Comprehensive nuclear proteome of Arabidopsis obtained by sequential extraction

Chieko Goto,Shoko Hashizume,Yoichiro Fukao,Ikuko Hara-Nishimura,Kentaro Tamura

doi:10.1080/19491034.2019.1603093

Chieko Goto, Shoko Hashizume + Show 3 more

Open Access

https://doi.org/10.1080/19491034.2019.1603093

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Abstract

ABSTRACTIn eukaryotes, the nucleus plays key roles in fundamental cellular processes, including DNA replication, chromatin maintenance, transcription, and translation. To better understand the functional diversity of nuclei, we developed a method for the comprehensive extraction of the nuclear proteome from Arabidopsis. We used a buffer with a high sucrose concentration to purify nuclei and then conducted solubility-based fractionation to increase proteome coverage. We identified 1539 proteins and two novel nuclear envelope (NE) proteins in the nuclear fraction of Arabidopsis cultured cells. The localization of 25 proteins was determined by GFP fusion analyses; 23 of these proteins were localized either in the nucleus or the NE-associated endoplasmic reticulum. This result was indicative of the high quality of the proteome. These findings will be useful for clarifying novel nuclear functions in plants.

Highlights

In plants and other organisms, the functions of the nucleus are crucial for cell proliferation and the regulation of gene expression during development and/or in response to biotic/abiotic stresses
Yeasts, and protists have focused on different components of the nuclear proteome, including the whole nucleus [1,2,3,4], the nucleolus [5,6,7], the nuclear matrix [8,9], interchromatin granule clusters [10], and the nuclear envelope (NE) [11,12]
To isolate sufficient quality of nuclei, Arabidopsis cultured cells were used as the starting materials

Summary

Introduction

In plants and other organisms, the functions of the nucleus are crucial for cell proliferation and the regulation of gene expression during development and/or in response to biotic/abiotic stresses. To improve the quality and quantity of the plant nuclear proteome, we used a buffer with a high concentration of sucrose to purify nuclei and conducted solubility-based fractionation to increase proteome coverage [43,44]. The isolated nuclei mentioned above were treated with DNase/RNase, and suspended in a salt solution (1 M NaCl, 25 mM MES [pH5.6], 5 mM MgCl2, 10 mM KCl, 0.35 M sucrose, 30% glycerol), followed by incubation for 10 minutes on ice with sometimes stirring.

Results

Conclusion