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Completing the heterotrimer: isolation and characterization of an Arabidopsis thaliana G protein gamma-subunit cDNA.

ABSTRACT: Heterotrimeric G proteins consist of three subunits (alpha, beta, and gamma). alpha- and beta- subunits have been previously cloned in plants, but the gamma-subunit has remained elusive. To isolate the gamma-subunit of a plant heterotrimeric G protein an Arabidopsis thaliana yeast two-hybrid library was screened by using a tobacco G-beta-subunit as the bait protein. One positive clone (AGG1) was isolated several times; it displays significant homology to the conserved domains of mammalian gamma-subunits. The predicted AGG1 protein sequence contains all of the typical characteristics of mammalian gamma-subunits such as small size (98 amino acids, 10.8 kDa), presence of a C-terminal CAAX box to direct isoprenyl modification, and an N-terminal alpha-helix region capable of forming a coiled-coil interaction with the beta-subunit. Northern and Southern analyses showed that AGG1 is a single-copy gene in Arabidopsis with a similar expression pattern to the Arabidopsis beta-subunit, AGB1 [Weiss, C. A., Garnaat, C. W., Mukai, K., Hu, Y. & Ma, H. (1994) Proc. Natl. Acad. Sci. USA 91, 9554-9558]. By using the yeast two-hybrid system, we show that AGG1 strongly interacts with tobacco and Arabidopsis beta-subunits. The in vivo results have been confirmed by using in vitro methods to prove the interaction between AGG1 and the Arabidopsis beta-subunit. As previously observed in mammalian systems, both the coiled-coil domain and the WD repeat regions of the beta-subunit are essential for AGG1 interaction. Also in agreement with previous observations, the removal of the N-terminal alpha-helix of the AGG1 greatly reduces but does not completely block the interaction.

SUBMITTER: Mason MG

PROVIDER: S-EPMC18996 | biostudies-literature | 2000 Dec

REPOSITORIES: biostudies-literature

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Completing the heterotrimer: isolation and characterization of an Arabidopsis thaliana G protein gamma-subunit cDNA.

Mason M G MG Botella J R JR

Proceedings of the National Academy of Sciences of the United States of America 20001201 26

Heterotrimeric G proteins consist of three subunits (alpha, beta, and gamma). alpha- and beta- subunits have been previously cloned in plants, but the gamma-subunit has remained elusive. To isolate the gamma-subunit of a plant heterotrimeric G protein an Arabidopsis thaliana yeast two-hybrid library was screened by using a tobacco G-beta-subunit as the bait protein. One positive clone (AGG1) was isolated several times; it displays significant homology to the conserved domains of mammalian gamma- ...[more]

PMID: 11121078

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Project description:BACKGROUND: In plants, proline synthesis occurs by two enzymatic steps starting from glutamate as a precursor. Some bacteria, including bacteria such as Agrobacterium rhizogenes have an Ornithine Cyclodeaminase (OCD) which can synthesize proline in a single step by deamination of ornithine. In A. rhizogenes, OCD is one of the genes transferred to the plant genome during the transformation process and plants expressing A. rhizogenes OCD have developmental phenotypes. One nuclear encoded gene of Arabidopsis thaliana has recently been annotated as an OCD (OCD-like; referred to here as AtOCD) but nothing is known of its function. As proline metabolism contributes to tolerance of low water potential during drought, it is of interest to determine if AtOCD affects proline accumulation or low water potential tolerance. RESULTS: Expression of AtOCD was induced by low water potential stress and by exogenous proline, but not by the putative substrate ornithine. The AtOCD protein was plastid localized. T-DNA mutants of atocd and AtOCD RNAi plants had approximately 15% higher proline accumulation at low water potential while p5cs1-4/atocd double mutants had 40% higher proline than p5cs1 at low water potential but no change in proline metabolism gene expression which could directly explain the higher proline level. AtOCD overexpression did not affect proline accumulation. Enzymatic assays with bacterially expressed AtOCD or AtOCD purified from AtOCD:Flag transgenic plants did not detect any activity using ornithine, proline or several other amino acids as substrates. Moreover, AtOCD mutant or over-expression lines had normal morphology and no difference in root elongation or flowering time, in contrast to previous report of transgenic plants expressing A. rhizogenes OCD. Metabolite analysis found few differences between AtOCD mutants and overexpression lines. CONCLUSIONS: The Arabidopsis OCD-like protein (AtOCD) may not catalyze ornithine to proline conversion and this is consistent with observation that three residues critical for activity of bacterial OCDs are not conserved in AtOCD. AtOCD was, however, stress and proline induced and lack of AtOCD expression increased proline accumulation by an unknown mechanism which did not require expression of P5CS1, the main enzyme responsible for stress-induced proline synthesis from glutamate. The results suggest that AtOCD may have function distinct from bacterial OCDs.

Dataset Information

Completing the heterotrimer: isolation and characterization of an Arabidopsis thaliana G protein gamma-subunit cDNA.

Publications

Completing the heterotrimer: isolation and characterization of an Arabidopsis thaliana G protein gamma-subunit cDNA.

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