title: 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.
authors: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warszawa, Poland.
authors: Kamil
authors: K
authors: Olejnik
authors: Maria
authors: M
authors: Bucholc
authors: Anna
authors: A
authors: Anielska-Mazur
authors: Agata
authors: A
authors: Lipko
authors: Martyna
authors: M
authors: Kujawa
authors: Marta
authors: M
authors: Modzelan
authors: Agnieszka
authors: A
authors: Augustyn
authors: Elzbieta
authors: E
authors: Kraszewska
mesh_terms: D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D057075:Enzyme Assays; D044388:GTP-Binding Protein gamma Subunits / Q000235:genetics / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism; D059828:Plant Cells / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011523:Protoplasts / Q000166:cytology / Q000378:metabolism; D011755:Pyrophosphatases / Q000235:genetics / Q000378:metabolism*; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D021241:Spectrometry, Mass, Electrospray Ionization; D020798:Two-Hybrid System Techniques; D000096964:Nudix Hydrolases
chemical_list: D029681:Arabidopsis Proteins; D018076:DNA, Complementary; D044388:GTP-Binding Protein gamma Subunits; D046912:Multiprotein Complexes; D011755:Pyrophosphatases; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D000096964:Nudix Hydrolases; C521249:AGG1 protein, Arabidopsis; C521250:AGG2 protein, Arabidopsis; C515163:RACK1 protein, Arabidopsis