1 Department of Molecular Biology, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan
2 Yale University, Department of Molecular Biophysics and Biochemistry, 266 Whitney Avenue, New Haven, CT 06520-8114, USA
3 CREST, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan
Author for correspondence (e-mail: hkobaya{at}molbiol.med.kyushu-u.ac.jp)
Accepted 1 October 2004
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Summary |
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Key words: Protein degradation, Dsk2, Ubiquitin receptor, Sem1, Rpn10, Proteasome stability
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Introduction |
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Sem1 is a small acidic protein that is conserved among eukaryotic species. SEM1 was originally isolated as a multi-copy suppressor of an exocyst mutant in budding yeast (Jantti et al., 1999). Mutations in Sem1 family proteins lead to pleiotropic phenotypes such as defects in exocytosis, pseudohyphal growth and the cell cycle in yeast (Jantti et al., 1999
; Marston et al., 1999
) and split hand/split foot malformation in mammals (Crackower et al., 1996
). Human DSS1 (Deleted-in-Split-Hand/Split-Foot-1), the ortholog of yeast Sem1, interacts with the product of the breast cancer susceptibility gene BRCA2 (Marston et al., 1999
). BRCA2 is a component of the large BRCC complex, which displays E3 ubiquitin-ligase activity (Dong et al., 2003
). DSS1, with BRCA2, appears to participate in the recombinational DNA repair pathway (Yang et al., 2002
; Kojic et al., 2003
; Shin et al., 2003
). It is not clear from these varied phenotypes whether Sem1 functions in a single or multiple protein complexes, and the mechanism of Sem1 action for any of its physiological functions remains obscure.
Taking advantage of the growth arrest caused by DSK2 overexpression in budding yeast (Funakoshi et al., 2002), we have identified sem1 mutants as extragenic suppressors of Dsk2-mediated lethality. sem1 mutants accumulate polyubiquitinated proteins, show impaired ubiquitin-dependent protein degradation and lose viability when combined with various proteasome mutations. We found that Sem1 is a tightly bound component of the RP, specifically the lid subcomplex. The latter conclusion was also reported recently (Sone et al., 2004
), while the present work was being prepared for publication. Loss of Sem1 impaired the integrity of the proteasomal RP. The RP stability defect was greatly enhanced by simultaneous deletion of both the SEM1 and RPN10 genes, the latter encoding another subunit of the RP known to stabilize lid-base interactions. Interestingly, this double mutant also displayed a striking sensitivity to DNA-damaging agents such as UV irradiation and hydroxyurea. Thus, our results indicate that two nonessential RP subunits, Sem1 and Rpn10, function synergistically in stabilizing the lid-base interaction in the proteasomal RP, and loss of this stability correlates with defects in the response to DNA damage.
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Materials and Methods |
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Protein degradation assays, immunoblotting and antibodies
Degradation assays using a model N-end rule substrate were carried out as described previously (Funakoshi et al., 2002); the plasmids carrying the ubiquitin-lacZ fusion genes, Ub-Leu-ßgal and Ub-Ala-ßgal, were provided by Dr A. Varshavsky. For protein-binding studies, cell extracts were prepared in lysis buffer (20 mM HEPES, pH 7.5, 100 mM NaCl, 0.5% NP-40, 1 mM EDTA, 1 mM APMSF and 1 µg/ml each of leupeptin, pepstatin and chymostatin) (Runder et al., 2000
). For immunoblotting, anti-GST (Santa Cruz, B-14), anti-T7 (Novagen), anti-polyubiquitin (FK1, Nippon Bio-Test Lab), anti-Cdc28 (R49-4, a gift of Dr K. Nasmyth), anti-Rpt1 (Affiniti), anti-FLAG (M2, Sigma), anti-ß-gal (Promega), anti-HA (Babco), anti-CP
subunit (MCP231, Affiniti) and various anti-RP subunit (gifts of Drs D. Finley or C. Mann, or made in the M.H. laboratory) antibodies were used. An anti-Sem1 polyclonal antiserum was raised in rabbits using purified Sem1 protein prepared from thrombin-digested recombinant GST-Sem1 as immunogen.
