Genetic Interaction of doa4Δ and doa3-1
As a first step toward evaluating whether Doa4 can associate with the proteasome, we investigated the effects of combining a deletion of the nonessential DOA4
gene with a partial loss-of-function mutation in one of the essential 20S proteasome subunit genes, DOA3
(Chen and Hochstrasser, 1995
). If Doa4 functions in the proteasomal pathway, then mutant doa3-1
cells, in which the catalytic core of the proteasome is compromised, might be sensitized to loss of the deubiquitinating enzyme and vice versa. We have found that doa4Δ
spores arising from a sporulated doa4Δ
heterozygous diploid have a spore viability/germination defect that becomes more severe with extended incubation under sporulation conditions. Sporulation for 3 d at 30°C resulted in ~30% of doa4Δ
spores failing to germinate. Strikingly, with the doa3-1
/+ double heterozygote, which sporulated normally, no germination of doa4Δ
spores was observed even when the spore carried a wild-type DOA3
gene (nine full tetrads). This unusual germination defect was circumvented if the double heterozygote carried a low-copy DOA4
plasmid, pDOA4-8, when it was sporulated. The pDOA4-8 plasmid could be lost from the doa3-1 doa4Δ
segregants at 30°C, demonstrating that the double mutant was viable; however, doa3-1 doa4Δ
cells grew much slower than either single mutant, and they failed to form colonies at 35°C, a temperature that allowed growth of both single mutants (Figure A). These mutual synthetic enhancement effects suggest that Doa4 and proteasome activities are functionally linked.
Figure 2 Genetic interaction between doa4Δ and doa3. (A) Enhanced growth defect of a doa3-1 doa4Δ double mutant. Doubly mutant doa3-1 doa4Δ cells, as well as the corresponding single mutants, were grown at 30°C and 35°C (more ...)
cells accumulate (poly)ubiquitinated species that are slightly larger than ubiquitin and ubiquitin multimers; these species might represent ubiquitin or ubiquitin chains attached to small remnant peptides derived from the hydrolysis of ubiquitinated proteins by the proteasome (Papa and Hochstrasser, 1993
). If this were true, then mutational impairment of the proteasome in doa4Δ
cells might suppress the accumulation of these conjugates. Antiubiquitin immunoblot analysis of extracts from doa3-1 doa4Δ
cells (Figure B) demonstrated that the level of the low molecular mass conjugates was indeed greatly reduced relative to the doa4Δ
single mutant; the residual accumulation might be attributable to the leaky nature of the doa3-1
mutation. These data support the possibility that Doa4 and proteasome activities are closely coupled.
Association of Doa4 with 26S Proteasomes
The genetic data detailed above suggested that Doa4 might associate with or even be a component of the yeast 26 proteasome. To examine these ideas, a purification procedure for yeast 26S proteasomes was developed that yielded active enzyme of high purity. Purification of 26S proteasomes from yeast has proven to be more difficult than from other sources (Fischer et al., 1994
; Fujimuro et al., 1998
). For our purification, we followed cleavage of a fluorigenic proteasome substrate, suc-LLVY-AMC, to identify proteasome-containing fractions through different chromatographic steps, and a fully functional HA epitope-tagged Doa4 derivative was used in place of the wild-type protein to follow the partitioning of Doa4 throughout the purification using Western immunoblot analysis with an anti-HA monoclonal antibody. A doublet of bands at ~110 kDa is observed in HA-Doa4–expressing cells but not in cells expressing the untagged protein. In most experiments, we purified proteasomes from a doa4Δ pep4
strain (MHY832) that carried a low-copy vector encoding HA-tagged Doa4 [pDOA4-8(HA)]. To help stabilize the 26S complex, 20% glycerol and 2 mM ATP were maintained in all buffers, as has been done for purification of 26S proteasomes from other sources (Kanayama et al., 1992
; Ugai et al., 1993
). All chromatographic steps were performed on an FPLC system (Pharmacia). A preliminary account of our purification was published previously and used to demonstrate that Sen3 and Cim5 were both components of the yeast 26S proteasome (DeMarini et al., 1995
). Table describes a typical purification. Recently, another group developed a related but distinct method for purification of yeast 26S proteasomes (Glickman et al., 1998
; Rubin et al., 1998
). The purity and activity of the enzymes isolated by the two procedures appear to be quite similar.
