Dissecting beta-ring assembly pathway of the mammalian 20S proteasome

Abstract

The 20S proteasome is the catalytic core of the 26S proteasome. It comprises four stacked rings of seven subunits each, alpha(1-7)beta(1-7)beta(1-7)alpha(1-7). Recent studies indicated that proteasome-specific chaperones and beta-subunit appendages assist in the formation of alpha-rings and dimerization of half-proteasomes, but the process involved in the assembly of beta-rings is poorly understood. Here, we clarify the mechanism of beta-ring formation on alpha-rings by characterizing assembly intermediates accumulated in cells depleted of each beta-subunit. Starting from beta2, incorporation of beta-subunits occurs in an orderly manner dependent on the propeptides of beta2 and beta5, and the C-terminal tail of beta2. Unexpectedly, hUmp1, a chaperone functioning at the final assembly step, is incorporated as early as beta2 and is required for the structural integrity of early assembly intermediates. We propose a model in which beta-ring formation is assisted by both intramolecular and extrinsic chaperones, whose roles are partially different between yeast and mammals.

Figure 1

Accumulation of distinct assembly intermediates in each β-subunit knockdown cells. The cell extracts (40 μg) used in Supplementary Figure S1 were separated by native PAGE. Assembly intermediates were detected by immunoblotting using the indicated antibodies (A, C–I) The bands corresponding to α-ring and the 20S proteasome as well as the locations of molecular size markers are depicted by arrowheads. (B) The peptide-hydrolysing activity was assayed by active staining of the gel using Suc-LLVY-MCA in the presence of SDS. Note that the 26S proteasome did not move inside the native PAGE gel.

Figure 2

Release of PAC3 is coupled with β3 incorporation. The same panels in Figure 1 were probed with anti-PAC1 (A) and -PAC3 (B) antibodies. The arrow indicates PAC3 species dissociated from proteasome precursors (B).

Figure 3

Role of hUmp1 in the structural integrity of early assembly intermediates. (A) The same panel in Figure 1 was probed with anti-hUmp1 antibody. (B–E) Extracts of HEK293T cells transfected with the indicated combinations of siRNAs were separated by native PAGE. Intermediate complexes were detected by immunoblotting using the indicated antibodies. The left lane representing β7 RNAi serves as a positive control for immunoblotting (C, D). Asterisks indicate nonspecific bands (A). (F) Flag–hUmp1 and each 20S subunit were co-translated and radio-labelled in reticulocyte lysates, immunoprecipitated with M2 agarose, and analysed by SDS–PAGE and autoradiography. ‘ALL' represents co-translation of all β-subunits together with hUmp1.

Figure 4

Both the propeptide and C-terminal tail of β2 are indispensable for β3 incorporation. Stable cell lines expressing the indicated mutant β2-subunits were treated with the siRNA targeting endogenous β2. Intermediate complexes were detected by immunoblotting using the indicated antibodies following native PAGE (A–D). Intermediates observed in β2^*ΔTail cells can be divided into two species; faster migrating ones (arrowheads) and slower migrating ones (vertical bars). The free complex of PAC3 is depicted by an arrow (D).

Figure 5

β5 propeptide is required for β6 incorporation. Stable cell lines expressing the indicated mutant β5-subunits were treated with the siRNA(s) for endogenous β5 or β6 (A–D), or for the indicated combinations (E, F). Cell extracts were resolved by native PAGE, followed by immunoblot analysis for the indicated antibodies.

Figure 6

C-terminal tail of β7 is essential for the incorporation of β7 and dimerization of half-mers. Stable cell lines expressing the indicated mutant β7-subunits were treated with siRNA targeting endogenous β7. Cell extracts were resolved by native PAGE, followed by immunoblot analysis using the indicated antibodies. The free form of β7^*Δtail is depicted by an arrow (B). The ‘half-mer' assembly intermediates are depicted by arrowheads (A, C).

Figure 7

A model for β-ring formation in mammalian 20S proteasome assembly. The roles of PAC1–PAC2 and PAC3 in the formation of α-rings were described previously (Hirano et al, 2005, 2006). PAC4 was recently identified as a heterodimeric partner of PAC3 (Le Tallec et al, 2007). Sequential incorporation of β-subunits starts from the association of β2 and hUmp1 on the α-ring. hUmp1 is required for the association of β2 in the early assembly intermediates. PAC3–PAC4, whose release is coupled with association of β3, holds the structural integrity of the intermediates until β3 is incorporated on the α-ring. Subsequent orderly incorporation of other β-subunits is also assisted by intramolecular chaperones such as the propeptides of β2 and β5 and the C-terminal tail of β2. Although β1 can be incorporated at various steps (dotted lines), such incorporation most likely follows that of β6. Dimerization of half-mers is assisted by the C-terminal tail of β7. This is followed by removal of β-subunit propeptides (β1, β2, β5, β6, and β7) and hUmp1 degradation. Essential propeptides, non-essential propeptides, and essential C-terminal tails of β-subunits for mammalian 20S proteasome biogenesis are depicted in red, blue, and yellow, respectively. See text for more details.