Purification and native gel substrate overlay assay of proteasomes
Fractionation of yeast cell extracts by Superose 6 chromatography or glycerol density gradient centrifugation was done as described (Arendt and Hochstrasser, 1999; Velichutina et al., 2004
). Yeast strains were grown in synthetic medium to an A600 of
1.5. Proteasomes or proteasome subcomplexes were affinity-purified or immunoprecipitated as described previously (Verma et al., 2000
; Leggett et al., 2002
). For salt elution, the lid or base was eluted from RPs immobilized on IgG-Sepharose by increasing salt concentrations and temperatures (0.3 M NaCl at 4°C or 1 M NaCl at 24°C; 1 hour each). Protein remaining on the resin after salt treatment was eluted in SDS gel-loading buffer at 100°C. For in-gel substrate overlay assays, purified proteasome samples or yeast whole extracts from frozen cell powder (Verma et al., 2000
) were resolved by nondenaturing PAGE as described, and the gels were incubated with the fluorogenic Suc-LLVY-AMC peptide (Sigma) (Glickman et al., 1998a
). Proteasome bands were visualized by exposure to UV (362 nm).
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Results |
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Given the link between suppression of Dsk2-induced lethality and proteasome defects, we examined the effects of sem1 mutations on proteasomal function. SEM1 deletion enhanced the growth defects of proteasome mutants (Fig. 2A); the rpn1 and pre2 proteasome mutations tested had been identified previously as suppressors of Dsk2-induced lethality (Funakoshi et al., 2002). Like sem1
, pre9
also suppressed Dsk2-induced lethality and was temperature-sensitive for growth (Fig. 2B). Despite the genetic interactions of SEM1 and DSK2 alleles, Sem1 protein did not appear to interact physically with Dsk2 (data not shown). However, sem1 mutants accumulated polyubiquitin-protein species, as expected if Sem1 contributed to proteasome function (Fig. 2C). Consistent with this inference, degradation of the model proteasome substrate Leu-ß-gal was also inhibited in sem1
cells (Fig. 2D), and the mutants were hypersensitive to the amino-acid analog canavanine (see Fig. 5D).
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Sem1 is a tightly bound component of the RP lid
We asked if the impaired proteasomal function in sem1 mutants reflected a direct association between Sem1 and the proteasome. Proteasomes were immunoprecipitated with anti-Flag antibodies and examined by anti-Sem1 immunoblotting (Fig. 3A). Flag-tagged Pre1, which precipitates the RP-CP complex in the presence of ATP, co-immunoprecipitated Sem1 (lane 8). Sem1 precipitation was very efficient, suggesting that proteasomes contain a large, probably stoichiometric amount of Sem1. When the immunoprecipitation was done in a buffer lacking ATP, which results in CP-RP dissociation, Sem1 was still efficiently precipitated by anti-Flag if the RP subunit Rpt1 carried the Flag tag (Fig. 3A, lane 12) but not if the CP subunit Pre1 did (lane 11). Therefore, Sem1 is primarily associated with the RP, and its ability to interact with the CP depends on the ATP-dependent association of the RP and CP. This result is fully consistent with the recent observations of Sone et al. (Sone et al., 2004). Whole cell extracts were also size-fractionated by gel filtration under rapid isolation and fractionation conditions, and the fractions were analyzed by immunoblotting (Fig. 3B). Sem1 protein (
10 kDa) showed reproducible elution peaks at positions of 26S proteasomes and the roughly co-migrating RP (19S) and CP (20S) complexes. It also could be detected under these conditions in species of smaller size.
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The RP can be separated into lid and base subcomplexes by incubation of resin-bound particles at high salt concentrations (Saeki et al., 2000). If a lid subunit is used to tether the RP to the resin, then salt treatment will specifically elute base subcomplexes and vice versa. We bound RP-containing complexes to an IgG resin with the protein A-tagged lid subunit Rpn7 (Fig. 3C, lanes 5-8). After the base was removed from the resin by elution with salt (lane 6, anti-Rpn2), all detectable Sem1 remained bound (lane 8), as did virtually all of the lid complexes (lane 8, anti-Rpn12). Conversely, if the base was tethered to the resin with protein A-Rpn2 (lanes 1-4), a large fraction of Sem1, along with the lid, was eluted under moderate salt conditions (lane 2). Even after a more stringent salt wash, a significant fraction of Sem1 remained bound to the base (lane 4), indicating that Sem1 was more difficult to remove from the RP base than were other lid subunits under these conditions. Collectively, these data indicate that Sem1 is a tightly bound component of the RP lid subcomplex, but it probably makes strong contacts with the base as well.
