Degradation of polyubiquitinated proteins by the proteasome often requires accessory factors; these include receptor proteins that bind both polyubiquitin chains and the regulatory particle of the proteasome. Overproduction of one such factor, Dsk2, is lethal in Saccharomyces cerevisiae and we show here that this lethality can be suppressed by mutations in SEM1, a gene previously recognized as an ortholog of the human gene encoding DSS1, which binds the BRCA2 DNA repair protein. Yeast sem1 mutants accumulate polyubiquitinated proteins, are defective for proteasome-mediated degradation and cannot grow under various stress conditions. Moreover, sem1 is synthetically lethal with mutations in proteasome subunits. We show that Sem1 is a component of the regulatory particle of the proteasome, specifically the lid subcomplex. Loss of Sem1 impairs the stability of the 26S proteasome and sem1Δ defects are greatly enhanced by simultaneous deletion of RPN10. The Rpn10 proteasome subunit appears to function with Sem1 in maintaining the association of the lid and base subcomplexes of the regulatory particle. Our data suggest a potential mechanism for this protein-protein stabilization and also suggest that an intact proteasomal regulatory particle is required for responses to DNA damage.

The proteasome is crucial to many cellular processes. Most proteasome substrates are polyubiquitinated prior to their degradation by the 26S proteasome, which consists of a proteolytically active core particle (CP or 20S proteasome) and the regulatory particle (RP or 19S particle). Four heteroheptameric rings comprise the CP, with outer rings of α-type subunits and an inner pair of β-type subunits. Components of the RP have roles in recognition, unfolding, and deubiquitination of substrates and in their translocation into the CP (Hershko and Ciechanover, 1998; Hochstrasser, 2002). Additional proteins, including Rad23 and Dsk2 in Saccharomyces cerevisiae, are thought to act as receptors that help deliver polyubiquitinated proteins to the RP (Schauber et al., 1998; Wilkinson et al., 2001; Chen and Madura, 2002; Elsasser et al., 2002; Funakoshi et al., 2002; Elsasser et al., 2004; Verma et al., 2004). By contrast, the RP subunit Rpn10 binds polyubiquitinated proteins directly (van Nocker et al., 1996). Proteasomes have an RP on either one (RP1CP) or two (RP2CP) ends of the CP cylinder. The RP can be split into so-called `lid' and `base' subcomplexes under certain conditions. Purified RP and CP associate directly, but this may be facilitated or stabilized by other factors in vivo (Baumeister et al., 1998; Glickman et al., 1998a). One factor, Ecm29, can bind to both the RP and CP and has been suggested to stabilize the association of the two (Leggett et al., 2002). Several other proteasome-associated proteins also have been proposed to contribute to RP-CP interactions or 26S proteasome assembly/stability (Takeuchi et al., 1999; Tone and Toh-e, 2002; Fehlker et al., 2003; Santamaria et al., 2003).

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.

Strains and plasmids

Saccharomyces cerevisiae YPH499 or MHY501 were the parental wild-type strains (Sikorsky and Hieter, 1989; Chen et al., 1993). Isolation of DSK2-mediated suppressor mutants was described previously (Funakoshi et al., 2002). Temperature-sensitive sem1 alleles from mutants that suppressed Dsk2-mediated lethality were cloned by gap-repair and the mutations were determined by DNA sequencing (Orr-Weaver and Szostak, 1983). Yeast strains deleted for the SEM1 and PRE9 genes were generated by PCR-mediated gene disruption (Adams et al., 1997) and verified by PCR and immunoblot analyses. In brief, to disrupt SEM1, the HIS3 gene in pRS403 was amplified by PCR (primers: 5′-ATAGAGTGGCAATAAAAGTCGAATAATAGTACAAATACGCAAAAATTTGTACTGAGAGTGCAC-3′, and 5′-GTATAATTTCCTTTTGCGTTATTTAGAAATAATGTTATGTAGCCCCTGCGGTATTTCACACCG-3′). The amplified DNA fragment was used for yeast transformation. Disruption of the SEM1 gene was checked by colony PCR (primers: 5′-CTTTTAAAGTCCTAATGGAAAC-3′ and 5′-ATATACTAGTATGACAACAATG-3′). To disrupt PRE9, the HIS3 gene in pRS403 was amplified by PCR (primers: 5′-CTCTATTTTAATAATTGATTATTGGATATAGTTAGTAGTGTTAAACTTGTACTGAGAGTGCAC-3′, and 5′-GCGTACATATTTATATAAGCATGAAGTCAAACAATACTTTCCAACCTGCGGTATTTCACACCG-3′). Disruption of PRE9 was checked by colony PCR (primers: 5′-CGCAGAGCTCGATGTTGGAGACAGTGAGTG-3′, and 5′-TTTCAATGATCGATTCACTAGTGACGCAGA-3′). The YIplac211-based plasmids pJD416 and pJD522 were used for expression of Flag-His6-tagged Pre1 and Flag-His6-tagged Rpt1 in yeast strain RJD1144 and RJD1171, respectively (Verma et al., 2000). Yeast expressing protein A-tagged versions of proteasome subunits were obtained from Open Biosystems. Escherichia coli DH5α was used for DNA manipulation. Yeast strains used in this study are listed in Table 1.

