Arabidopsis thaliana Nudix hydrolase AtNUDT7 forms complexes with the regulatory RACK1A protein and gamma subunits of the signal transducing heterotrimeric G protein
Treść / Zawartość
Arabidopsis thaliana AtNUDT7 Nudix pyrophosphatase hydrolyzes NADH and ADP-ribose in vitro and is an important factor in the cellular response to diverse biotic and abiotic stresses. Several studies have shown that loss-of-function Atnudt7 mutant plants display many profound phenotypes. However the molecular mechanism of AtNUDT7 function remains elusive. To gain a better understanding of this hydrolase cellular role, proteins interacting with AtNUDT7 were identified. Using AtNUDT7 as a bait in an in vitro binding assay of proteins derived from cultured Arabidopsis cell extracts we identified the regulatory protein RACK1A as an AtNUDT7-interactor. RACK1A-AtNUDT7 interaction was confirmed in a yeast two-hybrid assay and in a pull-down assay and in Bimolecular Fluorescence Complementation (BiFC) analysis of the proteins transiently expressed in Arabidopsis protoplasts. However, no influence of RACK1A on AtNUDT7 hydrolase catalytic activity was observed. In vitro interaction between RACK1A and the AGG1 and AGG2 gamma subunits of the signal transducing heterotrimeric G protein was also detected and confirmed in BiFC assays. Moreover, association between AtNUDT7 and both AGG1 and AGG2 subunits was observed in Arabidopsis protoplasts, although binding of these proteins could not be detected in vitro. Based on the observed interactions we conclude that the AtNUDT7 Nudix hydrolase forms complexes in vitro and in vivo with regulatory proteins involved in signal transduction. Moreover, we provide the initial evidence that both signal transducing gamma subunits bind the regulatory RACK1A protein.
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