Purification of yeast 26S proteasomes
After a high-speed centrifugation of crude yeast lysate, the supernatant was chromatographed on a Sephacryl S-400 gel filtration column (Swaffield et al., 1995
). As seen in Figure A, suc-LLVY-AMC cleaving activity is present in a broad peak centered at ~1600 kDa based on size standards. Little activity is detected at the position of the 20S proteasome (~700 kDa; peak fraction 48, as determined by fractionation of purified 20S proteasomes), suggesting that in yeast most 20S proteasomes are complexed with 19S regulatory particles. Alternatively, 20S proteasome peptidase activity might have been latent; this has been reported for mammalian 20S proteasomes in which low concentrations of SDS can unmask a latent activity (Orlowski, 1990
). When 0.01% SDS was added to the assay mixture, a small shoulder of activity was uncovered in fractions eluting after the major 26S peak; however, the size of this activity was still larger than that of the 20S proteasome, suggesting that if it were due to the 20S proteasome, then other proteins were still associated with it (our unpublished results). In addition, anti-20S proteasome immunoblots revealed that most of the 20S subunits were present in fractions corresponding to the peak of suc-LLVY-AMC cleaving activity centered at ~1600 kDa.
Figure 3 Peptidase activity profile and anti–HA-Doa4 immunoblot analysis of yeast extracts fractionated by Sephacryl S-400 gel filtration. (A) Cleavage of suc-LLVY-AMC in fractions collected from an S-400 column fractionation of extracts from doa4Δ (more ...)
HA-Doa4 eluted in a biphasic pattern, with the first peak of protein coinciding with the peak of peptidase activity (26S position, fractions 38–40) and the second peak around a molecular mass of ~700 kDa (fraction 51) (Figure B). When the high-speed spin was omitted before S-400 gel filtration, HA-Doa4 was primarily in the 26S proteasome fractions and earlier fractions and not in the second peak, suggesting that a high molecular mass complex(es) that contained HA-Doa4 was broken up during the spin; however, the centrifugation was necessary for obtaining high-purity 26S proteasomes and was therefore retained. Immunoblot analysis using antibodies against the Cim5 subunit of PA700 (Ghislain et al., 1993
; DeMarini et al., 1995
) revealed a Cim5 distribution that coincided with the peptidase activity profile, suggesting that there is little if any free PA700 in yeast cells (our unpublished results). Frequently, a species of HA-Doa4 that migrated more slowly on SDS-PAGE gels could be detected (Figure B, fraction 54), but this varied from preparation to preparation. Immunoblotting of crude lysates always revealed this slower migrating band; it may represent a modification, e.g., phosphorylation, of Doa4 that is lost to varying extents during purification. In no case did we detect HA-Doa4 at a position in the gel filtration elution predicted for the monomer (110 kDa, approximately fractions 60–63); however, when HA-Doa4 was overexpressed from a high-copy vector, a small amount of HA-Doa4 protein was found at the position expected for a monomer (see DISCUSSION).
The S-400 fractions containing the bulk of peptidase activity (fractions 36–44) were pooled. At this stage, a ~40-fold purification of peptidase activity had been achieved (Table ). Interestingly, a 3.5-fold increase in total activity occurred in this gel filtration step, perhaps because of removal of a proteasome inhibitor. The pooled fractions were applied to a Mono Q anion exchange column, and proteins were eluted with a linear salt gradient. Peptidase activity eluted in a narrow peak (fractions 33 and 34) (Figure A). The anion exchange step probably led to some breakdown of the 26S proteasome complex as evidenced by a approximately twofold decline in total activity; however, this effect was compensated by an even greater reduction in total protein (more than sixfold), producing an overall approximately threefold purification over the initial sizing step (Table ).
