A Versatile Workflow for the Identification of Protein–Protein Interactions Using GFP-Trap Beads and Mass Spectrometry-Based Label-Free Quantification

Née, Guillaume; Tilak, Priyadarshini; Finkemeier, Iris

doi:10.1007/978-1-0716-0528-8_19

Cited by 5 publications

(1 citation statement)

References 34 publications

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“…The 35S::HDA5‐GFP construct was used for analysis, and cytosolic/nuclear‐GFP (GFPcyt/nucl) created in a Col‐0 background was used as a control. Pulldown was performed as described in (Née et al., 2020) in three biological replicates using GFP TrapA (Chromotek, Planegg‐Martinsried, Germany) trap beads. Eluted samples were further treated with in‐solution digestion.…”

Section: Methodsmentioning

confidence: 99%

Proteome‐wide lysine acetylation profiling to investigate the involvement of histone deacetylase HDA5 in the salt stress response of Arabidopsis leaves

et al. 2023

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Post-translational modifications (PTMs) of proteins play important roles in the acclimation of plants to environmental stress. Lysine acetylation is a dynamic and reversible PTM, which can be removed by histone deacetylases. Here we investigated the role of lysine acetylation in the response of Arabidopsis leaves to 1 week of salt stress. A quantitative mass spectrometry analysis revealed an increase in lysine acetylation of several proteins from cytosol and plastids, which was accompanied by altered histone deacetylase activities in the salt-treated leaves. While activities of HDA14 and HDA15 were decreased upon salt stress, HDA5 showed a mild and HDA19 a strong increase in activity. Since HDA5 is a cytosolic-nuclear enzyme from the class II histone deacetylase family with yet unknown protein substrates, we performed a lysine acetylome analysis on hda5 mutants and characterized its substrate proteins. Next to histone H2B, the salt stressresponsive transcription factor GT2L and the dehydration-related protein ERD7 were identified as HDA5 substrates. In addition, in protein-protein interaction studies, HDA18 was discovered, among other interacting proteins, to work in a complex together with HDA5. Altogether, this study revealed the substrate proteins of HDA5 and identified new lysine acetylation sites which are hyperacetylated upon salt stress. The identification of specific histone deacetylase substrate proteins, apart from histones, will be important to unravel the acclimation response of Arabidopsis to salt stress and their role in plant physiology.

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Section: Methodsmentioning

confidence: 99%