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. 2016 Jan 28;291(13):6813–6830. doi: 10.1074/jbc.M115.678953

FIGURE 1.

FIGURE 1.

Expression of RipAY, but not other ChaC family proteins from various organisms, causes growth inhibition of yeast. A, phylogenetic analysis of ChaC domain containing proteins from various organisms. The organisms, locus tag/gene name, and GenbankTM accession numbers (in parentheses) are as follows: E. coli ChaC (L28709.1); R. solanacearum RSc0782 (AL646052.1) and RSp1022/RipAY (AL646053.1); A. citrulli Aave_2801 (CP000512.1) and Aave_4606 (CP000512.1); P. syringae PSPT_05239 (AE016853.1); S. cerevisiae YER163c/Gcg1 (NM_001179053.3); Aspergillus oryzae ChaC (XM_001818295.2); Homo sapiens ChaC1 (NM_001142776.1) and ChaC2 (NM_001008708.2); Mus musculus ChaC1 (NM_026929.4) and ChaC2 (NM_026527.3); Xenopus tropicalis ChaC1 (NM_001130284.1) and ChaC2 (NM_001017137.2); Gallus gallus ChaC1 (NM_001199656.1) and ChaC2 (AJ720918.1); Danio rerio ChaC1 (NM_001110126.1) and ChaC2 (BC154137.1); A. thaliana AT5G26220/GGCT2;1 (BT005234.1), AT4G31290/GGCT2;2 (BT006411.1), and AT1G44790/GGCT2;3 (BT006192.1). B, schematic representation of putative domain organization of ChaC domain-containing proteins from various organisms. C, yeast growth inhibition assay showing serial dilutions of S. cerevisiae BY4743 cells grown under inducing (galactose) or noninducing (glucose) conditions that are carrying plasmids expressing the indicated C-terminal GFP-tagged proteins under the control of the GAL1 promoter. The cells were grown at 26 °C for 2 days for SD (−Ura) and 3 days for SGal (−Ura). D and E, detections of the ChaC domain-containing proteins expressed in yeast. Yeast cells carrying the plasmids expressing GFP-tagged RipAY or other ChaC proteins under the control of the GAL1 promoter were grown in SD (−Ura) liquid medium to mid-log phase and then shifted to SGal (−Ura) liquid medium to induce the protein expression, further cultured for 12 h, and analyzed by immunoblotting with anti-GFP antibody (D) or fluorescence microscopy with native GFP fluorescence (E). Glucose 6-phosphate dehydrogenase (G6PDH) was used as a loading control. Scale bar, 5 μm. DIC, differential interference contrast.