Figure 4 Fractionation of pooled 26S proteasome-containing fractions from a Sephacryl S-400 column by Mono Q anion exchange chromatography. (A) Peptidase activities in fractions collected from a Mono Q HR (5/5) anion exchange column. Elution of proteins was with (more ...)
Both HA-Doa4, which has a basic pI, and Cim5 coeluted from the Mono Q column in the same fractions as the suc-LLVY-AMC cleaving activity, consistent with an association with the 26S proteasome complex (Figure ). The salt concentration at the point of elution of the 26S proteasome was estimated to be ~0.6 M. Because the 26S proteasome is known to be labile at this high ionic strength, the buffer was immediately replaced with low ionic strength buffer.
Pooled fractions 33–34 were concentrated and run on a Superose 6 gel filtration column. Proteasomal peptidase activity again eluted at ~1600 kDa (Figure A). Both Cim5 and HA-Doa4 eluted in the same fractions as the peptidase activity, and the relative levels of each protein in these fractions closely paralleled the level of peptidase activity (Figure B). Proteolysis of polyubiquitinated 125
I-lysozyme was measured in the same fractions tested for suc-LLVY-AMC cleavage, and the two activities were found to coelute as well. 125
I-lysozyme–conjugate degradation by the yeast protein fractions was fully ATP dependent (Table ). An ATP regenerating system was required to achieve maximal activity, and degradation in ATP-depleted reactions with the yeast proteasome fractions was identical to a reaction containing only buffer; hence, the ATP-independent degradation that was observed could be traced to the substrate preparation, which had been partially purified from rabbit reticulocyte lysate (Hoffman et al., 1992
). SDS-PAGE of the reaction products, followed by autoradiography and densitometric analysis demonstrated that in the ATP-supplemented reaction, polyubiquitinated 125
I-lysozyme was consumed without regeneration of free 125
I-lysozyme, indicating proteolysis of the substrate rather than deubiquitination (our unpublished results; other substrates for deubiquitination were not assayed).
Figure 5 Proteasome fractionation by Superose 6 gel filtration. (A) Peptidase activity and polyubiquitin(Ub)–[125I]-lysozyme degradation in fractions from a Superose 6 gel filtration column. The column was loaded with the pooled and concentrated (more ...)
ATP-dependence of ubiquitin–[125I]-lysozyme conjugate degradation by purified yeast 26S proteasomes
Superose 6 fractions containing maximal proteasome activity (32–36) were pooled. The specific peptidase activity of this pool was more than 500-fold higher than that of the crude extract (Table ). Considering that the proteasome is a highly abundant protein, estimated at 0.5–1% of total soluble protein (Orlowski, 1990
; Chen and Hochstrasser, 1995
), this proteasome preparation would be essentially homogeneous; however, because an apparent inhibitory activity was present before the S-400 gel filtration step (see above), it is difficult to gauge purity by this criterion alone. Nevertheless, both the pattern of subunits visualized on SDS gels and the morphology of particles seen by electron microscopy are very similar to what has been observed with highly purified 26S proteasomes from other organisms (Figure ).
Figure 6 Characterization of purified yeast 26S proteasomes. (A) Analysis of purified 20S and 26S proteasomes by SDS-PAGE followed by Coomassie Blue staining. Protein size standards (in kilodaltons) are indicated. (B) Electron micrograph of purified 26S proteasome (more ...)
