that steady-state levels represent, at least in part, an assessment of stability, PacC appears unstable in preB2 cells. Two highly speculative, not mutually exclusive and, as yet, untested possibilities could explain this. One would be that cleavage events leading to PacC27a and -b, impaired by preB2, would facilitate release of the polypeptide chain from the proteasome, whereas cleavage by the other catalytic β-subunits would favor degradation. In the second, PreBK101R would lead to structural changes in the proteasome affecting accessibility and/or orientation of the PacC substrate to/at the catalytic chamber. The second possibility is supported by the growth defect of S. cerevisiae Pre2pK108R mutants, which probably results from impaired proteasome maturation (36, 45, 55). A. nidulans preB2 leads to marked cryosensitivity (supplementary Fig. S5), which would be consistent with a partial defect in the assembly of the proteasome complex.
The occurrence of four relatively close cleavage sites at the C terminus of PacC27 most likely reflects the stochastic nature of the processing reaction, where the processing site depends on a distance dictated by a stably folded domain. The inability of the proteasome to unfold this domain cannot be absolute, and one unwanted lateral effect of proteasome-mediated processing is, inevitably, some complete degradation. In a model substrate, about one-third of the proteasome-mediated proteolytic events give rise to a processing product rather than to complete degradation (49). Hence, it seems plausible that the preB2 proteasome mutation showing altered specificity also alters the efficiency of processing to the benefit of degradation, particularly if it leads to changes in the orientation of the PacC substrate within the proteasome CP.
pacC910 and the Role of the Processing Efficiency Determinant—Additional support for a direct role of the proteasome in PacC processing came from the finding that the processing-impairing pacC910 mutation significantly ameliorates the low steady state levels of PacC resulting from preB2, but paradoxically, it does so by increasing levels of PacC27. Hence, PacC910 is recalcitrant to the processing protease in preB+ cells but leads to relatively elevated levels of the processed PacC27 product(s) in preB2 cells. Since the pattern of cleavage sites of these products is characteristic of preB2 mutants, these results are strong evidence that the proteasome is the processing protease.
Conversion of pacC910 PacC53, lacking the PED, to PacC27 is blocked both in preB+ and in preB2 backgrounds (Fig. 5). Thus, the PED contains one or more essential elements for PacC53 processing to PacC27. These might include a recruitment signal and a loosely folded domain, where the proteasome is engaged. Interactive regions A and B within the PED (Fig. 1A) are most likely unstructured in PacC53 and would provide a suitable domain(s) for productive proteasome engagement (28, 52). Proteins are usually recruited to the proteasome after their polyubiquitination (1). Thus, we investigated the role in processing of Lys337 and Lys267 within the PED. A quadruple Lys-to-Arg substitution involving these residues plus otherwise processing-silent Lys252 and Lys253 largely prevents processing to PacC27, which would agree with a hypothetical role of these Lys residues in ubiquitin-mediated recruitme
