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. 2017 Sep 21;6:e25012. doi: 10.7554/eLife.25012

Figure 3. Interaction of CNGC.IVA N-termini in yeast and channel activity in Xenopus oocytes.

(A) Yeast two-hybrid interaction of the soluble BRUSH N-terminus (NT) fused to the GAL4-binding domain (BD) and the NT of the indicated CNGC.IVA proteins fused to the GAL4 activation domain (AD). Yeast cells were resuspended in water (OD600= 0.5 and 0.05) and spotted onto -LW (leucine, tryptophan) and -LWAH (leucine, tryptophan, adenine, histidine) solid media. (B) Confocal fluorescence images of oocytes expressing either BRUSH-YFP or brush-YFP fusion proteins. (C) Plasma membrane currents of oocytes expressing BRUSH or brush in the presence of 30 mM CaCl2. Voltage steps ranged from +60 to −160 mV in 20 mV increments, starting from a holding potential of −40 mV. Dashed lines indicate 0 µA. (D) Current-voltage relations of oocytes injected with water or YFP (▲, n=15), BRUSH or BRUSH-YFP (▽, n=17), and brush or brush-YFP (●, n=26) . (E) Relative current-voltage relations for oocytes expressing brush and brush-YFP in the presence of 5 mM CaCl2 (◇ , n = 3), 15 mM CaCl2 (■, n = 3), and 30 mM CaCl2 (●, n = 6). Currents are shown relative to the current at −120 mV in the presence of 30 mM CaCl2. Data in (D) and (E) represent mean values ± standard deviations. Scale bars in images (B) represent 250 µm.

Figure 3—source data 1. Figure 3D source data.
elife-25012-fig3-data1.xlsx (34.3KB, xlsx)
DOI: 10.7554/eLife.25012.025
Figure 3—source data 2. Figure 3E source data.
elife-25012-fig3-data2.xlsx (35.7KB, xlsx)
DOI: 10.7554/eLife.25012.026
Figure 3—source data 3. Figure 3—figure supplement 2E source data.
elife-25012-fig3-data3.xlsx (38.1KB, xlsx)
DOI: 10.7554/eLife.25012.027
Figure 3—source data 4. Figure 3—figure supplement 2F source data.
elife-25012-fig3-data4.xlsx (40.2KB, xlsx)
DOI: 10.7554/eLife.25012.028

Figure 3.

Figure 3—figure supplement 1. BRUSH topology determination in yeast.

Figure 3—figure supplement 1.

(A) Yeast split-ubiquitin assay to determine the location of the BRUSH termini. A fusion of BRUSH to the C-terminal fragment of ubiquitin (BRUSH-Cub) was co-transformed with BRUSH fused C- or N-terminally to the N-terminal fragment of ubiquitin (NubI-BRUSH or BRUSH-NubI), NubI alone, or NubG. NubI (wild type, isoleucine at position 13) spontaneously associates with Cub and activates the reporters when both fragments are located in the cytoplasm. The mutated NubG variant (glycine at position 13) has no inherent affinity for Cub. Yeast cells were resuspended in water (OD600= 0.5 and 0.05) and spotted onto -LW (leucine, tryptophan) and -LWAH (leucine, tryptophan, adenine, histidine) solid media.

Figure 3—figure supplement 2. Brush interaction in oocytes and permeability to Ca2+ and K+.

Figure 3—figure supplement 2.

(A) Bimolecular fluorescence complementation (BiFC) analysis of BRUSH and brush fused to either a N-terminal (VN) or C-terminal (VC) fragment of Venus (YFP). Shown are representative oocytes 3 days after co-injection of the corresponding cRNAs. The bottom right image of each panel (VN fusion alone) displays the (non-detectable) background oocyte fluorescence. (B) Control BiFC experiments utilizing AtKAT1. Images are overlays of bright-field and fluorescent images. (C) Current responses of an oocyte expressing BRUSH-YFP in the presence of 30 mM CaCl2. (D) Currents of a brush-YFP injected oocyte in the presence of 30 mM CaCl2 (left) or 60 mM KCl (right). (E) Current-voltage relations of BRUSH-YFP (▽, n = 8), brush-YFP (●, n = 14), and oocytes injected with water or YFP (▲, n = 16). (F) Mean relative current-voltage relations (n = 4) in the presence of 30 mM CaCl2 (●) and 60 mM KCl (○) ±standard deviations. Currents were normalized to the value at −120 mV in the presence of 30 mM CaCl2. Scale bars (A,B) represent 250 µm.