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S5b Joins the Ranks of 26S Proteasome Assembly Chaperones
Molecular Cell
Previews Hsm3/S5b Joins the Ranks of 26S Proteasome Assembly Chaperones Daniela Go¨dderz1 and R. Ju¨rgen Dohmen1,* 1Institute for Genetics, University of Cologne, Zu ¨ lpicher Strasse 47, D-50674 Cologne, Germany *Correspondence: [email protected] DOI 10.1016/j.molcel.2009.02.007
In a recent issue of Molecular Cell, Le Tallec et al. (2009) characterize the yeast Hsm3 protein, an apparent ortholog of human S5b, as a dedicated chaperone that promotes assembly of the base subcomplex of the 19S proteasome activator. The 26S proteasome is an essential ATPdependent protease found in the cytosol and nucleus of eukaryotic cells that mediates degradation mainly of ubiquitylated proteins. It comprises 33 different proteins, which assemble into two distinct complexes, the 20S core particle (CP) and the 19S regulatory particle (RP). The 19S RP is a dynamic structure containing at least 19 subunits that are distributed between two subcomplexes, the base and the lid (Glickman et al., 1998; Murata et al., 2009). The base bears unfoldase activity that resides within a hexameric ATPase module and the non-ATPase subunits Rpn1, Rpn2, and Rpn13, which directly or indirectly participate in binding ubiquitylated substrates and deubiquitylating enzymes. The lid contains five PCI domain-containing and two MPN domaincontaining subunits that are related to subunits of the COP9 signalosome and eIF3. One of the MPN domain-containing subunits, Rpn11, provides the 19S RP with an intrinsic deubiquitylating activity (Murata et al., 2009, and references therein). 20S CP assembly has been intensely studied. Its formation from 14 distinct a and b subunits is a multistep process involving several dedicated chaperones (Ramos and Dohmen, 2008, and references therein) (Figure 1). By contrast, comparably little was known about 19S RP assembly (Murata et al., 2009). Peyroche and colleagues now provide a major contribution to filling in this gap with their identification of Hsm3/S5b as the first chaperone known to be dedicated to 19S RP assembly (Le Tallec et al., 2009). This and a previous study are based upon a genetic screen in which they analyzed a library of Saccharomyces
cerevisiae gene deletion mutants to identify mutations that suppress the lethal effect of a dominant-negative mutant of the DNA checkpoint protein kinase Rad53 (Le Tallec et al., 2007). Surprisingly, many of the suppressor mutants were directly linked to the 26S proteasome: they encoded either nonessential proteasome subunits such as Pre9/a3 or Rpn15/Sem1 (ortholog of human DSS1), or chaperones that promote the generation of the 26S proteasome (Le Tallec et al., 2007, 2009). Excitingly, one of the genes that emerged from this screen, HSM3, is now shown to encode the fourth known proteasome assembly chaperone (Le Tallec et al., 2009). hsm3 mutants were originally identified owing to their slightly higher spontaneous mutagenesis rates (Fedorova et al., 1998), but HSM3 is now clearly identified as a member of the proteasome epistasis group. The observed suppression of the dominant RAD53 mutation and the weak mutator phenotype caused by hsm3D have not been further investigated but presumably relate to a function of the proteasome in DNA damage repair processes (Reed and Gillette, 2007). Biochemical experiments revealed that Hsm3 associates mainly with base intermediates, and to a lesser extent with 19S RPs, suggesting that Hsm3 association is weakened when base and lid subcomplexes come together. Although no apparent effects on proteasome levels were observed in extracts from hsm3D cells grown at 30 C, severe defects were detected after shifting these cells to 37 C for 6 hr. Interestingly, whereas lid complex assembly appeared to be unaffected, base complexes and 19S RPs were not formed efficiently under these
