Tagging the proteasome active site \u03b25 causes tag specific phenotypes in yeast

Kenrick A Waite,Alicia Burris,Jeroen Roelofs

doi:10.1038/s41598-020-75126-1

Kenrick A Waite, Alicia Burris + Show 1 more

Open Access

https://doi.org/10.1038/s41598-020-75126-1

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Abstract

The efficient and timely degradation of proteins is crucial for many cellular processes and to maintain general proteostasis. The proteasome, a complex multisubunit protease, plays a critical role in protein degradation. Therefore, it is important to understand the assembly, regulation, and localization of proteasome complexes in the cell under different conditions. Fluorescent tags are often utilized to study proteasomes. A GFP-tag on the β5 subunit, one of the core particle (CP) subunits with catalytic activity, has been shown to be incorporated into proteasomes and commonly used by the field. We report here that a tag on this subunit results in aberrant phenotypes that are not observed when several other CP subunits are tagged. These phenotypes appear in combination with other proteasome mutations and include poor growth, and, more significantly, altered 26S proteasome localization. In strains defective for autophagy, β5-GFP tagged proteasomes, unlike other CP tags, localize to granules upon nitrogen starvation. These granules are reflective of previously described proteasome storage granules but display unique properties. This suggests proteasomes with a β5-GFP tag are specifically recognized and sequestered depending on physiological conditions. In all, our data indicate the intricacy of tagging proteasomes, and possibly, large complexes in general.

Highlights

Proteasomes are the major protease involved in the selective degradation of proteins in eukaryotic cells
We have previously reported that autophagy of the core particle (CP) and the regulatory particle (RP) occur through different pathways, a model that has been supported by subsequent data[19,24,25]
In part, based on subcomplex localization in autophagy mutants; RP remained largely nuclear in strains deleted for ATG7 or ATG17, while CP appeared in cytosolic punctate structures[19]

Summary

Introduction

Proteasomes are the major protease involved in the selective degradation of proteins in eukaryotic cells. Yeast harboring deletions of both chaperones are very sensitive to heat and other protein folding stresses These phenotypes are further exacerbated when the ability to upregulate transcription of proteasome subunits, as a response to a loss in proteasome activity, is eliminated. We report that tagging the active site subunit β5 (Pre2) resulted in tag specific phenotypes and poor cell growth when combined with other proteasome mutations. This was surprising as this tag has been used by a number of labs, including our own, and strains expressing β5-GFP show normal incorporation of this subunit into proteasomes, and exhibit normal behavior in several microscopic and biochemical a ssays[19,20,21]. Our data suggest that cells can recognize β5-GFP proteasomes as abnormal and sequester them away from the nucleus in stressful conditions

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