Xenbase Image ID: 170750

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.10.7554/eLife.25012.025Figure 3—source data 1. Figure 3D source data.Figure 3—source data 2. Figure 3E source data.Figure 3—source data 3. Figure 3—figure supplement 2E source data.Figure 3—source data 4. Figure 3—figure supplement 2F source data.Figure 3—figure supplement 1. BRUSH topology determination in yeast.(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. Image published in: Chiasson DM et al. (2017) © 2017, Chiasson et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View

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