Table 1.

Yeast strains used in this study

Strain Genotype Reference
YPH499  MATa ade2-101 leu2-Δ1 trp1-Δ63 ura3-52 lys2-801 his3-Δ200  Sikorski and Hieter, 1989  
des3  MATa ade2-101 leu2-Δ1 trp1-Δ63 ura3-52 lys2-801 his3-Δ200 pre2-75  
des16b  MATa ade2-101 leu2-Δ1 trp1-Δ63 ura3-52 lys2-801 his3-Δ200 rpn1-821  
des14  MATa ade2-101 leu2-Δ1 trp1-Δ63 ura3-52 lys2-801 his3-Δ200 sem1–60  
des19  MATa ade2-101 leu2-Δ1 trp1-Δ63 ura3-52 lys2-801 his3-Δ200 sem1–64  
YMF12-1a  MATa ade2-101 leu2-Δ1 trp1-Δ63 ura3-52 lys2-801 his3-Δ200 sem1Δ::HIS3  
YMF22-1a  MATa ade2-101 leu2-Δ1 trp1-Δ63 ura3-52 lys2-801 his3-Δ200 pre2-75 sem1Δ::HIS3  
YMF24-1b  MATa ade2-101 leu2-Δ1 trp1-Δ63 ura3-52 lys2-801 his3-Δ200 rpn1-821 sem1Δ::HIS3  
YMF27-1a  MATa ade2-101 leu2-Δ1 trp1-Δ63 ura3-52 lys2-801 his3-Δ200 pre9Δ::HIS3  
YMF31-3  MATa ade2-101 leu2-Δ1 trp1-Δ63 ura3-52 lys2-801 his3-Δ200 PRE1-FH::YIPlac211  
YMF32-1  MATa ade2-101 leu2-Δ1 trp1-Δ63 ura3-52 lys2-801 his3-Δ200 PRE1-FH::YIPlac211 sem1Δ::HIS3  
RJD1144/JD122  MATa leu2-3,112 trp1Δ63 ura3-52 lys2-801 his3Δ200 PRE1FH::YIPlac211 Verma et al., 2000  
RJD1171/JD165  MATa leu2-3,112 trp1Δ63 ura3-52 lys2-801 his3Δ200 RPT1FH::YIPlac211 Verma et al., 2000  
MHY501  MATα leu2-3,112 trp1-1 ura3-52 lys2-801 his3-Δ200 gal2 Chen et al., 1993  
MHY560  MATα ade2-101 leu2-Δ1 trp1-Δ63 ura3-52 lys2-801 his3-Δ200 rad6Δ::LEU2  
MHY961  MATα leu2-3,112 trp1-1 ura3-52 lys2-801 his3-Δ200 gal2 rpn10Δ::HIS3  
MHY1069  MATα leu2-3,112 trp1-1 ura3-52 lys2-801 his3-Δ200 gal2 pre9-Δ2::HIS3 Velichutina et al., 2004  
MHY1988  MATα leu2-3,112 trp1-1 ura3-52 lys2-801 his3-Δ200 gal2 doa3-Δ1::HIS3 ump1::pRS305-UMP1-HA2[YCp50-DOA3]  
MHY2963  MATα leu2-3,112 trp1-1 ura3-52 lys2-801 his3-Δ200 gal2 rpn10Δ::HIS3 sem1Δ::HIS3  
BY4741-Rpn2  MATa leu2Δ ura3Δ met15Δ1 his3Δ RPN2-TAP(HIS3) Yeast TAP fusion ORF Strain (Open Biosystems)   
BY4741-Rpn7  MATa leu2Δ ura3Δ met15Δ1 his3Δ RPN7-TAP(HIS3) Yeast TAP fusion ORF Strain (Open Biosystems)   
Strain Genotype Reference
YPH499  MATa ade2-101 leu2-Δ1 trp1-Δ63 ura3-52 lys2-801 his3-Δ200  Sikorski and Hieter, 1989  
des3  MATa ade2-101 leu2-Δ1 trp1-Δ63 ura3-52 lys2-801 his3-Δ200 pre2-75  
des16b  MATa ade2-101 leu2-Δ1 trp1-Δ63 ura3-52 lys2-801 his3-Δ200 rpn1-821  
des14  MATa ade2-101 leu2-Δ1 trp1-Δ63 ura3-52 lys2-801 his3-Δ200 sem1–60  
des19  MATa ade2-101 leu2-Δ1 trp1-Δ63 ura3-52 lys2-801 his3-Δ200 sem1–64  
YMF12-1a  MATa ade2-101 leu2-Δ1 trp1-Δ63 ura3-52 lys2-801 his3-Δ200 sem1Δ::HIS3  
YMF22-1a  MATa ade2-101 leu2-Δ1 trp1-Δ63 ura3-52 lys2-801 his3-Δ200 pre2-75 sem1Δ::HIS3  
YMF24-1b  MATa ade2-101 leu2-Δ1 trp1-Δ63 ura3-52 lys2-801 his3-Δ200 rpn1-821 sem1Δ::HIS3  
YMF27-1a  MATa ade2-101 leu2-Δ1 trp1-Δ63 ura3-52 lys2-801 his3-Δ200 pre9Δ::HIS3  
YMF31-3  MATa ade2-101 leu2-Δ1 trp1-Δ63 ura3-52 lys2-801 his3-Δ200 PRE1-FH::YIPlac211  
YMF32-1  MATa ade2-101 leu2-Δ1 trp1-Δ63 ura3-52 lys2-801 his3-Δ200 PRE1-FH::YIPlac211 sem1Δ::HIS3  
RJD1144/JD122  MATa leu2-3,112 trp1Δ63 ura3-52 lys2-801 his3Δ200 PRE1FH::YIPlac211 Verma et al., 2000  
RJD1171/JD165  MATa leu2-3,112 trp1Δ63 ura3-52 lys2-801 his3Δ200 RPT1FH::YIPlac211 Verma et al., 2000  
MHY501  MATα leu2-3,112 trp1-1 ura3-52 lys2-801 his3-Δ200 gal2 Chen et al., 1993  
MHY560  MATα ade2-101 leu2-Δ1 trp1-Δ63 ura3-52 lys2-801 his3-Δ200 rad6Δ::LEU2  
MHY961  MATα leu2-3,112 trp1-1 ura3-52 lys2-801 his3-Δ200 gal2 rpn10Δ::HIS3  
MHY1069  MATα leu2-3,112 trp1-1 ura3-52 lys2-801 his3-Δ200 gal2 pre9-Δ2::HIS3 Velichutina et al., 2004  
MHY1988  MATα leu2-3,112 trp1-1 ura3-52 lys2-801 his3-Δ200 gal2 doa3-Δ1::HIS3 ump1::pRS305-UMP1-HA2[YCp50-DOA3]  
MHY2963  MATα leu2-3,112 trp1-1 ura3-52 lys2-801 his3-Δ200 gal2 rpn10Δ::HIS3 sem1Δ::HIS3  
BY4741-Rpn2  MATa leu2Δ ura3Δ met15Δ1 his3Δ RPN2-TAP(HIS3) Yeast TAP fusion ORF Strain (Open Biosystems)   
BY4741-Rpn7  MATa leu2Δ ura3Δ met15Δ1 his3Δ RPN7-TAP(HIS3) Yeast TAP fusion ORF Strain (Open Biosystems)   

