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J Exp Bot. 2017 Jun 15;68(13):3487-3499. doi: 10.1093/jxb/erx183.

Affinity purification with metabolomic and proteomic analysis unravels diverse roles of nucleoside diphosphate kinases.

Author information

1
Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, D-14476 Potsdam-Golm, Germany.

Abstract

Interactions between metabolites and proteins play an integral role in all cellular functions. Here we describe an affinity purification (AP) approach in combination with LC/MS-based metabolomics and proteomics that allows, to our knowledge for the first time, analysis of protein-metabolite and protein-protein interactions simultaneously in plant systems. More specifically, we examined protein and small-molecule partners of the three (of five) nucleoside diphosphate kinases present in the Arabidopsis genome (NDPK1-NDPK3). The bona fide role of NDPKs is the exchange of terminal phosphate groups between nucleoside diphosphates (NDPs) and triphosphates (NTPs). However, other functions have been reported, which probably depend on both the proteins and small molecules specifically interacting with the NDPK. Using our approach we identified 23, 17, and 8 novel protein partners of NDPK1, NDPK2, and NDPK3, respectively, with nucleotide-dependent proteins such as actin and adenosine kinase 2 being enriched. Particularly interesting, however, was the co-elution of glutathione S-transferases (GSTs) and reduced glutathione (GSH) with the affinity-purified NDPK1 complexes. Following up on this finding, we could demonstrate that NDPK1 undergoes glutathionylation, opening a new paradigm of NDPK regulation in plants. The described results extend our knowledge of NDPKs, the key enzymes regulating NDP/NTP homeostasis.

KEYWORDS:

Affinity purification; Arabidopsis thaliana; NDPK; glutathionylation; metabolites; plant; protein–metabolite interactions; protein–protein interactions

PMID:
28586477
PMCID:
PMC5853561
DOI:
10.1093/jxb/erx183
[Indexed for MEDLINE]
Free PMC Article

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