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First published online December 11, 2006
doi: 10.1242/10.1242/jcs.03284

Journal of Cell Science 119, 5087-5097 (2006)
Published by The Company of Biologists 2006
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Plant G protein heterotrimers require dual lipidation motifs of G{alpha} and G{gamma} and do not dissociate upon activation

Merel J. W. Adjobo-Hermans, Joachim Goedhart and Theodorus W. J. Gadella, Jr*

Swammerdam Institute for Life Sciences, Section of Molecular Cytology, Centre for Advanced Microscopy, University of Amsterdam, Kruislaan 316, 1098 SM, Amsterdam, The Netherlands


Figure 1
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  		Fig. 1. Subcellular location and dynamics of GP{alpha}1-YFP and its lipid motif mutants in cowpea protoplasts. Protoplasts expressing (A) GP{alpha}1-YFP, (B) GP{alpha}1(G2A)-YFP, (C) GP{alpha}1(C5S)-YFP, (D) GP{alpha}1(G2A/C5S)-YFP, (E) unfused YFP. N, nucleus; V, vacuole. (F) FRAP curves of YFP (triangles); GP{alpha}1-YFP (open diamonds); GP{alpha}1(G2A)-YFP (circles), GP{alpha}1(C5S)-YFP (squares) and GP{alpha}1(G2A/C5S)-YFP (filled diamonds). Bars, 10 µm.

 

Figure 2
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  		Fig. 2. Subcellular location of plant Gß and G{gamma} subunits in cowpea protoplasts. (A-C) Protoplasts expressing (A) YFP-AGß1, (B) YFP-AGG1 and (C) YFP-AGG2. (D-F) Protoplast co-expressing (D) YFP-AGG1 and (E) ST-RFP, as seen in F which is the overlaid images of D and E. (G-I) Protoplast co-expressing (G) CFP-AGß1 and (H) YFP-AGG1; (I) the overlaid images of G and H. Arrows indicate Golgi-like structures. Bars, 10 µm.

 

Figure 3
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  		Fig. 3. The influence of AGG1 expression on the amount of YFP-AGß1 expressors. (A,B) Protoplasts transfected with YFP-AGß1. (C,D) Protoplasts transfected with both YFP-AGß1 and AGG1. A and C show yellow fluorescence of protoplasts expressing YFP-AGß1, B and D show yellow fluorescence of YFP-AGß1-expressing protoplasts combined with the red chlorophyll autofluorescence of all protoplasts. Bar, 20 µm.

 

Figure 4
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  		Fig. 4. Subcellular location of YFP-AGG2 lipidation mutants and the effect on localization of AGß1. Protoplasts expressing (A) the prenylation-deficient construct YFP-AGG2mut and (B) YFP-AGG2(C95S). (C,D) Protoplasts co-expressing (C) CFP-AGß1 and (D) YFP-AGG2mut. Bars, 10 µm.

 

Figure 5
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  		Fig. 5. The effect of co-expression on the localization of lipidation mutants. (A,B) Protoplast co-expressing (A) GP{alpha}1(C5S)-YFP and non-fluorescent AGG1 and (B) CFP-AGß1 and non-fluorescent AGG1. (C,D) Protoplasts co-expressing (C) GP{alpha}1-YFP and non-fluorescent AGß1 and (D) CFP-AGG2mut and non-fluorescent AGß1. Bars, 10 µm.

 

Figure 6
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  		Fig. 6. Heterotrimer formation at the plant plasma membrane requires dual lipidation of G{alpha} and G{gamma}. The model shows the formation of Gß{gamma} dimers in the cytosol. The waste-basket symbolizes the breakdown of Gß that occurs in the absence of G{gamma}. Dimerization does not require lipidation. Dual lipidation of G{gamma} targets the Gß{gamma} dimer to the plasma membrane where it can form a heterotrimer with G{alpha} that has been myristoylated in the cytosol and palmitoylated, probably at the plasma membrane. The heterotrimer does not fall apart upon activation of G{alpha} ({alpha}*). The arrows indicate the putative direction(s) of the reactions. Heterodimer and heterotrimer formation might be regulated by de/repalmitoylation cycles.

 

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© The Company of Biologists Ltd 2006