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First published online November 19, 2007
Journal of Experimental Biology 210, 4213-4223 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.005132
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Phosducin interacts with the G-protein β{gamma}-dimer of ciliate protozoan Blepharisma japonicum upon illumination

Katarzyna Sobierajska, Hanna Fabczak and Stanislaw Fabczak*

Department of Cell Biology, Nencki Institute of Experimental Biology, 3 Pasteur Street, PL-02-093 Warsaw, Poland


Figure 1
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  		Fig. 1. Immunodetection of phosphorylation level of ciliate Pdc and interaction of Pdc with Gβ{gamma} in Blepharisma. The precipitated proteins with specific antibody against Pdc in lysates from dark-adapted cells (lane 1), light-stimulated cells (lane 2) and cells incubated with okadaic acid prior to illumination (lane 3) were analyzed by western immunoblotting with antibodies against phosphoserine (A) or Gβ (B). Lanes 4 and 5 show immunoblots identifying Gβ{gamma} in a whole cell lysate from Blepharisma and the rod outer segment (ROS) of bovine photoreceptor cells, respectively (B). The blots are representative of the results of three independent experiments. Inserted densitometric diagrams show the levels of Pdc phosphorylation (A) and bound Gβ (B) in lysates from dark-adapted cells and cells exposed to light or okadaic acid. The levels of Pdc phosphorylation or bound Gβ in lysate from the control dark-adapted cells were taken as 100%. Bars represent mean values ± s.e.m. of three experiments.

 

Figure 2
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  		Fig. 2. Localization of Gβ{gamma} in dark-adapted and illuminated Blepharisma by cell fractionation and immunoblotting. Lysates from dark-adapted (lanes 1, 3 and 5) and illuminated (lanes 2, 4 and 6) cells were fractionated by centrifugation and Gβ was detected in the separate fractions by immunoblotting with specific antibody against Gβ. Results indicate interaction of the antibody with protein bands of ~32 kDa in the membranous fractions (lanes 3 and 4) and ~36 kDa in the cytoplasmic fractions (lanes 5 and 6). Lane 7 shows immunoblots identifying Gβ in whole cell lysate from the rod outer segment (ROS) of bovine photoreceptor cells. Blotting with β-tubulin antibody was used as a test of equal protein loading. Lower panel shows a densitometric quantification diagram of Gβ levels in lysate and cell fractions under different light conditions (filled and open bars correspond to 32 kDa and 36 kDa, respectively). The level of Gβ in lysate from the control dark-adapted cells was taken as 100%. Bars represent mean values ± s.e.m. of four experiments.

 

Figure 3
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  		Fig. 3. Two-dimensional electrophoresis gel of dark-adapted Blepharisma lysate and reaction with antibody against Gβ. Results indicate that only polypeptides at 32 kDa and 36 kDa with a pI of ~6 and 5.4, respectively, are immunoreactive. This gel is representative of the results of three independent experiments.

 

Figure 4
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  		Fig. 4. Coimmunoprecipitation of Pdc and 14-3-3 protein in Blepharisma. Total cell lysates were subjected to immunoprecipitation with antibody against Pdc. The precipitated proteins in lysates from dark-adapted cells (lane 1), light-stimulated cells (lane 2) and cells incubated with okadaic acid prior to illumination (lane 3) were resolved by SDS-PAGE and immunoblotted for 14-3-3 {epsilon} protein. Arrows mark a coimmunoprecipitated protein of ~33 kDa (lanes 1–3). Western blot analysis confirmed that this polypeptide represents a putative 14-3-3 protein (lane 4). Lower panel shows a densitometric quantification diagram of 14-3-3 {epsilon} protein levels in cell lysate under different experimental conditions (lysate from dark-adapted or illuminated cells and cells incubated with okadaic acid prior to illumination, respectively). The level of 14-3-3 {epsilon} protein in lysate from the control dark-adapted cells was taken as 100%. Bars represent the mean values ± s.e.m. of three experiments.

 

Figure 5
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  		Fig. 5. Colocalization of Pdc and Gβ{gamma} related to light conditions in cells of Blepharisma. Distribution of Pdc (red) and Gβ{gamma} (green) and their colocalization (yellow) close to the plasma membrane identified by immunocytochemistry. (A,B) A1–A3, staining of Pdc and Gβ in dark-adapted cells; B1–B3, Pdc and Gβ in cells exposed to light. (B) C1–C3, cells after 10 min incubation with 1 µmol l–1 okadaic acid followed by illumination.

 

Figure 6
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  		Fig. 6. Pdc and Gβ{gamma} interaction in Blepharisma examined by FRET. Cells were labeled and FRET values were generated as described in Materials and methods. Energy transfer efficiency was automatically calculated by the Leica software after subtracting the appropriate background value. The results are shown as the sensitized emission of the acceptor when the cells were excited at 488 nm.

 

Figure 7
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  		Fig. 7. Energy transfer between Pdc and Gβ{gamma} in Blepharisma detected by the FRET Sensitized Emission Method. Pixel by pixel FRET values between Pdc labeled with AlexaFluor 546 (A1, B1) and Gβ labeled with AlexaFluor 488 (A2, B2) in dark-adapted (A1–A4) and in light-stimulated cells (B1–B4). FRET images (A4, B4) are pseudocolored relative to the amplitude of FRET efficiency. The overlay is red where the overlap is significant (B4). The color code ranges between 0% (violet) and 100% (red).

 

Figure 8
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  		Fig. 8. Alignment of seven protein sequences (created by ClustalW; http://www.ebi.ac.uk/Tools/clustalw/index.html/) from the first subgroup of the phosducin family (Blaauw et al., 2003Go). In Blepharisma, serine residues of Pdc – the potential phosphorylation sites – are marked red, and residues highlighted in green form the characteristic amino acid motif (RSXSXP) that is believed to be involved in 14-3-3 protein binding to Pdc. Residues of human Pdc that contact Gβ are denoted by (+) above the aligned sequences. Protein accession numbers: Blepharisma japonicum Pdc, gi_124020703; Dictyostelium discoideum PhLP1, gi_33331889; Homo sapiens Pdc, gi_2967590; Homo sapiens PhLP1, gi_5430700; Danio rerio Pdc1, gi_38174547; Rattus norvegicus PhLP1, gi_66911418; Cryptonema parasitica Pdc, gi_6714949.

 

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© The Company of Biologists Ltd 2007