Use of the non-radioactive SUnSET method to detect decreased protein synthesis in proteasome inhibited Arabidopsis roots.

Doug Van Hoewyk

doi:10.1186/s13007-016-0120-z

Abstract

BackgroundIn eukaryotic cells, the proteasome maintains homeostasis by selectively degrading regulatory and misfolded proteins, and in doing so contributes to the amino acid pool. Inhibition of the proteasome in yeast and human cells decreases de novo protein synthesis. However, it is not know if proteasome inhibition in plants similarly suppresses protein synthesis. To address this gap in plant biology, protein synthesis in Arabidopsis roots was estimated using SUface SEnsing of Translation (SUnSET) techniques. This non-radioactive method has been validated in animal cells, but has not yet been applied to plants. The goal of this study was to investigate the suitability of SUnSET methodology to measure protein synthesis in plants, and to determine if proteasome inhibition decreases levels of newly synthesized proteins.ResultsThe SUnSET technique revealed that Arabidopsis plants treated with cycloheximide—an inhibitor of protein synthesis—severely decreased levels of newly synthesized proteins in root and shoot tissue, as detected on a Western Blot. Therefore, the non-radioactive method is suitable to detect changes in protein synthesis, and was subsequently used to monitor protein synthesis in proteasome-inhibited roots. The proteasome inhibitor MG132 decreased levels of newly synthesized proteins by 70–80 % after 4 and 16 h. Removal of MG132 from liquid media resulted in roots with increased levels of newly synthesized proteins compared to untreated plants, suggesting that recovery from proteasome inhibition results in elevated levels of protein synthesis. Additionally, SUnSET was used to detect a decrease in protein synthesis in the roots of plants subjected to salt stress or sulfur starvation.ConclusionsProteasome inhibition has been shown to decrease protein synthesis in yeast and human cells, and this study now shows that MG132’s inhibitory effects also applies to plants. These data represent the first time that SUnSET has been used to measure protein synthesis in plants. The study demonstrates that SUnSET is a suitable and robust technique to measure protein synthesis in plants. The use of this non-radioactive method to gauge protein synthesis offers a fast, safe, and cost-effective alternative compared to traditional techniques that rely upon radioactive material. The method is likely to have broad applicability to different disciplines in plant biology.

Highlights

In eukaryotic cells, the proteasome maintains homeostasis by selectively degrading regulatory and misfolded proteins, and in doing so contributes to the amino acid pool
SUnSET has successfully been used to estimate changes in protein synthesis in yeast and animal cells, but the technique has not been employed in plants
After 4 h, roots treated with 1 μM cycloheximide exhibited an 80 % decrease in protein synthesis compared to untreated plants; newly synthesized proteins were not detected in Arabidopsis treated with 20 μM cycloheximide (Fig. 1b)

Summary

Introduction

The proteasome maintains homeostasis by selectively degrading regulatory and misfolded proteins, and in doing so contributes to the amino acid pool. Inhibition of the proteasome in yeast and human cells decreases de novo protein synthesis. It is not know if proteasome inhibition in plants suppresses protein synthesis To address this gap in plant biology, protein synthesis in Arabidopsis roots was estimated using SUface SEnsing of Translation (SUnSET) techniques. This non-radioactive method has been validated in animal cells, but has not yet been applied to plants. Proteasome inhibition in nutrient-deprived human cells depleted the amino acid pool and decreased protein synthesis [6]. MG132 has been reported to decrease protein synthesis in neurons [9] and myoblasts [10]

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