Characterization of a G-protein β-subunit gene from the nematode Caenorhabditis elegans

Protein disulphide isomerase (PDI; EC 5.3.4.1) is a multifunctional polypeptide that is identical to the β subunit of prolyl 4-hydroxylases. We report here on the cloning and expression of the Caenorhabditis elegans PDI/β polypeptide and its isoform. The overall amino acid sequence identity and similarity between the processed human and C. elegans PDI/β polypeptides are 61% and 85% respectively, and those between the C. elegans PDI/β polypeptide and the PDI isoform 46% and 73%. The isoform differs from the PDI/β and ERp60 polypeptides in that its N-terminal thioredoxin-like domain has an unusual catalytic site sequence -CVHC-. Expression studies in insect cells demonstrated that the C. elegans PDI/β polypeptide forms an active prolyl 4-hydroxylase α2β2 tetramer with the human α subunit and an αβ dimer with the C. elegans α subunit, whereas the C. elegans PDI isoform formed no prolyl 4-hydroxylase with either α subunit. Removal of the 32-residue C-terminal extension from the C. elegans α subunit totally eliminated αβ dimer formation. The C. elegans PDI/β polypeptide formed less prolyl 4-hydroxylase with both the human and C. elegans α subunits than did the human PDI/β polypeptide, being particularly ineffective with the C. elegans α subunit. Experiments with hybrid polypeptides in which the C-terminal regions had been exchanged between the human and C. elegans PDI/β polypeptides indicated that differences in the C-terminal region are one reason, but not the only one, for the differences in prolyl 4-hydroxylase formation between the human and C. elegans PDI/β polypeptides. The catalytic properties of the C. elegans prolyl 4-hydroxylase αβ dimer were very similar to those of the vertebrate type II prolyl 4-hydroxylase tetramer, including the Km for the hydroxylation of long polypeptide substrates.

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Targeted Disruption of a Fungal G-Protein β Subunit Gene Results in Increased Vegetative Growth but Reduced Virulence

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.1997.10.8.984 ◽

1997 ◽

Vol 10 (8) ◽

pp. 984-993 ◽

Cited By ~ 106

Author(s):

Shin Kasahara ◽

Donald L. Nuss

Keyword(s):

Amino Acid ◽

G Protein ◽

Vegetative Growth ◽

Synthetic Medium ◽

Cryphonectria Parasitica ◽

Β Subunit ◽

Subunit Gene ◽

Targeted Disruption ◽

Fungal Virulence ◽

Α Subunit

Targeted disruption of two G-protein α subunit genes in the chestnut blight fungus Cryphonectria parasitica revealed roles for the Giα subunit CPG-1 in fungal reproduction, virulence, and vegetative growth. A second Gα subunit, CPG-2, was found to be dispensable for these functions. We now report the cloning and targeted disruption of a C. parasitica G-protein β subunit gene. The deduced amino acid sequence encoded by this gene, designated cpgb-1, was found to share 66.2, 65.9, and 66.7% amino acid identity with Gβ homologues from human, Drosophila, and Dictyostelium origins, respectively, but only 39.7% identity with the Saccharomyces cerevisiae Gβ homologue STE4 product. Low stringency Southern hybridization failed to detect any related Gβ subunit genes in C. parasitica. Targeted disruption of cpgb-1 resulted in several of the changes previously reported to accompany disruption of the C. parasitica Giα subunit gene cpg-1. These included very significant reductions in pigmentation, asexual sporulation, and virulence. In contrast to results obtained for Giα gene disruption, the reduction in virulence resulting from the disruption of a Gβ gene was accompanied by increased, rather than decreased, vegetative growth on synthetic medium. The relevance of these results to mechanisms of fungal virulence is considered.

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The maize heterotrimeric G protein β subunit controls shoot meristem development and immune responses

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1917577116 ◽

2019 ◽

Vol 117 (3) ◽

pp. 1799-1805 ◽

Cited By ~ 9

Author(s):

Qingyu Wu ◽

Fang Xu ◽

Lei Liu ◽

Si Nian Char ◽

Yezhang Ding ◽

...