Sem1 is required for the stability of the proteasome
We determined whether any physical changes in sem1 proteasomes could be detected when compared to wild-type particles. Reduced stability or assembly of 26S proteasomes in sem1
was suggested by anti-Flag immunoprecipitation, in the presence of ATP of proteasomes containing Flag-tagged Pre1 (a CP subunit) (Fig. 4A). Relative to the wild type (lane 5), reduced amounts of the Rpt1 RP subunit were precipitated from sem1
extracts (lane 6). Proteasome stability/assembly was also examined by preparing whole cell extracts in the presence of ATP and resolving particles by in-gel substrate overlay assays (Fig. 4B). In wild-type cells, most proteasomes were present as dyad-symmetric RP2CP complexes with a small amount of singly capped RP1CP (lane 1). By contrast, sem1
proteasomes fractionated as a much broader mixture of complexes, including complexes likely to be base-CP (RPB-CP) particles (Glickman et al., 1998a
) (lane 2). Affinity-purified proteasomes were also analyzed (lanes 3-6). In the presence of ATP, both wild-type and sem1
cells yielded RP1CP as a major fraction, but the relative amounts of RP1CP and RPB-CP were higher in sem1
(compare lanes 3 and 4). The amount of free CP was also relatively high in sem1
(lanes 4,6).
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Glycerol gradient analysis of proteasome profiles showed a sem1 profile with a modest shift of CP and RP base components to fractions smaller than full 26S particles (Fig. 4C); more free CP relative to intact 26S proteasome was present in sem1
cells as measured by CP activity assays as well (Fig. 4C, bottom). Lid subunits from the mutant cells, by contrast, broke up more drastically, and non-uniformly, into smaller species. These data were consistent with the native gel analyses, and suggested that lid stability and/or assembly was especially sensitive to the loss of Sem1.
Proteasome defects caused by sem1 are greatly exacerbated by rpn10
Rpn10 is a nonessential proteasome RP subunit, the deletion of which shows genetic interactions with mutations in the ubiquitin receptors Dsk2 and Rad23 (Chen and Madura, 2002) (our unpublished results). Because of these observations, we compared the sem1
or rpn10
single mutants to sem1
rpn10
double mutants. The double mutant cells showed a strong synthetic growth defect at high temperature (Fig. 5A). Proteasome profiles from these mutant strains were evaluated by gel filtration (Fig. 5B). As before, the sem1
single mutant displayed a modest shift of particles to species smaller than free RP. The rpn10
mutant looked similar to the wild-type when analyzed with either anti-lid (Rpn5; Fig. 5B, top) or anti-base (Rpn2; Fig. 5B, bottom) antibodies. In striking contrast, the sem1
rpn10
double mutant displayed a strong shift of the Rpn5 subunit to smaller particles (
300-400 kDa) even though most of the base still co-eluted with the CP (Fig. 5B). More free CP relative to intact 26S proteasome was present in both sem1
and sem1
rpn10
cells, as measured by CP activity assays (Fig. 5C).
We conclude that loss of Sem1, especially in an rpn10 background, impairs formation or stability of the 26S proteasome. The lid-base interaction in the RP is greatly reduced in the sem1
rpn10
double mutant, and the lid itself is not fully assembled (Fig. 5B and not shown). Interestingly, sem1
exhibited a mild sensitivity to UV irradiation, and this defect was greatly exacerbated in a sem1
rpn10
double mutant (Fig. 5D). The double mutant also displayed a striking sensitivity to a DNA damage-inducing concentration of hydroxyurea (HU), even though the single mutants were at least as resistant as the wild type. The correlation between DNA damage sensitivity and proteasome RP stability in the sem1
and rpn10
single and double mutants suggests that an intact RP is required for the response to and/or repair of DNA damage.
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Discussion |
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Sem1 as a modulator of protein-protein interactions in the proteasome
Identification of Sem1 as a proteasomal RP subunit was unexpected given the extensive proteomic analyses that have been done on proteasomes (Glickman et al., 1998b; Verma et al., 2000
). It was probably missed in earlier studies because of its small size. Sem1 associates tightly with the lid, but our data also suggest that it makes contacts with the base in the absence of the full lid, consistent with the idea that Sem1, like Rpn10, stabilizes lid-base interactions. This is congruent with the strong genetic interactions between the sem1 and rpn10 mutations seen by both growth and gel filtration assays (Fig. 5).
Sem1 can also associate directly with isolated subunits of the CP (our unpublished data). A lid-CP interaction is not inconsistent with previous EM analyses, which indicated that electron density seen at the side of the RP layer apposed to the CP was lost when the lid was dislodged from the base (Glickman et al., 1998a
). The mammalian DSS1-BRCA2 co-crystal structure revealed the BRCA2 region that binds DSS1 (Yang et al., 2002
); we compared this BRCA2 segment to all yeast proteins, and the closest sequence was a segment (residues 170-207) of the CP subunit Pre9/
3 (32% identity/61% similarity against human BRCA2 residues 2648-2685). This Pre9 element is on the surface of the CP in a position compatible with Sem1, as part of the RP, binding directly to the CP. The physiological significance of the Sem1-
subunit association is currently under investigation.