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-βgal and Ub-Ala-βgal, 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-β-gal (Promega), anti-HA (Babco), anti-CP α 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).

Yeast sem1 cells exhibit defects in proteasome function

Overexpression of Dsk2 is lethal to wild-type yeast (Funakoshi et al., 2002). Taking advantage of this inhibitory effect, we screened for extragenic suppressors of DSK2 overexpression-induced growth arrest. Seven temperature-sensitive mutants were isolated, three of which were identified with recessive mutations in SEM1. These mutants were temperature-sensitive for growth but at 30°C rescued the lethality caused by overexpression of Dsk2 (Fig. 1A). The mutations in sem1-60 and sem1-64 introduced premature stop codons in place of Trp60 and Trp64, respectively, in the 89-codon Sem1 ORF (Fig. 1B). As in sem1Δ, Sem1 protein was not detected in sem1-60 and sem1-64 cells by anti-Sem1 immunoblotting (Fig. 1C).

Fig. 1.

Identification of sem1 mutants in Saccharomyces cerevisiae. (A) Isolation of sem1 mutants. Two sem1 mutants were identified as suppressors of Dsk2-mediated lethality. Both the sem1 point mutants and sem1Δ were temperature-sensitive for growth. (B) Mutation sites of sem1 mutant alleles. Mutated sites (sem1-60 and sem1-64) are indicated in italics (60W and 64W). (C) Loss of Sem1 protein in sem1 mutants. Sem1 protein was detected in cell extracts by immunoblotting with anti-Sem1 antibody (top); Cdc28 was detected (bottom) as a loading control.

