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

2001 ◽  
Vol 58 (4) ◽  
Author(s):  
Kamil Olejnik ◽  
Maria Bucholc ◽  
Anna Anielska-Mazur ◽  
Agata Lipko ◽  
Martyna Kujawa ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
G Protein ◽  
Cellular Response ◽  
Regulatory Protein ◽  
Bimolecular Fluorescence Complementation ◽  
Nudix Hydrolase ◽  
Heterotrimeric G Protein ◽  
Cell Extracts ◽  
Gamma Subunits

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.

scholarly journals Rice heterotrimeric G-protein gamma subunits (RGG1 and RGG2) are differentially regulated under abiotic stress

2012 ◽  
Vol 7 (7) ◽  
pp. 733-740 ◽  
Author(s):  
Dinesh Kumar Yadav ◽  
S. M. Shahinul Islam ◽  
Narendra Tuteja
Keyword(s):  
Abiotic Stress ◽  
G Protein ◽  
Heterotrimeric G Protein ◽  
Gamma Subunits

scholarly journals Dominant-negative Gα subunits are a mechanism of dysregulated heterotrimeric G protein signaling in human disease

2016 ◽  
Vol 9 (423) ◽  
pp. ra37-ra37 ◽  
Author(s):  
Arthur Marivin ◽  
Anthony Leyme ◽  
Kshitij Parag-Sharma ◽  
Vincent DiGiacomo ◽  
Anthony Y. Cheung ◽  
...  
Keyword(s):  
G Protein ◽  
Mammalian Cells ◽  
Neural Crest Cell ◽  
Craniofacial Development ◽  
Dominant Negative ◽  
Protein Subunit ◽  
Protein Activity ◽  
Guanine Nucleotide Binding Protein ◽  
Heterotrimeric G Protein

Auriculo-condylar syndrome (ACS), a rare condition that impairs craniofacial development, is caused by mutations in a G protein–coupled receptor (GPCR) signaling pathway. In mice, disruption of signaling by the endothelin type A receptor (ETAR), which is mediated by the G protein (heterotrimeric guanine nucleotide–binding protein) subunit Gαq/11 and subsequently phospholipase C (PLC), impairs neural crest cell differentiation that is required for normal craniofacial development. Some ACS patients have mutations in GNAI3, which encodes Gαi3, but it is unknown whether this G protein has a role within the ETAR pathway. We used a Xenopus model of vertebrate development, in vitro biochemistry, and biosensors of G protein activity in mammalian cells to systematically characterize the phenotype and function of all known ACS-associated Gαi3 mutants. We found that ACS-associated mutations in GNAI3 produce dominant-negative Gαi3 mutant proteins that couple to ETAR but cannot bind and hydrolyze guanosine triphosphate, resulting in the prevention of endothelin-mediated activation of Gαq/11 and PLC. Thus, ACS is caused by functionally dominant-negative mutations in a heterotrimeric G protein subunit.

scholarly journals cpc-1, the general regulatory gene for genes of amino acid biosynthesis in Neurospora crassa, is differentially expressed during the asexual life cycle

1991 ◽  
Vol 11 (2) ◽  
pp. 928-934 ◽  
Author(s):  
D J Ebbole ◽  
J L Paluh ◽  
M Plamann ◽  
M S Sachs ◽  
C Yanofsky
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Neurospora Crassa ◽  
Regulatory Gene ◽  
Regulatory Protein ◽  
Binding Activity ◽  
Recognition Sequence ◽  
Dna Binding Activity ◽  
Cell Extracts

CPCI, the principal regulatory protein required for cross-pathway control of amino acid biosynthetic genes in Neurospora crassa, contains a domain similar to the DNA-binding domain of GCN4, the corresponding general regulator in Saccharomyces cerevisiae. We examined binding by CPC1 synthesized in vitro and by CPC1 present in N. crassa whole-cell extracts. CPCI from both sources was shown to bind to the DNA sequence 5'-ATGACTCAT-3', which is also the preferred recognition sequence of GCN4, CPC1 was confirmed as the source of DNA-binding activity in extracts by immunoblotting. Slightly mobility differences between DNA complexes containing CPCI synthesized in vitro and CPC1 in mycelial extracts were observed. Analyses of N. crassa extracts from different stages of asexual development revealed that CPC1 was abundant immediately following spore germination and through early mycelial growth but was scarce subsequently. CPC1 levels could be increased at any time by imposing amino acid starvation. Copies of the CPC1 response element are located upstream of several genes regulated by cross-pathway control, including cpc-1 itself.

