Integrity of the Saccharomyces cerevisiae Rpn11 Protein Is Critical for Formation of Proteasome Storage Granules (PSG) and Survival in Stationary Phase

Rémy Saunier,Agnès Delahodde,Emmanuel P Dassa,Michela Esposito

doi:10.1371/journal.pone.0070357

Rémy Saunier, Agnès Delahodde + Show 2 more

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https://doi.org/10.1371/journal.pone.0070357

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Abstract

Decline of proteasome activity has been reported in mammals, flies and yeasts during aging. In the yeast Saccharomyces cerevisiae, the reduction of proteolysis in stationary phase is correlated with disassembly of the 26S proteasomes into their 20S and 19S subcomplexes. However a recent report showed that upon entry into the stationary phase, proteasome subunits massively re-localize from the nucleus into mobile cytoplasmic structures called proteasome storage granules (PSGs). Whether proteasome subunits in PSG are assembled into active complexes remains an open question that we addressed in the present study. We showed that a particular mutant of the RPN11 gene (rpn11-m1), encoding a proteasome lid subunit already known to exhibit proteasome assembly/stability defect in vitro, is unable to form PSGs and displays a reduced viability in stationary phase. Full restoration of long-term survival and PSG formation in rpn11-m1 cells can be achieved by the expression in trans of the last 45 amino acids of the C-terminal domain of Rpn11, which was moreover found to co-localize with PSGs. In addition, another rpn11 mutant leading to seven amino acids change in the Rpn11 C-terminal domain, which exhibits assembled-26S proteasomes, is able to form PSGs but with a delay compared to the wild type situation. Altogether, our findings indicate that PSGs are formed of fully assembled 26S proteasomes and suggest a critical role for the Rpn11 protein in this process.

Highlights

The vast majority of cells from prokaryotes to multicellular organisms exist in a non-dividing state called quiescence, a cellular state defined as a temporary and reversible absence of proliferation
The rpn11-m1 mutation is a frameshift in position 276 that results in a truncated protein lacking its C-terminal last 31 amino acids replaced by nine non-native residues (Figure 1A)
We found that proteasome subcomplexes such as 20S core particles and base-CP subcomplexes cannot be relocalized into cytosolic dots in quiescent cells

Summary

Introduction

The vast majority of cells from prokaryotes to multicellular organisms exist in a non-dividing state called quiescence, a cellular state defined as a temporary and reversible absence of proliferation. The quiescent state can be induced by nutrient starvation as in stationary phase and quiescent cells can support long-term survival [1]. In addition to its role as a protease, the proteasome functions non-proteolytically in a variety of cellular processes, including transcription [14,15], DNA repair [16] and chromatin remodeling [17]. The 26S proteasome is composed of two sub-complexes: a 20S core particle (CP) carrying the proteolytic activity and the 19S regulatory particle (RP). The base made up of six homologous AAA-ATPases together with two non-ATPase subunits mediates a direct contact with the 20S core complex. The lid of the RP is made of nine non-ATPase subunits and contains a deubiquitinase activity carried by Rpn. The main functions of the 19S RP are to recognize ubiquitinated proteins, to cleave the ubiquitin moiety and to unfold and insert the substrates into the 20S core particle [19]

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