Heterotrimeric Gα subunit from wheat (Triticum aestivum), GA3, interacts with the calcium-binding protein, Clo3, and the phosphoinositide-specific phospholipase C, PI-PLC1
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The canonical Gα subunit of the heterotrimeric G protein complex from wheat (Triticum aestivum), GA3, and the calcium-binding protein, Clo3, were revealed to interact both in vivo and in vitro and Clo3 was shown to enhance the GTPase activity of GA3. Clo3 is a member of the caleosin gene family in wheat with a single EF-hand domain and is induced during cold acclimation. Bimolecular Fluorescent Complementation (BiFC) was used to localize the interaction between Clo3 and GA3 to the plasma membrane (PM). Even though heterotrimeric G-protein signaling and Ca2+ signaling have both been shown to play a role in the response to environmental stresses in plants, little is known about the interaction between calcium-binding proteins and Gα. The GAP activity of Clo3 towards GA3 suggests it may play a role in the inactivation of GA3 as part of the stress response in plants. GA3 was also shown to interact with the phosphoinositide-specific phospholipase C, PI-PLC1, not only in the PM but also in the endoplasmic reticulum (ER). Surprisingly, Clo3 was also shown to interact with PI-PLC1 in the PM and ER. In vitro analysis of the protein–protein interaction showed that the interaction of Clo3 with GA3 and PI-PLC1 is enhanced by high Ca2+ levels. Three-way affinity characterizations with GA3, Clo3 and PI-PLC1 showed the interaction with Clo3 to be competitive, which suggests that Clo3 may play a role in the Ca2+-triggered feedback regulation of both GA3 and PI-PLC1. This hypothesis was further supported by the demonstration that Clo3 has GAP activity with GA3.
KeywordsHeterotrimeric G protein alpha subunit Gα protein Calcium-binding protein Signal transduction GTPase-activating protein Phosphoinositide-specific phospholipase Protein–protein interaction
This work is supported by grants from the Natural Science and Engineering Research Council of Canada, and the Agricultural Bioproducts Innovation Program of Agriculture and Agri-Food Canada. We thank Alan Jones, North Carolina State University, for kindly providing clones and vectors for control expression and protein–protein interaction. We thank Hugo Zheng, McGill University, for providing control clones for protein–protein interaction. The sequences of the T. aestivum genes described in the manuscript were deposited in GenBank with the following accession numbers: Ga3, HQ020506; PI-PLC1, HM754654; PI-PLC2, HM754653; Clo3, HQ020505.
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