conditions. Together, these findings characterize Hsm3 as an assembly chaperone for the base subcomplex in yeast. By sequence, Hsm3 is remotely related to the human S5b protein (Le Tallec et al., 2009). S5b, which was identified in preparations of proteasome complexes from red blood cells, forms complexes with Rpt1, Rpt2, and Rpn1 in vitro (Deveraux et al., 1995; Gorbea et al., 2000). Similar to Hsm3, tagged S5b efficiently coprecipitates with subunits of the 19S RP, but not with those of the 20S CP (Le Tallec et al., 2009). Together, these findings support the notion that Hsm3 and S5b represent a conserved 19S RP assembly chaperone. The order of events in 26S proteasome biogenesis has remained somewhat puzzling. Does binding of the lid to the base occur prior to its association with the CP (as depicted in Figure 1), or vice versa? The observation that Hsm3 is absent from complexes containing CP subunits but is present in a fraction of complexes containing base and lid subunits on one hand argues in favor of 19S RP formation prior to CP docking (Le Tallec et al., 2009). Interestingly, both Hsm3 and S5b directly bind a C-terminal part of the Rpt1 subunit, and a C-terminal HbYX motif of this ATPase subunit was previously shown to be critical for stable interaction of 19S RP and 20S CP (Le Tallec et al., 2009; Smith et al., 2007). As Hsm3 release from the Rpt1 subunit seems to coincide with the association of base and lid, it is therefore a plausible idea that Hsm3 provides a checkpoint function by preventing the interaction of the Rpt1 C terminus with the CP until 19S RP formation is complete (Le Tallec et al., 2009). If true, base complexes which, for example, accumulate in rpn15D mutants
Molecular Cell 33, February 27, 2009 ª2009 Elsevier Inc. 415
Molecular Cell
Previews
Figure 1. Schematic Model of 26S Proteasome Assembly Assembly of 28 a and b subunits into the 20S CP involves the chaperone complexes PAC1-PAC2 and PAC3-PAC4 as well as UMP1 (for details, see Ramos and Dohmen, 2008). The 19S RP is formed by six ATPase (Rpt) subunits (t1–t6) and 12 non-ATPase (Rpn) subunits (n1–n3, n5–n13, and n15/Sem1/DSS1). The assembly of these subunits occurs via the formation of two subcomplexes, the base and the lid (Murata et al., 2009). Base subcomplex assembly is promoted by Hsm3 (ortholog of human S5b), a protein that interacts directly with the Rpt1 C terminus. Hsm3 is released when the lid binds to the base. Formation of the 26S proteasome from two RPs and one CP requires ATP binding, and it critically depends on C-terminal HbYX (Y) motifs present in Rpt subunits. Complexes that accumulate in hsm3D or rpn7-3 mutants are indicated. MPN or PCI domain subunits in the lid are shown in purple and blue, respectively. Note that the exact relative positions of subunits in the base and lid subcomplexes are unknown.
would be expected to exhibit a higher tendency to associate with the CP in the absence of Hsm3, a testable prediction, as base complexes form relatively efficiently in hsm3D cells grown at 30 C. On the other hand, the observation that mutants impaired in CP formation accumulate abnormal 19S RP subcomplexes suggests that the CP might serve as a platform to promote 19S RP assembly (Kusmierczyk et al., 2008). A scenario that could explain both observations is that fully assembled 19S RP complexes might be unstable in vivo in the absence of sufficient amounts of 20S CP, or that stabilizing events occur only after docking of the 19S RP to the CP. Indeed, it remains unclear when Rpn10, a subunit that can stabilize the base-lid association (Glickman et al., 1998), joins the complex. Rpn10 did not
copurify with Hsm3-containing base precursor complexes and is therefore expected to enter the assembly pathway at the step of subcomplex dimerization or later (Le Tallec et al., 2009). The discovery of Hsm3/S5b as a dedicated chaperone of 19S particle assembly represents a major step forward in gaining a complete understanding of 26S proteasome biogenesis.
REFERENCES Deveraux, Q., Jensen, C., and Rechsteiner, M. (1995). J. Biol. Chem. 270, 23726–23729.
Gorbea, C., Taillandier, D., and Rechsteiner, M. (2000). J. Biol. Chem. 275, 875–882. Kusmierczyk, A.R., Kunjappu, M.J., Funakoshi, M., and Hochstrasser, M. (2008). Nat. Struct. Mol. Biol. 15, 237–244. Le Tallec, B., Barrault, M.B., Courbeyrette, R., Gue´rois, R., Marsolier-Kergoat, M.C., and Peyroche, A. (2007). Mol. Cell 27, 660–674. Le Tallec, B., Barrault, M.-B., Gue´rois, R., Carre´, T., and Peyroche, A. (2009). Mol. Cell 33, 389–399. Murata, S., Yashiroda, H., and Tanaka, K. (2009). Nat. Rev. Mol. Cell Biol. 10, 104–115. Ramos, P.C., and Dohmen, R.J. (2008). Structure 16, 1296–1304.
Fedorova, I.V., Gracheva, L.M., Kovaltzova, S.V., Evstuhina, T.A., Alekseev, S.Y., and Korolev, V.G. (1998). Genetics 148, 963–973.
Reed, S.H., and Gillette, T.G. (2007). DNA Repair (Amst.) 6, 149–156.
Glickman, M.H., Rubin, D.M., Coux, O., Wefes, I., Pfeifer, G., Cjeka, Z., Baumeister, W., Fried, V.A., and Finley, D. (1998). Cell 94, 615–623.
Smith, D.M., Chang, S.C., Park, S., Finley, D., Cheng, Y., and Goldberg, A.L. (2007). Mol. Cell 27, 731–744.
