Yeast Nst1 is a novel component of P-bodies and is a specific suppressor of proteasome base assembly defects

Abstract

Proteasome assembly utilizes multiple dedicated assembly chaperones and is regulated by signaling pathways that respond to diverse stress conditions. To discover new factors influencing proteasome base assembly, we screened a tiled high-copy yeast genomic library to identify dosage suppressors of a temperature-sensitive proteasome regulatory particle (RP) base mutant. The screen identified Nst1, a protein that when overexpressed, specifically suppressed the temperature sensitivity and proteasome-assembly defects of multiple base mutants. Nst1 overexpression reduced cytosolic RP ATPase (Rpt) aggregates in nas6Δ rpn14Δ cells, which lack two RP assembly chaperones. Nst1 is highly polar and predicted to have numerous intrinsically disordered regions, characteristics commonly found in proteins that can segregate into membraneless condensates. In agreement with this, both endogenous and overexpressed Nst1 could form cytosolic puncta that co-localized with processing body (P-body) components. Consistent with the accumulation of translationally inactive mRNAs in P-bodies, Nst1 overexpression inhibited global protein translation in nas6Δ rpn14Δ cells. Translational inhibition is known to suppress aggregation and proteasome assembly defects in base mutants under heat stress . Our data indicate that Nst1 is a previously overlooked P-body component that when expressed at elevated levels inhibits translation, prevents Rpt subunit aggregation, and rescues proteasome assembly under stress conditions.

Assembly checkpoint of the proteasome regulatory particle is activated by coordinated actions of proteasomal ATPase chaperones

“…Throughout different stages of RP assembly, RP chaperones act by the same mechanism—binding to their cognate proteasomal ATPases. The chaperones may be able to satisfy different requirements for the accuracy of a given assembly event via acting with Not4 and other proteins as potential checkpoint factors ( Cheng et al, 2021 ; Zavodszky et al, 2021 ), thereby providing the first point of quality control for the proteasome holoenzyme.…”

Nst1, Densely Associated to P-Body in the Post-Exponential Phases of Saccharomyces cerevisiae, Shows an Intrinsic Potential of Producing Liquid-Like Condensates of P-Body Components in Cells

“…We investigated the function of Nst1 in PB accumulation because we observed that the antiproliferative phenotype of its overexpression was suppressed by well-known components of PBs, such as Psp2 and Pop2 ( Supplementary Figure S1 ). Although we are studying the function of Nst1 in PB formation, another group has recently reported it as a novel PB component [ 29 ]. When we recognized that the endogenous EGFP-tagged Nst1 is strongly self-condensed and co-localized with the Dcp2 and Edc3 condensates in the post-exponential growth phases, we expected that the endogenous EGFP-tagged Nst1 in exponentially growing cells would be observed as puncta similar to those of cells exposed to glucose deprivation.…”

“…Because both processes of PB accumulation from small to large bodies are not discrete but continuous, it is challenging to determine the degree of PB accumulation. In previous studies, PB accumulation was quantified by the number of cells containing intensified puncta [29]. Instead, to accurately measure the extent of accumulation during the process, we applied a systematic quantification method by image processing utilizing FIJI ImageJ (https://imagej.net/software/fiji/, accessed on 7 January 2022) with the BaSiC plugin [30].…”