scholarly journals Chloroplast DnaJ-like proteins 3 and 4 (CDJ3/4) from Chlamydomonas reinhardtii contain redox-active Fe–S clusters and interact with stromal HSP70B

2010 ◽  
Vol 427 (2) ◽  
pp. 205-215 ◽  
Author(s):  
Karolin V. Dorn ◽  
Felix Willmund ◽  
Christian Schwarz ◽  
Christine Henselmann ◽  
Thomas Pohl ◽  
...  
Keyword(s):  
Heat Shock ◽  
Chlamydomonas Reinhardtii ◽  
Higher Plants ◽  
Protein Complexes ◽  
Phylogenetic Analyses ◽  
Regulatory Protein ◽  
Transcript Levels ◽  
Cell Extracts ◽  
Redox Active

In the present study we report on the identification and characterization of three novel chloroplast-targeted DnaJ-like proteins CDJ3–5, which in addition to their J-domains contain bacterial-type ferredoxin domains. In sequence databases we could identify homologues of CDJ3–5 in green algae, moss and higher plants, but not in cyanobacteria. Phylogenetic analyses allowed us to distinguish two clades containing CDJ3/4 and CDJ5 that must have diverged early in the ancestor of the ‘green lineage’ and have further diversified later on. Molecular and biochemical analysis of CDJ3 and CDJ4 from Chlamydomonas reinhardtii revealed that both proteins are weakly expressed and appear to be localized to the stroma and to thylakoid membranes respectively. The low transcript levels of the CDJ3 and CDJ4 genes declined even further in the initial phase of heat shock, but CDJ3 transcript levels strongly increased after a dark-to-light shift. Accordingly, the Arabidopsis orthologue of CDJ5 was also found to be light-inducible and to be under strong circadian control. CDJ3 and CDJ4 proteins could both be expressed in Escherichia coli and had redox-active Fe–S clusters. In vitro cross-linking studies demonstrated that CDJ3 and CDJ4 interact with chloroplast ATP-bound HSP70B (heat-shock protein 70B), presumably as dimers, and immunoprecipitation studies showed that CDJ3/4 were also in a complex with HSP70B in Chlamydomonas cell extracts. Finally, CDJ3 was found in complexes with apparent molecular masses of approx. 550–2800 kDa, which appeared to contain RNA. We speculate that the CDJ3–5 proteins might represent redox switches that act by recruiting HSP70B for the reorganization of regulatory protein complexes.

scholarly journals Subcellular localization and quantitation of the major neutrophil pertussis toxin substrate, Gn.

1988 ◽  
Vol 106 (6) ◽  
pp. 1927-1936 ◽  
Author(s):  
G M Bokoch ◽  
K Bickford ◽  
B P Bohl
Keyword(s):  
G Protein ◽  
Pertussis Toxin ◽  
Specific Binding ◽  
Regulatory Protein ◽  
Alpha Subunit ◽  
Human Neutrophils ◽  
Adp Ribosylation ◽  
Gamma Subunits ◽  
G Protein Alpha Subunit ◽  
Protein Alpha Subunit

The subcellular distribution of G protein subunits in the neutrophil was examined. Cells were nitrogen cavitated and subcellular organelles fractionated on discontinuous sucrose gradients. The presence of GTP-binding regulatory protein (G protein) alpha and beta/gamma subunits in each organelle was determined using three methods of analysis: specific binding of guanine nucleotide, ADP ribosylation by pertussis toxin, and immunoblot analysis with subunit-specific G protein antibodies. Both plasma membrane and cytosolic G protein components were detected. In contrast, neither the specific nor the azurophilic granules contained detectable G protein. Based on the ability of exogenous G protein beta/gamma subunits to increase the ADP ribosylation of the cytosolic form of G protein and upon the hydrodynamic behavior of the cytosolic protein, it is likely that this represents an uncomplexed G protein alpha subunit. Proteolytic mapping with Staphylococcus aureus V8 protease suggests the soluble alpha subunit is from Gn, the major pertussis toxin substrate of human neutrophils. Using quantitative analysis, the levels of the 40-kD G protein alpha subunit and of the 35/36-kD beta subunit in the neutrophil membrane were determined.

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