Fig. 1.

Identification of sem1 mutants in Saccharomyces cerevisiae. (A) Isolation of sem1 mutants. Two sem1 mutants were identified as suppressors of Dsk2-mediated lethality. Both the sem1 point mutants and sem1Δ were temperature-sensitive for growth. (B) Mutation sites of sem1 mutant alleles. Mutated sites (sem1-60 and sem1-64) are indicated in italics (60W and 64W). (C) Loss of Sem1 protein in sem1 mutants. Sem1 protein was detected in cell extracts by immunoblotting with anti-Sem1 antibody (top); Cdc28 was detected (bottom) as a loading control.

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 sem1Δ, pre9Δ 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-β-gal was also inhibited in sem1Δ cells (Fig. 2D), and the mutants were hypersensitive to the amino-acid analog canavanine (see Fig. 5D).

Fig. 2.

Mutation in SEM1 causes defects in proteasome function. (A) Genetic interactions between sem1Δ and proteasome mutations. Synthetic lethality of sem1Δ with rpn1-821 at 34°C (top) and with pre2-75 at 34°C (bottom) is shown. (B) Suppression of Dsk2-mediated growth arrest by disruption of PRE9. pGAL1-DSK2 was introduced into wild-type, pre9Δ or congenic sem1Δ cells. The disruption of PRE9, SEM1 or both genes did not affect the interaction of Dsk2 with the 26S proteasome (data not shown). (C) Accumulation of polyubiquitinated proteins in sem1 mutants. Cell extracts were subjected to immunoblot analysis with anti-polyubiquitin antibody (top) or with anti-Cdc28 antibody (bottom) as a loading control. (D) Defective protein degradation in sem1Δ cells. The rapidly degraded model substrate Leu-β-gal and the stable Ala-β-gal control proteins were expressed as ubiquitin fusions from galactose-induced genes. Transcription was blocked with glucose (0 minute), and proteins were followed for 80 minutes by anti-β-gal immunoblotting.

Fig. 2.

Mutation in SEM1 causes defects in proteasome function. (A) Genetic interactions between sem1Δ and proteasome mutations. Synthetic lethality of sem1Δ with rpn1-821 at 34°C (top) and with pre2-75 at 34°C (bottom) is shown. (B) Suppression of Dsk2-mediated growth arrest by disruption of PRE9. pGAL1-DSK2 was introduced into wild-type, pre9Δ or congenic sem1Δ cells. The disruption of PRE9, SEM1 or both genes did not affect the interaction of Dsk2 with the 26S proteasome (data not shown). (C) Accumulation of polyubiquitinated proteins in sem1 mutants. Cell extracts were subjected to immunoblot analysis with anti-polyubiquitin antibody (top) or with anti-Cdc28 antibody (bottom) as a loading control. (D) Defective protein degradation in sem1Δ cells. The rapidly degraded model substrate Leu-β-gal and the stable Ala-β-gal control proteins were expressed as ubiquitin fusions from galactose-induced genes. Transcription was blocked with glucose (0 minute), and proteins were followed for 80 minutes by anti-β-gal immunoblotting.

Fig. 5.

The sem1Δ rpn10Δ double mutant has severe defects in RP lid integrity and DNA damage resistance. (A) Synthetic lethal interactions between sem1Δ and rpn10Δ at 36°C. (B) Disruption of the lid and dissociation of the lid from the base in sem1Δ rpn10Δ cells. Superose 6 fractions were subjected to immunoblot analysis with antibodies to the indicated proteins. (C) CP activity of the same column fractions used in B. (D) Growth defects of mutants exposed to canavanine, UV irradiation or hydroxyurea (HU). Cells were spotted onto plates in fivefold serial dilutions and were subjected to the indicated treatments at 30°C.

Fig. 5.

The sem1Δ rpn10Δ double mutant has severe defects in RP lid integrity and DNA damage resistance. (A) Synthetic lethal interactions between sem1Δ and rpn10Δ at 36°C. (B) Disruption of the lid and dissociation of the lid from the base in sem1Δ rpn10Δ cells. Superose 6 fractions were subjected to immunoblot analysis with antibodies to the indicated proteins. (C) CP activity of the same column fractions used in B. (D) Growth defects of mutants exposed to canavanine, UV irradiation or hydroxyurea (HU). Cells were spotted onto plates in fivefold serial dilutions and were subjected to the indicated treatments at 30°C.

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.

Fig. 3.