SDS-PAGE analysis of the pooled Superose 6 fractions followed by Coomassie Blue staining revealed a complex pattern of protein bands (Figure A). A cluster of characteristic 20S subunits was seen along with additional, primarily higher molecular mass species expected for subunits of the 26S proteasome. Electron microscopy (Figure B) demonstrated the presence of particles with the characteristic shapes seen previously with 26S proteasomes from other sources (Peters et al., 1991
; Yoshimura et al., 1993
). These particles appeared to have a PA700 complex attached at each end of the 20S proteasomal cylinder or sometimes at just one end. The mixture of singly and doubly capped 20S proteasomes would explain the slight asymmetry in the activity curve seen in the gel filtration column fractions in Figure . Some free 20S particles were also seen in the micrographs (Figure B); these may have formed during sample preparation for microscopy. Were this the case, dissociated PA700 regulatory complexes should also have been detected; an additional particle (particle 3), which was probably free PA700, was in fact observed in the samples.
Collectively, these data indicate that a significant percentage of Doa4 protein in yeast cells is associated with active yeast 26S proteasomes. In a later section, we describe additional evidence supporting this inference as well as the idea that proteasome interaction is important for Doa4 function.
Ubp5, a Deubiquitinating Enzyme Related to Doa4
An S. cerevisiae
ORF first described during the yeast genome sequencing project, YER144c, encodes a protein more highly related to Doa4 than is any other available protein sequence. The protein, which was named Ubp5 (Xiao et al., 1994
), is 44% identical (62% similar) to Doa4 over the entire length of the two ORFs (Figure A). In the C-terminal regions of the two proteins beginning at their respective Cys boxes, the degree of similarity is especially high (62% identical; 76% similar). Ubp5 is slightly smaller than Doa4 (805 vs. 926 residues), lacking several peptide segments present in the N-terminal domain of Doa4.
Figure 7 An enzyme closely related to Doa4 encoded by the yeast UBP5 (YER144c) gene. (A) Sequence alignment of Doa4 and Ubp5. The two proteins were aligned with the ClustalW algorithm followed by manual adjustment. Identical residues are boxed in black, and structurally (more ...)
Ubp5 was first tested for deubiquitinating activity. UBP5
and a reporter gene encoding a ubiquitin–Met–β-galactosidase fusion (Ub-M-βgal) were coexpressed in E. coli
MC1061 cells (Papa and Hochstrasser, 1993
). As measured by anti-βgal immunoblot analysis (Figure B, lane 2), approximately half of the Ub-M-βgal was deubiquitinated at steady state in cells expressing Ubp5, indicating that Ubp5 can cleave ubiquitin in peptide linkage with another protein. Using a Lys48-linked diubiquitin molecule as substrate, Ubp5 was also shown to have a ubiquitin isopeptidase activity (Figure C). Therefore, like Doa4, Ubp5 is a deubiquitinating enzyme with activity against both peptide and isopeptide-linked ubiquitin moieties.
To create a yeast strain deleted for UBP5, one of the two copies in a diploid yeast strain was replaced with the HIS3 gene by homologous recombination. The deleted sequences (codons 442–716) included the Cys box coding region. When the resulting heterozygotes were sporulated and tetrads were dissected, all four meiotic segregants grew at the same rate in all tetrads, and histidine prototrophy segregated 2:2. The mutant cells exhibited no obvious defects characteristic of mutants in the ubiquitin–proteasome pathway. Mutant ubp5 cells degraded the test substrates Deg1-βgal, L-βgal, and Ub-P-βgal at wild-type rates. Also unlike doa4Δ cells, the mutant was neither sensitive to heat shock nor hypersensitive to the arginine analog canavanine, and a ubp5/ubp5 homozygous diploid sporulated normally. A doa4Δ ubp5Δ double mutant was constructed to test the possibility that Ubp5 has an overlapping function with Doa4. The doa4Δ ubp5Δ strain could not be distinguished from a doa4Δ single mutant: no further stabilization of Deg1-βgal was seen, nor was the double mutant any more sensitive to heat shock or canavanine. Moreover, the ubiquitin conjugate profile of ubp5Δ cells could not be distinguished from wild-type cells, whereas the doa4Δ ubp5Δ double mutant displayed the same characteristic spectrum of ubiquitinated species seen in doa4Δ cells. Finally, high-copy expression of UBP5 in doa4Δ cells did not rescue the block to degradation of Deg1-βgal (expression of an HA epitope-tagged Ubp5 from a high-copy plasmid led to a large increase in protein level compared with expression of the same derivative from a low-copy vector). We conclude that Ubp5, despite its sequence similarity to Doa4, has little or no overlap in function with Doa4.
