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Science 9 November 2007:
Vol. 318. no. 5852, p. 914
DOI: 10.1126/science.1143320
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Technical Comments

Response to Comment on "A G Protein–Coupled Receptor Is a Plasma Membrane Receptor for the Plant Hormone Abscisic Acid"

Xigang Liu1, Yanling Yue1, Wei Li1,2 and Ligeng Ma1*

1 National Institute of Biological Sciences, 7 Science Park Road, Zhongguancun Life Science Park, Beijing 102206, China.
2 Laboratory of Molecular Cellular Biology, Hebei Normal University, Shijiazhuang, Hebei 050016, China.


Figure 1 Fig. 1. GCR2 subcellular localization. (A) GCR2 is localized in the plasma membrane in the protoplast expressing 35S::GFP (top) and dex-inductive promotor::GCR2-YFP (bottom). I, YFP fluorescence; II, chloroplast fluorescence; III, bright field; IV, merge of I and II. (B) GCR2 is predominantly associated with the membrane fraction. Total protein (T) was isolated from transgenic plants expressing dex-inductive promotor::GCR2-YFP. Proteins were fractionated into soluble (S) or membrane (M) fractions. Equal amounts of protein were separated on SDS-PAGE and subjected to immunoblotting using antibodies to GFP. The samples were isolated from the transgenic plant under dex-inductive conditions. [View Larger Version of this Image (59K GIF file)]
 

Figure 2 Fig. 2. Physical interaction between GCR2 and GPA1, and between GCR2 and Gß subunit. (A) The dependence of intrinsic GTPase activity for physical interaction between GPA1 and GCR2 or GCR1 shown by Yeast growth assay (left) and LacZ activity assay (right). KAT1-NubG + KAT1-Cub, positive control; SUC2-NubG + KAT1-Cub, negative control; cGPA1, Q222L-mutated GPA1 defect in GTPase activity. (B) GCR2 and GCR1 interact with the Gß subunit (AGB1) by split-ubiquitin assay in yeast. (Left) Yeast growth assay. (Middle) X-gal overlay assay. (Right) Corresponding LacZ activity for each yeast strain. I, GCR1-NubG + AGB1-Cub; II, NubG-GCR1 + AGB1-Cub; III, GCR2-NubG + AGB1-Cub; IV, NubG-GCR2 + AGB1-Cub; V, NubG + KAT1-Cub, positive control; VI, SUC2-NubG + KAT1-Cub, negative control. ß-galactosidase activity unit: Miller unit. [View Larger Version of this Image (64K GIF file)]
 

Figure 3 Fig. 3. Physical interaction between GCR2 and GPA1 by SPR assay. Representative SPR experiments show (A) the binding of GPA1 to immobilized GCR2, (B) nonbinding of GPA1 to immobilized BSA, (C) merged image from (A) and (B), (D) specific binding of GPA1 to GCR2, (E) nonbinding of BSA to immobilized GCR2, and (F) nonbinding of GCR2 to immobilized BSA. Representative SPR experiments show the binding of series concentrations of GPA1 to immobilized GCR2 (G). (H) Simulated SPR binding curves for a 2.1 nM affinity interaction between GPA1 and GCR2 using the data from (G) (the maximum inputs for the simulation in the software is 5). (I) Representative image for the surface regeneration of the sensor chip. [View Larger Version of this Image (23K GIF file)]
 

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