Sem1 is a component of the RP lid. (A) Sem1 is tightly associated with the RP. Endogenous PRE1 (CP) or RPT1 (RP) genes were replaced with alleles that expressed the corresponding proteins with Flag-His6 tags. Extracts from yeast expressing Flag-His6-Pre1 or Flag-His6-Rpt1 were precipitated with anti-Flag antibody, followed by immunoblotting with antibodies to the indicated antigens. Inputs (lanes 1-6) represent 15% (for α-Sem1) or 45% (for α-Rpt1, α-20S and α-Flag) of the amounts used for precipitation (lanes 7-12). (B) Co-elution of Sem1 in proteasome-containing gel-filtration-column fractions. Anti-Sem1 (top), anti-Rpt6 (middle), and anti-CP α subunit (MCP231) plus anti-HA antibodies (bottom) were used for immunoblotting. Size standards are shown at the bottom. (C) Sem1 is tightly associated with the lid. The lid and base were salt eluted from RPs immobilized with protein-A tags on either the base (lanes 1-4) or the lid (lanes 5-8), followed by immunoblotting with antibodies as indicated. Proteins run on the gel were bound to the IgG resin prior to batch salt washes (lanes 1, 5); eluted with 0.3 M NaCl (lanes 2, 6); eluted with 1 M NaCl (lanes 3, 7); or remained after salt washes (lanes 4, 8). Positions of molecular weight standards are indicated on the left.

Fig. 3.

Sem1 is a component of the RP lid. (A) Sem1 is tightly associated with the RP. Endogenous PRE1 (CP) or RPT1 (RP) genes were replaced with alleles that expressed the corresponding proteins with Flag-His6 tags. Extracts from yeast expressing Flag-His6-Pre1 or Flag-His6-Rpt1 were precipitated with anti-Flag antibody, followed by immunoblotting with antibodies to the indicated antigens. Inputs (lanes 1-6) represent 15% (for α-Sem1) or 45% (for α-Rpt1, α-20S and α-Flag) of the amounts used for precipitation (lanes 7-12). (B) Co-elution of Sem1 in proteasome-containing gel-filtration-column fractions. Anti-Sem1 (top), anti-Rpt6 (middle), and anti-CP α subunit (MCP231) plus anti-HA antibodies (bottom) were used for immunoblotting. Size standards are shown at the bottom. (C) Sem1 is tightly associated with the lid. The lid and base were salt eluted from RPs immobilized with protein-A tags on either the base (lanes 1-4) or the lid (lanes 5-8), followed by immunoblotting with antibodies as indicated. Proteins run on the gel were bound to the IgG resin prior to batch salt washes (lanes 1, 5); eluted with 0.3 M NaCl (lanes 2, 6); eluted with 1 M NaCl (lanes 3, 7); or remained after salt washes (lanes 4, 8). Positions of molecular weight standards are indicated on the left.

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 sem1Δ proteasomes could be detected when compared to wild-type particles. Reduced stability or assembly of 26S proteasomes in sem1Δ 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 sem1Δ 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, sem1Δ 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 sem1Δ cells yielded RP1CP as a major fraction, but the relative amounts of RP1CP and RPB-CP were higher in sem1Δ (compare lanes 3 and 4). The amount of free CP was also relatively high in sem1Δ (lanes 4,6).

Fig. 4.

Sem1 contributes to RP stability. (A) Immunoprecipitation analysis of proteasomes. Flag-His6-Pre1 was expressed in YPH499 or sem1Δ cells. Extracts were prepared after cells were incubated at 37°C for 6 hours, and were immunoblotted with the indicated antibodies (lanes 1-3). In lanes 4-6, anti-Flag immunoprecipitation was followed by immunoblotting. Inputs (lanes 1-3) were 1/3 to 1/2 of the amounts used in lanes 4-6. (B) Proteasome stability in sem1Δ cells. Cell extracts at 30°C were resolved by nondenaturing PAGE either directly (lanes 1, 2) or following affinity purification of Flag-Pre1-containing proteasomes (lanes 3-6). CP-containing species were detected by an in-gel peptidase assay using the Suc-LLVY-AMC fluorogenic substrate and UV illumination of the gel. Extracts from cells grown at 37°C showed a similar pattern. (C) Glycerol gradient analysis of sem1Δ proteasomes. Glycerol gradient fractions were evaluated for the proteasomes by immunoblot analysis with antibodies against the indicated proteins (top) and by CP peptidase activity assays (bottom). The anti-CP antibody was MCP231, which recognizes multiple α subunits. Asterisk indicates cross-reacting protein.

Fig. 4.