Keyword(s):

G Protein ◽

Cross Talk ◽

Normal Growth ◽

Shoot Meristem ◽

Β Subunit ◽

Subunit Gene ◽

Α Subunit ◽

Knock Out ◽

Meristem Size ◽

Meristem Development

Heterotrimeric G proteins are important transducers of receptor signaling, functioning in plants with CLAVATA receptors in controlling shoot meristem size and with pathogen-associated molecular pattern receptors in basal immunity. However, whether specific members of the heterotrimeric complex potentiate cross-talk between development and defense, and the extent to which these functions are conserved across species, have not yet been addressed. Here we used CRISPR/Cas9 to knock out the maize G protein β subunit gene (Gβ) and found that the mutants are lethal, differing from those in Arabidopsis, in which homologous mutants have normal growth and fertility. We show that lethality is caused not by a specific developmental arrest, but by autoimmunity. We used a genetic diversity screen to suppress the lethal Gβ phenotype and also identified a maize Gβ allele with weak autoimmune responses but strong development phenotypes. Using these tools, we show that Gβ controls meristem size in maize, acting epistatically with G protein α subunit gene (Gα), suggesting that Gβ and Gα function in a common signaling complex. Furthermore, we used an association study to show that natural variation in Gβ influences maize kernel row number, an important agronomic trait. Our results demonstrate the dual role of Gβ in immunity and development in a cereal crop and suggest that it functions in cross-talk between these competing signaling networks. Therefore, modification of Gβ has the potential to optimize the trade-off between growth and defense signaling to improve agronomic production.

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Isolation and characterization of a G protein γ subunit gene responsive to plant hormones and abiotic stresses in Brassica napus L

Acta Physiologiae Plantarum ◽

10.1007/s11738-010-0558-y ◽

2010 ◽

Vol 33 (2) ◽

pp. 391-399 ◽

Cited By ~ 6

Author(s):

Yong Gao ◽

Tingting Li ◽

Yun Zhao ◽

Caixia Ren ◽

Yiqiong Zhang ◽

...

Keyword(s):

Brassica Napus ◽

G Protein ◽

Plant Hormones ◽

Abiotic Stresses ◽

Subunit Gene ◽

Brassica Napus L ◽

Isolation And Characterization ◽

Γ Subunit

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Cloning and biochemical characterization of the cyclophilin homologues from the free-living nematode Caenorhabditis elegans

Biochemical Journal ◽

10.1042/bj3170179 ◽

1996 ◽

Vol 317 (1) ◽

pp. 179-185 ◽

Cited By ~ 43

Author(s):

Antony P. PAGE ◽

Kenneth MacNIVEN ◽

Michael O. HENGARTNER

Keyword(s):

Signal Transduction ◽

Protein Folding ◽

Caenorhabditis Elegans ◽

Biochemical Characterization ◽

Single Species ◽

Immunosuppressive Agent ◽

Specific Substrate ◽

Nematode Caenorhabditis Elegans ◽

Isomerase Activity

Cyclosporin A (CsA) is the most widely used immunosuppressive agent, whose properties are exerted via an interaction with cyclophilin, resulting in down-regulation of signal-transduction events in the T-cell. Cyclophilin is identical with peptidylprolyl cis–trans isomerase (PPI; EC 5.2.1.8), an enzyme which catalyses the isomerization between the two proline conformations in proteins, thereby acting as a catalyst in protein-folding events. Several reports indicate that CsA has potent anti-parasitic activity, effective against both protozoan and helminth species. In order to understand the various biological roles that cyclophilins play we have initiated a study of these proteins in the genetically tractable nematode Caenorhabditis elegans. Here we describe the cloning and characterization of 11 cyclophilin genes (cyp-1 to -11) derived from this nematode; this is currently the greatest number of isoforms described in a single species. Southern blotting and physical mapping indicated that these genes are dispersed throughout the nematode genome. A high degree of conservation exists between several isoforms, which also share characteristics with the ubiquitous isoforms previously described. The remaining isoforms are divergent, having altered CsA-binding domains and additional non-cyclophilin domains, which may impart compartmental specificity. Ten of these isoforms have been expressed in Escherichia coli, and the resultant fusion proteins have been examined biochemically for PPI activity, which they all possess. Isomerase activity is highest in the conserved and lowest in divergent isoforms, perhaps indicating a more specific substrate for the latter. Analysis of the C. elegans cyp genes will provide answers as to the roles played by cyclophilins in protein folding and signal transduction.

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