Using a proteomic approach to identify proteasome interacting proteins in mammalian

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1 Supplementary Discussion Using a proteomic approach to identify proteasome interacting proteins in mammalian cells, we describe in the present study a novel chaperone complex that plays a key role in the 5 assembly of mammalian 20S proteasomes, named PAC1 and PAC2. The assembly of 20S proteasomes is a multistep-ordered process, and presumptive α-ring formation represents the initial step. At present, the process of α-ring formation is considered spontaneous. This is based on the observations that the α subunit of archaebacteria 1, Trypanosoma brucei 2 and human 3 can form homomeric seven-membered 10 rings when expressed in E. coli. However, no homoheptameric rings occur in vivo in eukaryotic proteasomes, whose α-ring is invariably composed of seven different α subunits. In addition, in yeast all α subunits except α3 are essential for life 4-6. These observations imply that there is a mechanism that correctly arranges the seven α subunits into a heteroheptamer, but such mechanism had remained entirely unknown. 15 In the present study, we identified the complex containing all seven α subunits but no β subunits with the size of approximately 230 kda, which is most likely to represent α-rings associated with PAC1/PAC2 heterodimer (Fig. 1). The PAC complex was most abundantly associated with α-rings, indicating its role in α-ring assembly. Indeed, IVTT experiment suggested that the PAC complex promoted the gathering of α subunits (Supplementary Fig. 20 4). In cells, we demonstrated that it was also bound to early α subunit assembly intermediates that contained a restricted subset of α subunits. These results indicate that the formation of α-rings of mammalian 20S proteasomes is chaperoned by the PAC1/PAC2 complex.

2 Knockdown of the PAC complex revealed its additional but important role in proteasome assembly. Glycerol gradient analysis demonstrated accumulation of α subunits in 25 fractions corresponding to half-proteasomes without any accompanying increase in pro-β subunits or hump1 (Fig. 3b), which are hallmarks of half-proteasomes. Indeed, this fraction in PAC-knockdown cells contained much smaller amounts of pro-β subunits and hump1 relative to α subunits (Fig. 3c). Considering its size and content, it is most likely that this fraction results from dimerization of α-rings. It is uncertain whether the ring is normally 30 organized or not in the absence of the PAC complex, but considering the milder defect in proteasome function in PAC-knockdown cells than hump1-knockdown cells, at least part of the α-rings was normal, which could be incorporated into functional half-proteasomes and 20S proteasomes. Since human α subunits expressed in E. coli tend to form a double ring-like structure 3, prevention of formation of double α-ring can be important for proper assembly of 35 20S proteasomes (see our model, Fig. 4d). Our data indicate that the PAC complex is responsible for suppressing the formation of off-pathway, non-productive α-ring dimers. As for chaperones for the 20S proteasome assembly, it has been shown that Ump1 is included in half-proteasomes and is required for the coordination between correct maturation of β subunits and dimerization of half-proteasomes in yeast 7. Several studies have suggested 40 that the mammalian homolog of Ump1 operates in a similar fashion. We investigated the relationship between the PAC complex and hump1. The PAC complex was directly associated with at least some of the α subunits (Supplementary Fig. 4a), whereas hump1 is reported to bind to certain β subunits but not to any of α subunits in yeast two hybrid analysis 8. Glycerol gradient analysis of cell extracts revealed that hump1 specifically 45 associates with half-proteasomes and is not associated with α-rings, unlike the PAC complex

3 (Fig. 1). Though both the PAC complex and hump1 were included in half-proteasomes, they did not directly associate with each other in vitro (Supplementary Fig. 2c). RNAi experiments further distinguished their functional differences. Knockdown of PACs resulted in loss of normal α-rings, whereas knockdown of hump1 was associated with defective dimerization of 50 half-proteasomes though the α-rings and half-proteasomes were maintained. These results demonstrate that the PAC complex is a chaperone devoted to α-rings, whereas hump1 is dedicated to half-proteasome formation, taking over the duty of the PAC complex (Fig. 4d). MG132 treatment elicited another difference. The PAC complex accumulated in 20S proteasome fractions, whereas hump1 did not. hump1 is likely to be located at the interface 55 between two β-rings, as predicted in yeast analysis 7, and its degradation is tightly coupled with 20S proteasome formation. On the other hand, the PAC complex is likely to be located at the side of α-ring opposite to hump1 and β-ring, since it directly binds to α subunits and is still a component of half-proteasomes without inhibiting the assembly of hump1 and β subunits on α-rings. This indicates that the PAC complex constitutively binds α subunits 60 whether in precursor or mature proteasomes and that it is continually degraded by 20S proteasomes. Therefore, when proteasome activity is inhibited, PACs still associate with the ends of 20S proteasomes (Figs. 1 and 4d). The precise mechanism of PAC1/PAC2 degradation by 20S proteasomes is not clear at present, but it is plausible that 20S proteasomes directly degrade PAC1/PAC2 through a mechanism similar to that employed for 65 the degradation of naturally unfolded polypeptides 9 such as α-synuclein 10, tau 11 and p21 Cip1 (refs 12, 13), or localization of the PAC complex to the proteasome may be sufficient for degradation 14. In this context, it is intriguing whether triplication of PAC1 gene in Down

4 syndrome overloads proteasomes and contributes to the pathogenesis of various disease entities. 70 Another question is whether this system is conserved throughout eukaryotes. PAC2 seems to have a yeast homolog, YKL206C, with 19% identity to the mammalian gene. YKL206C is known to interact with proteasomes, but its null mutant is viable and showed no obvious phenotypes (data not shown). Thus, YKL206C might be the ancestral gene of PAC2 but does not appear to be a functional homolog. As for PAC1, we could find its orthologous 75 genes only in vertebrates. Whereas the individual α subunits and β subunits are approximately 50-60% identical in humans and yeast, Ump1 and PAC2 are only 27% and 19% identical, respectively. Therefore, the apparent lack of yeast or invertebrate orthologues of human PAC1 may simply reflect low amino acid sequence conservation. Recent proteomic approaches in yeast have identified many proteasome interacting proteins with yet unknown 80 functions, among which there may be a functional orthologue of PAC1. Alternatively, it is possible that PAC1 is an invention of vertebrates because there are significant differences between human and yeast in the assembly of 20S proteasomes. As we reported here, hump1 did not accumulate in 20S proteasomes upon inhibition of proteasome activity (Fig. 1b), whereas yeast Ump1 was 7. Instead, hump1 appeared in lighter fractions (fractions 2, 4, and 6 85 of Supplementary Fig. 1b), presumably in a free form. In addition, in human cell lines, disturbance of the assembly pathway did not cause accumulation of unprocessed β subunits as free forms (Figs. 1a and 3), which was clearly observed in yeast 7. In human cells, unassembled subunits may be subjected to rapid degradation by protein quality control system. Structural differences have also been identified in each α subunit between mammals 90 and yeast 15, though the subunit composition and arrangement of mammalian 20S proteasomes

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