Sem1 contributes to RP stability. (A) Immunoprecipitation analysis of proteasomes. Flag-His6-Pre1 was expressed in YPH499 or sem1Δ cells. Extracts were prepared after cells were incubated at 37°C for 6 hours, and were immunoblotted with the indicated antibodies (lanes 1-3). In lanes 4-6, anti-Flag immunoprecipitation was followed by immunoblotting. Inputs (lanes 1-3) were 1/3 to 1/2 of the amounts used in lanes 4-6. (B) Proteasome stability in sem1Δ cells. Cell extracts at 30°C were resolved by nondenaturing PAGE either directly (lanes 1, 2) or following affinity purification of Flag-Pre1-containing proteasomes (lanes 3-6). CP-containing species were detected by an in-gel peptidase assay using the Suc-LLVY-AMC fluorogenic substrate and UV illumination of the gel. Extracts from cells grown at 37°C showed a similar pattern. (C) Glycerol gradient analysis of sem1Δ proteasomes. Glycerol gradient fractions were evaluated for the proteasomes by immunoblot analysis with antibodies against the indicated proteins (top) and by CP peptidase activity assays (bottom). The anti-CP antibody was MCP231, which recognizes multiple α subunits. Asterisk indicates cross-reacting protein.

Glycerol gradient analysis of proteasome profiles showed a sem1Δ 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 sem1Δ 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 sem1Δ are greatly exacerbated by rpn10Δ

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 sem1Δ or rpn10Δ single mutants to sem1Δ rpn10Δ 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 sem1Δ single mutant displayed a modest shift of particles to species smaller than free RP. The rpn10Δ 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 sem1Δrpn10Δ 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 sem1Δ and sem1Δ rpn10Δ cells, as measured by CP activity assays (Fig. 5C).

We conclude that loss of Sem1, especially in an rpn10Δ background, impairs formation or stability of the 26S proteasome. The lid-base interaction in the RP is greatly reduced in the sem1Δ rpn10Δ double mutant, and the lid itself is not fully assembled (Fig. 5B and not shown). Interestingly, sem1Δ exhibited a mild sensitivity to UV irradiation, and this defect was greatly exacerbated in a sem1Δ rpn10Δ 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 sem1Δ and rpn10Δ single and double mutants suggests that an intact RP is required for the response to and/or repair of DNA damage.

By a genetic analysis of suppressors of Dsk2 overexpression-induced cell death, we have found that the S. cerevisiae Sem1 protein is a previously overlooked component of the lid subcomplex within the proteasome RP. While this paper was in preparation, another report showing that Sem1 is a lid subunit of the 26S proteasome was published (Sone et al., 2004); the biochemical work by these authors is fully consistent with our results. Our data also indicate that Sem1 contributes to proteasome function at least in part through its ability to stabilize the lid subcomplex and the lid-base interaction within the RP complex. Moreover, Sem1 and the Rpn10 RP subunit appear to act synergistically in both RP stability and DNA repair, indicating a requirement for an intact RP in the DNA damage response.

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 α 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/α3 (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-α 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 sem1Δ 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 β5 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 sem1Δ (Fig. 5D). In an rpn10Δ background, sem1Δ 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 sem1Δ rpn10Δ 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 sem1Δ rpn10Δ 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.

We thank R. Deshaies for plasmids and yeast strains, and D. Finley and C. Mann for antibodies. This work was supported by grants from the Ministry of Education, Science, Sports and Technology of Japan and in part by CREST, JST to H.K., and a grant from the NIH (GM46904) to M.H.