416 Molecular Cell 33, February 27, 2009 ª2009 Elsevier Inc.
Previews Hsm3/S5b Joins the Ranks of 26S Proteasome Assembly Chaperones Daniela Go¨dderz1 and R. Ju¨rgen Dohmen1,* 1Institute for Genetics, University of Cologne, Zu ¨ lpicher Strasse 47, D-50674 Cologne, Germany *Correspondence: [email protected] DOI 10.1016/j.molcel.2009.02.007
In a recent issue of Molecular Cell, Le Tallec et al. (2009) characterize the yeast Hsm3 protein, an apparent ortholog of human S5b, as a dedicated chaperone that promotes assembly of the base subcomplex of the 19S proteasome activator. The 26S proteasome is an essential ATPdependent protease found in the cytosol and nucleus of eukaryotic cells that mediates degradation mainly of ubiquitylated proteins. It comprises 33 different proteins, which assemble into two distinct complexes, the 20S core particle (CP) and the 19S regulatory particle (RP). The 19S RP is a dynamic structure containing at least 19 subunits that are distributed between two subcomplexes, the base and the lid (Glickman et al., 1998; Murata et al., 2009). The base bears unfoldase activity that resides within a hexameric ATPase module and the non-ATPase subunits Rpn1, Rpn2, and Rpn13, which directly or indirectly participate in binding ubiquitylated substrates and deubiquitylating enzymes. The lid contains five PCI domain-containing and two MPN domaincontaining subunits that are related to subunits of the COP9 signalosome and eIF3. One of the MPN domain-containing subunits, Rpn11, provides the 19S RP with an intrinsic deubiquitylating activity (Murata et al., 2009, and references therein). 20S CP assembly has been intensely studied. Its formation from 14 distinct a and b subunits is a multistep process involving several dedicated chaperones (Ramos and Dohmen, 2008, and references therein) (Figure 1). By contrast, comparably little was known about 19S RP assembly (Murata et al., 2009). Peyroche and colleagues now provide a major contribution to filling in this gap with their identification of Hsm3/S5b as the first chaperone known to be dedicated to 19S RP assembly (Le Tallec et al., 2009). This and a previous study are based upon a genetic screen in which they analyzed a library of Saccharomyces
cerevisiae gene deletion mutants to identify mutations that suppress the lethal effect of a dominant-negative mutant of the DNA checkpoint protein kinase Rad53 (Le Tallec et al., 2007). Surprisingly, many of the suppressor mutants were directly linked to the 26S proteasome: they encoded either nonessential proteasome subunits such as Pre9/a3 or Rpn15/Sem1 (ortholog of human DSS1), or chaperones that promote the generation of the 26S proteasome (Le Tallec et al., 2007, 2009). Excitingly, one of the genes that emerged from this screen, HSM3, is now shown to encode the fourth known proteasome assembly chaperone (Le Tallec et al., 2009). hsm3 mutants were originally identified owing to their slightly higher spontaneous mutagenesis rates (Fedorova et al., 1998), but HSM3 is now clearly identified as a member of the proteasome epistasis group. The observed suppression of the dominant RAD53 mutation and the weak mutator phenotype caused by hsm3D have not been further investigated but presumably relate to a function of the proteasome in DNA damage repair processes (Reed and Gillette, 2007). Biochemical experiments revealed that Hsm3 associates mainly with base intermediates, and to a lesser extent with 19S RPs, suggesting that Hsm3 association is weakened when base and lid subcomplexes come together. Although no apparent effects on proteasome levels were observed in extracts from hsm3D cells grown at 30 C, severe defects were detected after shifting these cells to 37 C for 6 hr. Interestingly, whereas lid complex assembly appeared to be unaffected, base complexes and 19S RPs were not formed efficiently under these