An N-Terminal Region of Doa4 Confers Doa4 Function on Ubp5
There are 15 other UBP genes in S. cerevisiae, yet none of several of these genes that were tested could compensate for loss of DOA4 even when they were present on high-copy plasmids (F.R. Papa, S. Swaminathan, M. Hochstrasser, unpublished data). This suggested that the Doa4 protein has some unique structural features that impart specificity to its activity in vivo. Given the evidence that Doa4 can associate with proteasomes, one such “specificity element” might be a domain(s) that mediates proteasome binding. We therefore sought to localize a Doa4 specificity determinant(s) using chimeric proteins that fused segments of Doa4 to another Ubp. Ubp5 was chosen for this analysis because segments with similarity to Doa4 are present along the length of the Ubp5 sequence, providing logical positions to place joints in the chimeras that should minimize general structural perturbations.
An initial chimera between DOA4 and UBP5 was made by traditional cloning methods to fuse the DOA4 gene promoter plus coding sequence for the N-terminal region of Doa4 that extended almost to its Cys box (residues 1–560) to a segment of UBP5 encoding a domain that included the Cys box and extended beyond sequences encoding the Ubp5 C terminus (residues 443–805) (Figure A, construct 1). The chimeric gene, when expressed from a YEplac195-based plasmid in a doa4Δ strain, provided full Doa4 function as judged by its ability to allow growth of a doa4Δ haploid on canavanine-containing medium and to restore normal sporulation to a doa4Δ/doa4Δ diploid. In the same cells, Deg1-βgal levels, as measured by activity assays, were reduced to an amount ~30% higher than that seen in wild-type cells, suggesting that the protein encoded by the chimeric gene provided substantial albeit not quite wild-type Doa4 function (Figure A).
Figure 9 Close correlation between Deg1-βgal accumulation and levels of low molecular mass (poly)ubiquitin conjugates in doa4Δ cells expressing Doa4-Ubp5 chimeras. (A) βgal activity assays of extracts made from yeast cells with an integrated (more ...)
Six additional Doa4-Ubp5 chimeras were constructed (Figure A). Because of the lack of convenient restriction sites in DOA4 and UBP5, we devised a convenient method for constructing gene fusions. This method, which we call PCR–GR, has general utility and is described in detail in MATERIALS AND METHODS (Figure ). PCR–GR was performed in strain MHY905, a doa4Δ mutant that carried an integrated Deg1-lacZ reporter gene. We recovered the recombinant plasmids and transformed them back into MHY905 cells to confirm that any changes in phenotype were due to the plasmid and not to chromosomal mutations. The plasmids were also transformed into bacteria that expressed a Ub-L-βgal reporter; all recombinant proteins had deubiquitinating activity against this substrate. The UBP5 gene used in the gap repair constructions had a sequence encoding a triple-HA tag at the 3′ end of the ORF, allowing us to check protein levels of the chimeric enzymes. All chimeras were expressed in yeast except construct 3, which was undetectable by immunoblot analysis (Figure B).