Adams, A., Gottschling, D. E., Kaiser, C. A. and Stearns, T. (
1997
). Gene replacement. In
Methods in Yeast Genetics
, pp.
59
-70. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Arendt, C. and Hochstrasser, M. (
1999
). Eukaryotic 20S proteasome catalytic subunit propeptides prevent active-site inactivation by N-terminal acetylation and promote particle assembly.
EMBO J.
18
,
3575
-3585.
Baumeister, W., Walz, J., Zuhl, F. and Seemuller, E. (
1998
). The proteasome: paradigm of a self-compartmentalizing protease.
Cell
92
,
367
-389.
Chen, L. and Madura, K. (
2002
). Rad23 promotes the targeting of proteolytic substrates to the proteasome.
Mol. Cell. Biol.
22
,
4902
-4913.
Chen, P., Johnson, P., Sommer, T., Jentsch, S. and Hochstrasser, M. (
1993
). Multiple ubiquitin-conjugating enzymes participate in the in vivo degradation of the yeast MATα2 repressor.
Cell
74
,
357
-369.
Crackower, M. A., Scherer, S. W., Rommens, J. M., Hui, C.-C., Poorkaj, P., Soder, S., Cobben, J. M., Hudgins, L., Evans, J. P. and Tsui, L.-C. (
1996
). Characterization of the split hand/split foot malformation locus SHFM1 at 7q21.3-q22.1 and analysis of a candidate gene for its expression during limb development.
Hum. Mol. Genet.
5
,
571
-579.
Dong, Y., Hakimi, M.-A., Chen, X., Kumaraswamy, E., Cooch, N. S., Godwin, A. K. and Shiekhattar, R. (
2003
). Regulation of BRCC, a holoenzyme complex containing BRCA1 and BRCA2, by a signalosome-like subunit and its role in DNA repair.
Mol. Cell
12
,
1087
-1099.
Dor, Y., Raboy, B. and Kulka, R. G. (
1996
). Role of the conserved carboxy-terminal alpha-helix of Rad6p in ubiquitination and DNA repair.
Mol. Microbiol.
21
,
1197
-1206.
Dreveny, I., Kondo, H., Uchiyama, K., Shaw, A., Zhang, X. and Freemont, P. S. (
2004
). Structural basis of the interaction between the AAA ATPase p97/VCP and its adaptor protein p47.
EMBO J.
23
,
1030
-1039.
Elsasser, S., Gali, R. R., Schwickart, M., Larsen, C. N., Leggett, D. S., Muller, B., Feng, M. T., Tubing, F., Dittmar, G. A. G. and Finley, D. (
2002
). Proteasome subunit Rpn1 binds ubiquitin-like protein domains.
Nat. Cell Biol.
4
,
725
-730.
Elsasser, S., Chandler-Militello, D., Muller, B., Hanna, J. and Finley, D. (
2004
). Rad23 and Rpn10 serve as alternative ubiquitin receptors for the proteasome.
J. Biol. Chem.
279
,
26817
-26822.
Fehlker, M., Wendler, P., Lehman, A. and Enenkel, C. (
2003
). Blm3 is part of nascent proteasomes and is involved in a late stage of nuclear proteasome assembly.
EMBO Rep.
4
,
1
-5.
Funakoshi, M., Sasaki, T., Nishimoto, T. and Kobayashi, H. (
2002
). Budding yeast Dsk2p is polyubiquitin-binding protein that can interact with the proteasome.
Proc. Natl. Acad. Sci. USA
99
,
745
-750.
Gillete, T. G., Huang, W., Russell, S. J., Reed, S. H., Johnston, S. A. and Friedberg, E. C. (
2001
). The 19S complex of the proteasome regulates nucleotide excision repair in yeast.
Genes Dev.
15
,
1528
-1539.
Glickman, M. H., Rubin, D. M., Coux, O., Wefes, I., Pfeifer, G., Cjeka, Z., Baumeister, W., Fried, V. A. and Finley, D. (
1998a
). A subcomplex of the proteasome regulatory particle required for ubiquitin-conjugate degradation and related to the COP9-signalosome and eIF3.
Cell
94
,
615
-623.
Glickman, M. H., Rubin, D. M., Fried, V. A. and Finley, D. (
1998b
). The regulatory particle of the Saccharomyces serevisiae proteasome.
Mol. Cell. Biol.
18
,
3149
-3162.
Hershko, A. and Ciechanover, A. (
1998
). The ubiquitin system.
Annu. Rev. Biochem.
67
,
425
-479.
Hochstrasser, M. (
2002
). New proteases in a ubiquitin stew.
Science
298
,
549
-552.
Jantti, J., Lahdenranta, J., Olkkonen, V. M., Soderlund, H. and Keranen, S. (
1999
). SEM1, a homologue of the split hand/split foot malformation candidate gene Dss1, regulates exocytosis and pseudohyphal differentiation in yeast.
Proc. Natl. Acad. Sci. USA
96
,
909
-914.
Kojic, M., Yang, H., Kostrub, C. F., Pavletich, N. P. and Holloman, W. K. (
2003
). The BRCA2-interacting protein DSS1 is vital for DNA repair, recombination, and genome stability in Ustilago maydis.
Mol. Cell
12
,
1043
-1049.
Leggett, D. S., Hanna, J., Borodovsky, A., Crosas, B., Schmidt, M., Baker, R. T., Walz, T., Ploegh, H. and Finley, D. (
2002
). Multiple associated proteins regulate proteasome structure and function.
