Yamaguchi et al. 10.1073/pnas.0504437102. |
Fig. 6. Affinity-purified GST, GST-AtCaM15, His6-S-b-gal, and His6-S-X1CT were incubated with glutathione–Shepharose at pH 5.5, 6.5, and 7.5 in the presence of 1 mM Ca2+ or 5 mM EGTA as indicated. Eluted proteins were analyzed by Western blot using S protein horseradish peroxidase (HRP) conjugate (S-protein-HRP) and anti-GST antibody-HRP conjugate (a-GST-HRP). No binding was detected with His6, S-tag, or X1CT to GST or glutathione beads, and S-tag did not bind to AtCaM15, given that no bands were observed in the sample containing His6-S-X1CT, GST and glutathione-Sepharose, or His6-S-b-gal and GST-AtCaM15, respectively. The X1CT protein was pulled down only in the presence of both glutathione–Sepharose and Ca2+, with interaction showing pH dependence, confirming the results of the coimmunoprecipitation using yeast whole lysates (Fig. 3).
Fig. 7. Localization of AtCaM15-EGFP and EGFP alone within Arabidopsis protoplasts. A Z-series of successive confocal images of Arabidopsis protoplasts that were transiently transformed with AtCaM15-EGFP (A) and EGFP control (B) constructs shows the respective vacuolar localization of the CaM15-EGFP construct and the cytosolic staining pattern of EGFP expressed alone. Images were taken with an Olympus FV1000 at ´60 magnification. An argon, 488-nm laser was used to visualize EGFP fluorescence with emissions observed at 500-515 nm. Positions of the focal planes with respect to the first plane (indicated as 0) were shown (+2 mm ≈ +8 mm) toward the middle of the protoplasts. The interval between the images is 2 mm.
Supporting Materials and Methods
Pull-Down Assay Using Purified Proteins. Arabidopsis thaliana calmodulin (CaM)-like protein 15 (AtCaM15) was cloned into BamHI/SalI sites of pGEX-6P-2 (Amersham Biosciences) and pGEX-6P-2-AtCaM15 and pGEX6-P-2 were introduced into Escherichia coli BL21 (Novagen). Purification of GST protein from BL21/pGEX-6P-2 and GST-AtCaM15 protein from BL21/pGEX-6P-2-AtCaM15 was achieved by using glutathione-Sepharose (Amersham Biosciences) according to the manufacturer’s instructions.
The A. thaliana Na+/H+ exchanger 1 (AtNHX1) C terminus (X1CT, 94 aa from C terminus) was cloned into BamHI/EcoRI sites of pET30b(+) and the resulting plasmid was introduced into E. coli BL21(DE3). To obtain His6-S-tagged X1CT (His6-S-X1CT) protein, BL21(DE3)/ pET30b(+)-X1CT were grown overnight in LB media with 50 mg/liter of kanamycin and diluted 15 times with 5 liters of fresh media containing 1% glucose, and incubated for 4 h at 30°C. Purification of His6-S-tag X1CT and His6-S-tagged b-galactosidase (His6-S-b-gal) from induction control G [pET-30(b) + b-gal in BL21(DE3), Novagen, WI] was accomplished with S protein agarose according to the manufacturer’s protocols.
Pull down assays were performed with the same buffers as those used for coimmunoprecipitation using yeast. Equal amounts (200 ng) of GST, GST-AtCaM15, His6-S-b-gal, and His6-S-tag X1CT protein were mixed in solubilization buffer at the indicated pH values, and incubated with 20 ml of 50% glutathione–Sepharose slurry for 2 h at 4°C. After incubation, resins are precipitated by centrifugation and washed four times with the appropriate buffers. After the last wash, the resins were incubated with 50 ml of glutathione elution buffer (20 mM glutathione in 50 mM Tris·HCl, pH 8.0) on ice for 10 min to elute protein complexes. Eluted proteins were recovered by centrifugation. S-tagged protein and GST-tagged proteins were detected by Western blots using S protein horseradish peroxidase conjugate (Novagen) and anti-GST horseradish peroxidase conjugate (Amersham Biosciences), respectively. EGTA (5 mM) and 1 mM CaCl2 were included in the solubilization buffer as indicated.
Confocal Microscopy.
An Olympus FV1000 confocal microscope was used to further visualize the localization of the CaM15-GFP construct. An argon laser (488 nm) was used for excitation of GFP, and image florescence was analyzed from 500–515 nm (GFP emission peaks around 510 nm). Protoplasts were visualized at ´60 magnification.