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. 2018 Dec 26;115(52):13246-13251.
doi: 10.1073/pnas.1805353115. Epub 2018 Dec 10.

Ubiquitin-dependent switch during assembly of the proteasomal ATPases mediated by Not4 ubiquitin ligase

Affiliations

Ubiquitin-dependent switch during assembly of the proteasomal ATPases mediated by Not4 ubiquitin ligase

Xinyi Fu et al. Proc Natl Acad Sci U S A. .

Abstract

In the proteasome holoenzyme, the hexameric ATPases (Rpt1-Rpt6) enable degradation of ubiquitinated proteins by unfolding and translocating them into the proteolytic core particle. During early-stage proteasome assembly, individual Rpt proteins assemble into the hexameric "Rpt ring" through binding to their cognate chaperones: Nas2, Hsm3, Nas6, and Rpn14. Here, we show that Rpt ring assembly employs a specific ubiquitination-mediated control. An E3 ligase, Not4, selectively ubiquitinates Rpt5 during Rpt ring assembly. To access Rpt5, Not4 competes with Nas2 until the penultimate step and then with Hsm3 at the final step of Rpt ring completion. Using the known Rpt-chaperone cocrystal structures, we show that Not4-mediated ubiquitination sites in Rpt5 are obstructed by Nas2 and Hsm3. Thus, Not4 can distinguish a Rpt ring that matures without these chaperones, based on its accessibility to Rpt5. Rpt5 ubiquitination does not destabilize the ring but hinders incorporation of incoming subunits-Rpn1 ubiquitin receptor and Ubp6 deubiquitinase-thereby blocking progression of proteasome assembly and ubiquitin regeneration from proteasome substrates. Our findings reveal an assembly checkpoint where Not4 monitors chaperone actions during hexameric ATPase ring assembly, thereby ensuring the accuracy of proteasome holoenzyme maturation.

Keywords: AAA+ ATPase; Not4; assembly chaperone; checkpoint; proteasome.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Not4 ubiquitin ligase regulates the progression of base assembly. (A) The not4-L35A catalytic mutants affect base assembly, as reflected in the Rpt5-Rpt4 module. Whole-cell extracts (80 μg) were analyzed by 3.5% native PAGE and immunoblotting for Rpt5, a subunit of the base. The not4-L35A allele is integrated into the chromosomal locus of NOT4. The plus (+) indicates the wild-type NOT4. Pgk1, loading control. (B) The not4 catalytic mutants exhibit enhanced progression of base assembly, as indicated by increased RP (base–lid) relative to the Rpt5-Rpt4 module. Assembly intermediates were affinity-purified via 3× FLAG-tagged Nas2 and were analyzed by 3.5% native PAGE and immunoblotting for the indicated Rpt proteins (i and ii). Nas2 serves as a loading control.
Fig. 2.
Fig. 2.
Not4 ubiquitin ligase provides inhibitory control during base assembly, as indicated by phenotypic suppression of the chaperone mutants by the not4 mutants. (A and B) Growth assays showing that the not4-L35A allele restores the growth of the chaperone-deletion mutants upon heat stress (Top). Threefold serial dilutions of indicated yeast cells were spotted onto yeast extract–peptone–dextrose plates and grown at the indicated temperature for 2–4 d.
Fig. 3.
Fig. 3.
Not4 ubiquitinates Rpt5 by competing with both Nas2 and Hsm3 in the base. (A) An experimental scheme for in vitro ubiquitination reactions. The heterohexameric Rpt ring is shown with specific positioning of individual Rpt subunits (Left) and their cognate chaperones (Right) (4, 20). (B) Not4 ubiquitinates Rpt5 in the base and RP. The base and RP (2 pmol each) were subjected to ubiquitination reactions and then analyzed by 10% Bis-Tris SDS/PAGE and immunoblotting for each Rpt subunit. Asterisk, nonspecific signal (SI Appendix, Fig. S5). (C and D) Nas2 and Hsm3 block Not4-mediated ubiquitination of Rpt5, whereas Nas6 and Rpn14 do not. Each chaperone was added at the indicated molar excess over the base (2 pmol) during ubiquitination reactions and then analyzed as in B. Rpt1, a loading control. (E and F) Not4 ubiquitinates the Rpt5 C-domain (Rpt5C). Ubiquitination reactions were conducted using the recombinant Rpt5C, full-length Rpt5, Rpt5-Rpt4 cocomplex and Rpt5ΔC lacking the C-domain (75 pmol each). In all cases, Rpt5 is His6-tagged. “None” in F indicates substrates only, without ubiquitination reaction. (G) Rpt5 (orange) in the heterohexameric Rpt ring [Protein Data Bank (PDB) ID code 4CR2] (42); Rpt1, gray; the other Rpt proteins, beige. Rpt5C is indicated by the dotted box. Arrowheads indicate Not4-mediated ubiquitination sites: K411, K426, and K428 (SI Appendix, Fig. S7). GI were generated by University of California, San Francisco Chimera (43). (H and I) Rpt5 ubiquitination sites (yellow highlights) are obstructed by Nas2 C-domain (magenta, PDB ID code 4O06; SI Appendix, Fig. S8) (21, 44) in H and Hsm3 from Rpt1-Hsm3 cocrystal structure (PDB ID code 4JPO) (21), which was superimposed onto the heterohexameric Rpt ring structure (PDB ID code 4CR2) (42) in I. Rpt1 is shown in gray.
Fig. 4.
Fig. 4.
Not4-mediated ubiquitination of Rpt5 blocks recruitment of Ubp6 and Rpn1 during endogenous base assembly. (A and B) Not4-mediated ubiquitination of Rpt5 blocks incorporation of Rpn1 and Ubp6 into the base. Assembly intermediates were isolated using 3× FLAG-tagged Nas6 and were analyzed by 4–12% Bis-Tris SDS/PAGE and immunoblotting for indicated proteins in i and iii (See SI Appendix, Supplementary Materials and Methods for details). Rpn1-containing species were detected by 3.5% native PAGE and immunoblotting (ii). Nas6, a loading control (iii). (C) Not4-mediated control of proteasome assembly regulates the free ubiquitin pool. Whole-cell lysates (20 μg) were subjected to 10% Bis-Tris SDS/PAGE and immunoblotted for ubiquitin (SI Appendix, Supplementary Materials and Methods). Pgk1, loading control. Relative signal intensities of free ubiquitin bands were quantified (n = 4; mean ± SEM; ns, not significant; *P < 0.05; **P < 0.01).

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