Mol. Cell
10
,
495
-507.
Marston, N. J., Richards, W. J., Hughes, D., Bertwistle, D., Marshall, C. J. and Ashworth, A. (
1999
). Interaction between the product of the breast cancer susceptibility gene BRCA2 and DSS1, a protein functionally conserved from yeast to mammals.
Mol. Cell. Biol.
19
,
4633
-4642.
Orr-Weaver, L. T. and Szostak, W. J. (
1983
). Yeast Recombination: the association between double-strand gap repair and crossing-over.
Proc. Natl. Acad. Sci. USA
80
,
4417
-4421.
Podlaska, A., McIntyre, J., Skoneczna, A. and Sledziewska-Gojska, E. (
2003
). The link between 20S proteasome activity and post-replication DNA repair in Saccharomyces serevisiae.
Mol. Microbiol.
49
,
1321
-1332.
Runder, A. D., Hardwick, K. G. and Murray, A. (
2000
). Cdc28 activates exit from mitosis in budding yeast.
J. Cell Biol.
149
,
1361
-1376.
Russell, S. J., Reed, S. H., Huang, W., Friedberg, E. C. and Johnston, S. A. (
1999
). The 19S regulatory complex of the proteasome functions independently of proteolysis in nucleotide excision repair.
Mol. Cell
3
,
687
-695.
Saeki, Y., Toh-e, A. and Yokosawa, H. (
2000
). Rapid isolation and characterization of the yeast proteasome regulatory complex.
Biochem. Biophys. Res. Commun.
273
,
509
-515.
Santamaria, P. G., Finley, D., Ballesta, J. P. G. and Remacha, M. (
2003
). Rpn6, a proteasome subunit from Saccharomyces cerevisiae, is essential for the assembly and activity of the 26S proteasome.
J. Biol. Chem.
278
,
6687
-6695.
Schauber, C., Chen, L., Tongaonkar, P., Vega, I., Lambertson, D., Potts, W. and Madura, K. (
1998
). Rad23 links DNA repair to the ubiquitin/proteasome pathway.
Nature
391
,
715
-718.
Shin, D. S., Pellegrini, L., Daniels, D. S., Yelent, B., Craig, L., Bates, D., Yu, D. S., Shivji, M. K., Hitomi, C., Arvai, A. et al. (
2003
). Full-length archaeal Rad51 structure and mutants: mechanisms for RAD51 assembly and control by BRCA2.
EMBO J.
22
,
4566
-4576.
Sikorsky, R. S. and Hieter, P. (
1989
). A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces serevisiae.
Genetics
122
,
19
-27.
Sone, T., Saeki, Y., Toh-E, A., and Yokosawa, H. (
2004
). Sem1p is a novel subunit of the 26S proteasome from Saccharomyces cerevisiae.
J. Biol. Chem.
279
,
28807
-28816.
Takeuchi, J., Fujimuro, M., Yokosawa, H., Tanaka, K. and Toh-e, A. (
1999
). Rpn9 is required for efficient assembly of the yeast 26S proteasome.
Mol. Cell. Biol.
19
,
6575
-6584.
Tone, Y. and Toh-e, A. (
2002
). Nob1p is required for biogenesis of the 26S proteasome and degraded upon its maturation in Saccharomyces cerevisiae.
Genes Dev.
16
,
3142
-3157.
van Nocker, S., Sadis, S., Rubin, D. M., Glickman, M., Fu, H., Coux, O., Wefes, I., Finley, D. and Vierstra, R. D. (
1996
). The multiubiquitin-chain-binding protein Mcb1 is a component of the 26S proteasome in Saccharomyces cerevisiae and plays a nonessential, substrate-specific role in protein turnover.
Mol. Cell. Biol.
16
,
6020
-6028.
Velichutina, I., Connerly, P. L., Arendt, C. S., Li, X. and Hochstrasser, M. (
2004
). Plasticity in eucaryotic 20S proteasome ring assembly revealed by a subunit deletion in yeast.
EMBO J.
23
,
500
-510.
Verma, R., Chen, S., Feldman, R., Schieltz, D., Yates, J., Dohmen, J. and Deshaies, R. J. (
2000
). Proteasome proteomics: identification of nucleotide-sensitive proteasome-interacting proteins, by mass spectrometric analysis of affinity-purified proteasomes.
Mol. Biol. Cell
11
,
3425
-3439.
Verma, R., Oania, R., Graumann, J. and Deshaies, R. J. (
2004
). Multiubiquitin chain receptors define a layer of substrate selectivity in the ubiquitin-proteasome system.
Cell
118
,
99
-110.
Wilkinson, C. R. M., Seeger, M., Hartmann-Petersen, R., Stone, M., Wallace, M., Semple, C. and Gordon, C. (
2001
). Proteins containing the UBA domain are able to bind to multi-ubiquitin chains.
Nat. Cell Biol.
3
,
939
-943.
Xie, Y. and Varshavsky, A. (
2000
). Physical association of ubiquitin ligases and the 26S proteasome.
Proc. Natl. Acad. Sci. USA
97
,
2497
-2502.
Yang, H., Jeffrey, P. D., Miller, J., Kinnucan, E., Sun, Y., Thoma, N. H., Zheng, N., Chen, P.-L., Lee, W.-H. and Pavletich, N. P. (
2002
). BRCA2 function in DNA binding and recombination from a BRCA2-DSS1-ssDNA structure.
Science
297
,
1837
-1848.