DSS1 from mammals can replace Sem1 in S. cerevisiae. It acts as a cofactor in DNA repair for BRCA2 in mammalian cells and in certain fungi (Marston et al., 1999; Kojic et al., 2003
). (DSS1)-BRCA2 remodels the Rad51 AAA-type ATPase, apparently by mimicking a Rad51-Rad51subunit interaction, from inactive rings into helical filaments that assemble onto single-stranded DNA (Shin et al., 2003
). By analogy, we speculate that the Sem1-lid complex helps induce changes in the AAA-ATPase ring of the RP that facilitates lid binding and other aspects of RP function. Interestingly, Sem1 is weakly similar over almost its full length to a region (residues 424-503) of another AAA ATPase, Cdc48 (24% identity/51% similarity; P=2.0e-4); this region also shows some similarity to proteasomal ATPases, especially Rpt1 (residues 103-196) and Rpt3 (residues 139-233). In the crystal structure of the mouse p97 (Cdc48) hexamer (Dreveny et al., 2004
) (Protein Data Bank entry 1OZ4), the Sem1-related region runs along the outer surface of the ring, close to the ATP-binding site. Using the structure of p97 as a guide, we suggest that Sem1 binds along the outside of the proteasomal ATPase heterohexamer, which might allow it to link the lid directly to CP. The structural model also can account for a loss of RP stability in sem1
strains, particularly the dissociation of the lid from the base.
Sem1 and Dsk2 in the ubiquitin-proteasome pathway
Dsk2 has been characterized as one of the non-proteasomal ubiquitin receptors that mediate the delivery of polyubiquitinated proteins to the proteasome (Wilkinson et al., 2001; Funakoshi et al., 2002
; Elsasser et al., 2004
; Verma et al., 2004
). In addition to SEM1 (and PRE9), we identified RPN1 and PRE2 as Dsk2 suppressors (Funakoshi et al., 2002
). Rpn1 is the largest base subunit of the 19S RP, whereas PRE2/DOA3 encodes the ß5 catalytic subunit of the CP. Rpn1 can interact with both the non-proteasomal ubiquitin receptor Rad23 (Elsasser et al., 2004
) and the proteasomal ubiquitin receptor Rpn10 (Xie and Varshavsky, 2000
). Rpn10 interacts genetically with Rad23 and Dsk2 (Chen and Madura, 2002
) (our unpublished data). Therefore, the largest RP subunit Rpn1 might act as a scaffolding protein to assemble these polyubiquitin receptors. The molecular basis of Dsk2 overexpression lethality remains to be determined, but our data indicate that reducing function of the 26S proteasome in multiple ways can suppress this growth defect. This suggests that high levels of Dsk2 cause an aberrantly high rate of degradation of one or more proteins required for viability.
Sem1 and the proteasome in the DNA damage response
Sem1 is nonessential under optimal growth conditions but becomes essential under conditions that place an extra burden on proteasome activity. Many proteasome mutants are hypersensitive to the amino acid analog canavanine, and the same is true for sem1 (Fig. 5D). In an rpn10
background, sem1
also exhibited strong sensitivity to UV irradiation and DNA damage-inducing concentrations of hydroxyurea. Orthologs of Sem1 bind BRCA2 in species that encode this latter factor, and Sem1/DSS1 is thought to participate as part of a BRCA2 complex in double-stranded DNA break repair in these organisms. Our data showing high sensitivity of sem1
rpn10
double mutants, but not single mutants, to DNA-damaging agents indicate that Sem1 also participates in the context of the proteasome or RP in certain DNA repair responses.
The 26S proteasome probably affects DNA repair in multiple ways in vivo, but many of the details remain controversial. In yeast, the proteasomal RP has been suggested to function as a negative regulator of nucleotide excision repair (NER) independently of CP-dependent proteolysis (Gillete et al., 2001); however, in vitro the RP seemed to have a positive role in NER (Russell et al., 1999
). Participation of CP proteolytic activity in post-replication repair was reported recently (Podlaska et al., 2003
), but this was not observed in an earlier study (Dor et al., 1996
). As with the proteasomal contribution to transcription, the issue of whether the entire proteasome or even an intact RP is required for DNA repair has not been settled. The greatly enhanced damage sensitivity of sem1
rpn10
double mutants correlates closely with a severe drop in intact RP levels (Fig. 5), strongly suggesting that loss of intact RP impairs at least some aspects of the DNA damage response. It will be of interest to investigate how the proteasome is involved in DNA damage repair and to determine if Sem1/DSS1 has mechanistically similar functions in both BRCA2-containing complexes and proteasomes.
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Acknowledgments |
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Footnotes |
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