The Assembly Pathway of the 19S Regulatory Particle of the Yeast 26S Proteasome

Abstract

The 26S proteasome consists of the 20S proteasome (core particle) and the 19S regulatory particle made of the base and lid substructures, and it is mainly localized in the nucleus in yeast. To examine how and where this huge enzyme complex is assembled, we performed biochemical and microscopic characterization of proteasomes produced in two lid mutants, rpn5-1 and rpn7-3, and a base mutant ⌬N rpn2, of the yeast Saccharomyces cerevisiae. We found that, although lid formation was abolished in rpn5-1 mutant cells at the restrictive temperature, an apparently intact base was produced and localized in the nucleus. In contrast, in ⌬N rpn2 cells, a free lid was formed and localized in the nucleus even at the restrictive temperature. These results indicate that the modules of the 26S proteasome, namely, the core particle, base, and lid, can be formed and imported into the nucleus independently of each other. Based on these observations, we propose a model for the assembly process of the yeast 26S proteasome.

Intracellular Dynamics of the Ubiquitin-Proteasome-System

“…At permissive temperatures, neither the Rpn2 nor the Rpt2 NLS deletion had severe impact on nuclear proteasome localization suggesting a redundancy of proteasomal NLSs ( Wendler et al , 2004). Isono et al (2007) later confirmed that Rpn2 provides a crucial NLS to aid nuclear import of the RP base and that the lid is separately imported. The nuclear import of the RP lid also requires importin α, though no classical NLS has been identified within RP lid subunits; rather Sts1, a short-lived protein that itself contains a classical NLS, associates with Rpn11 to facilitate nuclear import of the RP lid by importin αβ ( Chen et al , 2011).…”

Quantitative live-cell imaging reveals spatio-temporal dynamics and cytoplasmic assembly of the 26S proteasome

“…1). We first investigated whether these methods could be applied to macromolecules in extracts from cells expressing chromosomally green fluorescent protein (GFP)-tagged proteasome subunit (Pre6) 14,21 , Hsp104, or ribosome subunit Rpl19a (Fig. 2) [31][32][33] .…”

“…In the rpn2 mutant, the 26S proteasome was almost completely disassembled into the lid, base (lacking Rpn2) and CP even at the permissive temperature 21 . Indeed, the FCF curves of Pre6-GFP, Rpn1-GFP and Rpn7-GFP were dramatically altered ( Fig.…”

“…Our FCS analysis did not detect proteasome assembly intermediates in wild-type cells, probably because such intermediates are not abundant [37][38][39][40] . Previous studies using a specific importin-a mutant, srp1-49, suggested that nucleocytoplasmic transport mediated by importin a/b is coupled to proteasome biogenesis [19][20][21]23 . We hypothesized that proteasome intermediates might be detected in the srp1-49 mutant.…”

“…In support of this notion, previous studies have shown that CP components are imported into the nucleus as inactive precursor complexes via the importin-a/b pathway 20 . Additional evidence suggests that the CP and the lid and base subcomplexes are imported separately 19,21 . These observations imply that the yeast 26S proteasome is assembled inside the nucleus from independently imported modules.…”