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

Minghui Bai,Kazutaka Sahara,Yuki Ohte,Xian Zhao,Yuko Hirano,Shigeo Murata,Hideki Yashiroda,Takeumi Kaneko

doi:10.3390/biom9060213

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.

Highlights

Protein degradation exerted by the ubiquitin–proteasome system starts with the conjugation of ubiquitin chains to target proteins
We investigated the biogenesis of the mammalian lid subcomplex using a combination of RNA interference and mass spectrometry. siRNA knockdown of each lid subunit caused a characteristic change in distributions of the other lid subunits separated by glycerol gradient centrifugation
To investigate the assembly pathway of the human lid subcomplex, we performed the knockdown of each lid subunit using siRNAs targeting Rpn3, Rpn5–Rpn9, Rpn11, Rpn12, and Rpn15 in HEK293T

Summary

Introduction

Protein degradation exerted by the ubiquitin–proteasome system starts with the conjugation of ubiquitin chains to target proteins. Polyubiquitinated proteins are recognized and captured by the 26S proteasome and digested to short peptide fragments [1]. The ubiquitin–proteasome system is required for various cellular processes such as DNA damage repair, cell cycle progression, signal transduction, and immune response [2]. The 26S proteasome is made up of the 20S core particle (CP) and the 19S regulatory particle (RP) [3]. As a prerequisite for protein degradation by the CP, substrate proteins need to be recruited, deubiquitinated, and unfolded by the RP [4]. The RP can be further divided into a “base” and a “lid” subcomplex. The base subcomplex is composed of six ATPase subunits (Rpt1–Rpt6) and three non-ATPase subunits (Rpn, Rpn, and Rpn13), whereas the lid subcomplex is composed of nine non-ATPase subunits (Rpn, Rpn5–Rpn, Rpn, Rpn, and Rpn15/Sem1), and Rpn appears to mainly bind the lid subunits [5,6,7,8]

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Results

Discussion

Conclusion