Arabidopsis thaliana Nudix hydrolase AtNUDT7 forms complexes with the regulatory RACK1A protein and Ggamma subunits of the signal transducing heterotrimeric G protein.

Abstract

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.

The Receptor for Activated C Kinase in Plant Signaling: Tale of a Promiscuous Little Molecule

“…The data indicates that GPA1does not directly interact with RACK1 in any of the implemented assays (YTH, suYTH, Co-IP, in planta BiFC, SFLC, etc.). Moreover, although the interaction between RACK1 and the AGG1/AGG2 subunits was not analyzed in these reports, it was previously shown that AtRACK1A consistently interacted with both AGG1 and AGG2 using in vitro (pull-down), and in vivo (YTH and BiFC in protoplasts) assays (Olejnik et al, 2011 ). Therefore, the accumulating evidence leads to the notion that plant RACK1 can form complexes with the Gβ and Gγ subunits, but not with Gα.…”

“…More recently, Olejnik et al ( 2011 ) found that AtRACK1A binds to AtNUDT7. AtNUDT7 is a pyrophosphatase that hydrolyzes NADH and ADP-ribose in vitro , and plays a role in the response to biotic and abiotic stress.…”

“…This further supported the central role of RACK1 in innate immunity, as previously shown for O. sativa (Nakashima et al, 2008 ). In addition, Olejnik et al ( 2011 ) later reported that AtRACK1A bound to AGG1, AGG2, and AtNUDT7. The latter is a pyrophosphatase capable of hydrolyzing NADH and ADP-ribose in vitro , and plays a role in the response to biotic and abiotic stresses.…”

The ammonium/nitrate ratio is an input signal in the temperature‐modulated, SNC1‐mediated and EDS1‐dependent autoimmunity of nudt6‐2 nudt7

“…Such examples include GTPases and R proteins, which function as signaling components, rather than modulators of cellular GTP and ATP levels, respectively (Li and Zhang, 2004;Takken and Tameling, 2009). Interestingly, At-NUDT7 was reported to interact with 14-3-3 proteins (Olejnik et al, 2009), RACK1A and the gamma subunit of G proteins (Olejnik et al, 2011), but the physiological effects of these interactions are still unknown. Some point mutations in AtNUDT7 may change its quaternary structures and hamper its interaction with other proteins (Olejnik et al, 2009), but the physiological consequences of these point mutations have not been assessed.…”

The Nucleotide‐Dependent Interactome of Rice Heterotrimeric G‐Protein α ‐Subunit

“…Hydrolases and oxidoreductases covered 16.5 and 12.5% of the interactors, respectively. A. thaliana Nudix hydrolase AtNUDT7 plays important role in cellular response to diverse biotic and abiotic stresses by forming complexes with RACK1A protein and Gγ subunits of the signal transducing heterotrimeric G‐protein . Certain oxidoreductase proteins are located at the membrane/cytoskeleton area where they act as adaptors to receive signals from the cell surface .…”