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. 2019 May 31;9(6):213.
doi: 10.3390/biom9060213.

In-depth Analysis of the Lid Subunits Assembly Mechanism in Mammals

Minghui Bai  1 , Xian Zhao  2 , Kazutaka Sahara  3 , Yuki Ohte  4 , Yuko Hirano  5 , Takeumi Kaneko  6 , Hideki Yashiroda  7 , Shigeo Murata  8
Affiliations

In-depth Analysis of the Lid Subunits Assembly Mechanism in Mammals

Minghui Bai et al. Biomolecules. .

Abstract

The 26S proteasome is a key player in the degradation of ubiquitinated proteins, comprising a 20S core particle (CP) and a 19S regulatory particle (RP). The RP is further divided into base and lid subcomplexes, which are assembled independently from each other. We have previously demonstrated the assembly pathway of the CP and the base by observing assembly intermediates resulting from knockdowns of each proteasome subunit and the assembly chaperones. In this study, we examine the assembly pathway of the mammalian lid, which remains to be elucidated. We show that the lid assembly pathway is conserved between humans and yeast. The final step is the incorporation of Rpn12 into the assembly intermediate consisting of two modular complexes, Rpn3-7-15 and Rpn5-6-8-9-11, in both humans and yeast. Furthermore, we dissect the assembly pathways of the two modular complexes by the knockdown of each lid subunit.

Keywords: 19S regulatory particle; 26S proteasome; Rpn proteins; assembly; lid subcomplex.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Interdependence between the lid subunits for protein expression. (A) siRNAs targeting Rpn3, Rpn5–Rpn9, Rpn11, Rpn12, and Rpn15 were transfected into HEK293T cells. The cell extracts (20 μg) were then separated by native PAGE and were detected by immunoblot (IB) using antibodies against Rpn3, Rpn5–Rpn9, Rpn11, Rpn12, Rpt6, and α6. (B) The Suc-LLVY-AMC hydrolyzing activity of the knockdown of Rpn3, Rpn5–Rpn9, Rpn11, Rpn12, and Rpn15 was measured without added SDS (mean ± SD, n = 3).
Figure 2
Figure 2
Rpn12 is incorporated at the final step of lid formation. (A) HEK293T cells stably expressing Rpn7-Flag were lysed and subjected to 4%–24% glycerol gradient centrifugation. The fractions were analyzed by immunoblotting with anti-Rpn3, Rpn5–Rpn9, Rpn11, Rpn12, Rpt6, and α6 antibodies. Fractions corresponding to the accumulated complexes (boxed in red) were immunoprecipitated (IP) with the anti-Flag antibody. The precipitated complexes were analyzed by LC-MALDI followed by tandem mass spectrometry. The table shows the sequence coverage of the identified subunits. (B) siRNA targeting Rpn12 was transfected into HEK293T cells for 48 h and then analyzed as described in Figure 2A.
Figure 3
Figure 3
Rpn6 is required for the interaction between Rpn3-7-15 and Rpn5-8-9-11, and for Rpn11 stability. HEK293T cells stably expressing Rpn5-Flag and Rpn7-Flag treated with siRNA targeting Rpn6 for 48 h were analyzed in the same way as described in Figure 2. The table shows the sequence coverage of the identified subunits.
Figure 4
Figure 4
Loss of Rpn11 does not affect the assembly of other lid subunits but affects lid–base joining. HEK293T cells stably expressing Rpn5-Flag and Rpn7-Flag treated with siRNA targeting Rpn11 were analyzed for 48 h in the same way as described in Figure 2. The table shows the sequence coverage of the identified subunits.
Figure 5
Figure 5
Interdependent assembly of the Rpn5-8-9 complex. HEK293T cells stably expressing Rpn7-Flag and Rpn9-Flag treated with siRNA targeting Rpn5 (A), cells stably expressing Rpn5-Flag and Rpn7-Flag treated with siRNA targeting Rpn8 (B), and cells stably expressing Rpn5-Flag and Rpn7-Flag treated with siRNA targeting Rpn9 (C) were analyzed in the same way as described in Figure 2. The tables show the sequence coverage of the identified subunits.
Figure 6
Figure 6
The Rpn7–Rpn6 interaction connects the Rpn3-7-15 module with the Rpn5-6-8-9-11 module. HEK293T cells stably expressing Rpn7-Flag treated with siRNA targeting Rpn3 (A), cells stably expressing Rpn3-Flag and Rpn5-Flag treated with siRNA targeting Rpn7 (B), and cells stably expressing Rpn5-Flag and Rpn7-Flag treated with siRNA targeting Rpn15 (C) were analyzed in the same way as described in Figure 2. The tables show the sequence coverage of the identified subunits.
Figure 7
Figure 7
Rpn15 mediates the association of Rpn3 with Rpn7. siRNA targeting Rpn7, Rpn3, and Rpn6 was performed using HEK293T cells stably expressing Rpn15-GFP. Accumulated intermediates were collected and immunoprecipitated with anti-GFP antibody. When Rpn7 was knocked down, only Rpn3 was coimmunoprecipitated with Rpn15. None of the lid subunits was identified when Rpn3 was knocked down. Intermediates of Rpn3-7-15 were detected when Rpn6 was knocked down.
Figure 8
Figure 8
Rpn15 directly binds to Rpn3 and promotes Rpn3–Rpn7 association in vitro. Flag-Rpn3, Rpn7, and GFP-Rpn15 (A) or Flag-Rpn15, Rpn3, and Rpn7 (B) were cotranscribed/cotranslated and 35S-radiolabeled in reticulocyte lysates in various combinations as indicated. Anti-Flag agarose beads were added to the reaction mixture, and the immunoprecipitates were run on an SDS-PAGE gel. Coimmunoprecipitated proteins with Flag-Rpn3 (A) or Flag-Rpn15 (B) were visualized using autoradiography.
Figure 9
Figure 9
Assembly pathway of the mammalian proteasome lid subcomplex. The assembly of the lid subcomplex starts with the formation of two independent intermediates: Rpn3-15 and Rpn5-8-9. Then, Rpn7 and Rpn6 are incorporated into Rpn3-15 and Rpn5-8-9, forming Rpn3-7-15 and Rpn5-6-8-9, respectively. The Rpn5-6-8-9 complex is a prerequisite for incorporation of Rpn11. Rpn12 is the last subunit to be incorporated.
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