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First published online December 11, 2006
doi: 10.1242/10.1242/jcs.03284
Research Article |
and G
and do not dissociate upon activation
Swammerdam Institute for Life Sciences, Section of Molecular Cytology, Centre for Advanced Microscopy, University of Amsterdam, Kruislaan 316, 1098 SM, Amsterdam, The Netherlands
* Author for correspondence (e-mail: gadella{at}science.uva.nl)
Accepted 28 September 2006
In plants one bona fide G
subunit has been identified, as well as a single Gß and two G
subunits. To study the roles of lipidation motifs in the regulation of subcellular location and heterotrimer formation in living plant cells, GFP-tagged versions of the Arabidopsis thaliana heterotrimeric G protein subunits were constructed. Mutational analysis showed that the Arabidopsis G subunit, GP1, contains two lipidation motifs that were essential for plasma membrane localization. The Arabidopsis Gß subunit, AGß1, and the G subunit, AGG1, were dependent upon each other for tethering to the plasma membrane. The second G subunit, AGG2, did not require AGß1 for localization to the plasma membrane. Like AGG1, AGG2 contains two putative lipidation motifs, both of which were necessary for membrane localization. Interaction between the subunits was studied using fluorescence resonance energy transfer (FRET) imaging by means of fluorescence lifetime imaging microscopy (FLIM). The results suggest that AGß1 and AGG1 or AGß1 and AGG2 can form heterodimers independent of lipidation. In addition, FLIM-FRET revealed the existence of GP1-AGß1-AGG1 heterotrimers at the plasma membrane. Importantly, rendering GP1 constitutively active did not cause a FRET decrease in the heterotrimer, suggesting no dissociation upon GP1 activation.
Key words: heterotrimeric G protein, Arabidopsis thaliana, GFP, lipid modification, FRET, FRAP
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