conditions. Together, these findings characterize Hsm3 as an assembly chaperone for the base subcomplex in yeast. By sequence, Hsm3 is remotely related to the human S5b protein (Le Tallec et al., 2009). S5b, which was identified in preparations of proteasome complexes from red blood cells, forms complexes with Rpt1, Rpt2, and Rpn1 in vitro (Deveraux et al., 1995; Gorbea et al., 2000). Similar to Hsm3, tagged S5b efficiently coprecipitates with subunits of the 19S RP, but not with those of the 20S CP (Le Tallec et al., 2009). Together, these findings support the notion that Hsm3 and S5b represent a conserved 19S RP assembly chaperone. The order of events in 26S proteasome biogenesis has remained somewhat puzzling. Does binding of the lid to the base occur prior to its association with the CP (as depicted in Figure 1), or vice versa? The observation that Hsm3 is absent from complexes containing CP subunits but is present in a fraction of complexes containing base and lid subunits on one hand argues in favor of 19S RP formation prior to CP docking (Le Tallec et al., 2009). Interestingly, both Hsm3 and S5b directly bind a C-terminal part of the Rpt1 subunit, and a C-terminal HbYX motif of this ATPase subunit was previously shown to be critical for stable interaction of 19S RP and 20S CP (Le Tallec et al., 2009; Smith et al., 2007). As Hsm3 release from the Rpt1 subunit seems to coincide with the association of base and lid, it is therefore a plausible idea that Hsm3 provides a checkpoint function by preventing the interaction of the Rpt1 C terminus with the CP until 19S RP formation is complete (Le Tallec et al., 2009). If true, base complexes which, for example, accumulate in rpn15D mutants
Molecular Cell 33, February 27, 2009 ª2009 Elsevier Inc. 415
Molecular Cell
Previews
Figure 1. Schematic Model of 26S Proteasome Assembly Assembly of 28 a and b subunits into the 20S CP involves the chaperone complexes PAC1-PAC2 and PAC3-PAC4 as well as UMP1 (for details, see Ramos and Dohmen, 2008). The 19S RP is formed by six ATPase (Rpt) subunits (t1–t6) and 12 non-ATPase (Rpn) subunits (n1–n3, n5–n13, and n15/Sem1/DSS1). The assembly of these subunits occurs via the formation of two subcomplexes, the base and the lid (Murata et al., 2009). Base subcomplex assembly is promoted by Hsm3 (ortholog of human S5b), a protein that interacts directly with the Rpt1 C terminus. Hsm3 is released when the lid binds to the base. Formation of the 26S proteasome from two RPs and one CP requires ATP binding, and it critically depends on C-terminal HbYX (Y) motifs present in Rpt subunits. Complexes that accumulate in hsm3D or rpn7-3 mutants are indicated. MPN or PCI domain subunits in the lid are shown in purple and blue, respectively. Note that the exact relative positions of subunits in the base and lid subcomplexes are unknown.
would be expected to exhibit a higher tendency to associate with the CP in the absence of Hsm3, a testable prediction, as base complexes form relatively efficiently in hsm3D cells grown at 30 C. On the other hand, the observation that mutants impaired in CP formation accumulate abnormal 19S RP subcomplexes suggests that the CP might serve as a platform to promote 19S RP assembly (Kusmierczyk et al., 2008). A scenario that could explain both observations is that fully assembled 19S RP complexes might be unstable in vivo in the absence of sufficient amounts of 20S CP, or that stabilizing events occur only after docking of the 19S RP to the CP. Indeed, it remains unclear when Rpn10, a subunit that can stabilize the base-lid association (Glickman et al., 1998), joins the complex. Rpn10 did not
copurify with Hsm3-containing base precursor complexes and is therefore expected to enter the assembly pathway at the step of subcomplex dimerization or later (Le Tallec et al., 2009). The discovery of Hsm3/S5b as a dedicated chaperone of 19S particle assembly represents a major step forward in gaining a complete understanding of 26S proteasome biogenesis.
REFERENCES Deveraux, Q., Jensen, C., and Rechsteiner, M. (1995). J. Biol. Chem. 270, 23726–23729.
Gorbea, C., Taillandier, D., and Rechsteiner, M. (2000). J. Biol. Chem. 275, 875–882. Kusmierczyk, A.R., Kunjappu, M.J., Funakoshi, M., and Hochstrasser, M. (2008). Nat. Struct. Mol. Biol. 15, 237–244. Le Tallec, B., Barrault, M.B., Courbeyrette, R., Gue´rois, R., Marsolier-Kergoat, M.C., and Peyroche, A. (2007). Mol. Cell 27, 660–674. Le Tallec, B., Barrault, M.-B., Gue´rois, R., Carre´, T., and Peyroche, A. (2009). Mol. Cell 33, 389–399. Murata, S., Yashiroda, H., and Tanaka, K. (2009). Nat. Rev. Mol. Cell Biol. 10, 104–115. Ramos, P.C., and Dohmen, R.J. (2008). Structure 16, 1296–1304.
Fedorova, I.V., Gracheva, L.M., Kovaltzova, S.V., Evstuhina, T.A., Alekseev, S.Y., and Korolev, V.G. (1998). Genetics 148, 963–973.
Reed, S.H., and Gillette, T.G. (2007). DNA Repair (Amst.) 6, 149–156.
Glickman, M.H., Rubin, D.M., Coux, O., Wefes, I., Pfeifer, G., Cjeka, Z., Baumeister, W., Fried, V.A., and Finley, D. (1998). Cell 94, 615–623.
Smith, D.M., Chang, S.C., Park, S., Finley, D., Cheng, Y., and Goldberg, A.L. (2007). Mol. Cell 27, 731–744.
416 Molecular Cell 33, February 27, 2009 ª2009 Elsevier Inc.