TY - JOUR
T1 - Affinity purification with metabolomic and proteomic analysis unravels diverse roles of nucleoside diphosphate kinases
AU - Luzarowski, Marcin
AU - Kosmacz, Monika
AU - Sokolowska, Ewelina
AU - Jasinska, Weronika
AU - Willmitzer, Lothar
AU - Veyel, Daniel
AU - Skirycz, Aleksandra
N1 - Generated from Scopus record by KAUST IRTS on 2021-02-18
PY - 2017/6/15
Y1 - 2017/6/15
N2 - 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.
AB - 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.
UR - https://academic.oup.com/jxb/article/68/13/3487/3861624
UR - http://www.scopus.com/inward/record.url?scp=85042463677&partnerID=8YFLogxK
U2 - 10.1093/jxb/erx183
DO - 10.1093/jxb/erx183
M3 - Article
SN - 1460-2431
VL - 68
SP - 3487
EP - 3499
JO - Journal of Experimental Botany
JF - Journal of Experimental Botany
IS - 13
ER -