The chimeras were tested for their ability to restore growth of doa4Δ mutant cells on canavanine; in addition, the ability of the chimeras to support sporulation of a doa4Δ/doa4Δ diploid was tested. By both tests, constructs 1, 2, and 5 were fully active (Figure A). In addition, steady-state Deg1-βgal levels were measured by quantitative βgal activity assays (Figure A). Constructs 1, 2, and 5 all caused a reduction of Deg1-βgal levels relative to that seen in doa4Δ cells. In contrast, neither a high-copy UBP5 plasmid nor plasmids encoding Doa4-Ubp5 chimeras 3, 4, 6, or 7 had doa4Δ-complementing activity by any of the above assays. Finally, we examined extracts from all of these cells by antiubiquitin immunoblot analysis (Figure B). Mutant doa4Δ cells that harbored constructs with doa4Δ-complementing activity, but not those with noncomplementing plasmids, contained greatly reduced levels of the small (poly)ubiquitinated species seen in doa4Δ cells. The levels were still slightly above those observed in wild-type cells, again indicating that Doa4 function was not quite completely restored. As is evident in Figure , levels of the apparent ubiquitin–peptide conjugates correlated very closely with steady-state levels of the Deg1-βgal reporter (and therefore should correlate inversely with rates of Deg1-βgal degradation).
The complementing DOA4-UBP5 construct with the shortest DOA4 sequence was chimera 5, in which the only DOA4-derived sequences are those encoding the N-terminal 310 residues of Doa4. All of the signature sequences that are thought to define the catalytic domain of the Ubp enzyme family in this chimera were derived from Ubp5. The Doa4-Ubp5 chimera with the next smallest N-terminal Doa4 segment (#6, Doa4 residues 1–232) lacked Doa4 activity in vivo, as did construct 4, with Doa4 residues 113–310. Therefore, the N-terminal 310 residues of Doa4 were sufficient for imparting Doa4 function to the heterologous Ubp5 catalytic domain, and Doa4 residues 233–310 and 1–112 were necessary in this context. The N-terminal 310 residues of Doa4 also appeared to be necessary for in vivo activity in the context of the intact Doa4 protein because a Doa4 variant lacking these residues failed to complement a doa4Δ mutant (our unpublished results).
A Doa4-Ubp5 Chimera Can Associate with the 26S Proteasome
One hypothesis for the function of the specificity element(s) contained in the N-terminal domain of Doa4 is that it participates in Doa4 binding to the 26S proteasome. To test this idea, we determined whether a doa4Δ-complementing Doa4-Ubp5 chimera could also associate with proteasomes (Figure ). We chose Doa41–560–Ubp5444–805 (chimera 2) for this analysis because levels of this chimera were slightly higher than those of Doa41–310–Ubp5265–805, and the doa4Δ-complementing capabilities of these two constructs did not differ significantly. After a high speed centrifugation, extracts from doa4Δ cells expressing either the HA–Doa4-Ubp5 chimera or HA–Ubp5 were fractionated by S-400 gel filtration. Ubp5 eluted over a broad range of fractions, including some of the proteasome-containing fractions, (Figure , Input), so this chromatographic step was insufficient to distinguish Ubp5 from the Doa4-Ubp5 chimera. Therefore, the peak proteasome fractions from each run were pooled and chromatographed on a Mono Q column (Figure ). As had been found with full-length Doa4, the functional Doa4-Ubp5 chimera cofractionated precisely with the 26S proteasome-containing fractions, the latter followed by means of peptidase activity assays and anti-Cim5 immunoblot analysis. In contrast, Ubp5 appeared not to bind the anion exchange matrix under the conditions used and was undetectable in the proteasome-containing fractions. The correlation between doa4Δ-complementing activity and ability to cofractionate with the proteasome demonstrated by these data suggests that physical association of Doa4 with the protease is physiologically relevant.
Figure 10 Cofractionation of a Doa4-Ubp5 chimera with 26S proteasomes. Top, suc-LLVY-AMC–cleaving activity of Mono Q column fractions. Pooled 26S proteasome-containing fractions from an S-400 gel filtration provided the input. Lysate was made from doa4Δ (more ...)