Effect of leucine on the expression of the UGA4-lacZ fusion gene in wild-type, ssy1Δ, stp1Δ, stp2Δ, and stp1Δ stp2Δ cells. β-Galactosidase activity was determined for wild-type (23344c), ssy1Δ (30995b), stp1Δ (KW018), stp2Δ (KW021), and stp1Δ stp2Δ (KW023) cells carrying the UGA4-lacZ fusion gene. Cells were grown in minimal medium and preincubated with 1.3 mM leucine for 30 min (gray bars) or not preincubated (black bars). Then, cells were incubated with 0.1 mM GABA for 60 min, and samples were taken out for β-galactosidase activity measurements. The results shown, expressed in Miller Units, are the means ± standard deviations for duplicates within a representative assay. The numbers above the bars are the ratios between the Miller units calculated for untreated cells and the Miller units calculated for treated cells of each strain.

Effect of leucine on transcription driven by different promoter constructions in wild-type and ssy1Δ cells. (A) Scheme of the fusion genes used. (B to G) β-Galactosidase activity was determined for wild-type (23344c) (B, D, E, F, and G) and ssy1Δ (30995b) (C) cells carrying the UGA4-lacZ (B and C), the UAS_GATAΔ-lacZ (D), the UASΔ-lacZ (E), the UAS_GABAmut-lacZ (F), or the UAS_GABAdel-lacZ (G) fusion gene. Cells were grown in minimal medium and preincubated with 1.3 mM leucine for 30 min before the addition of 0.1 mM GABA (squares) or not preincubated (circles). Then, samples were taken out at the indicated time points, and β-galactosidase activity was measured. The results shown, expressed in Miller Units, are the means for duplicates within a representative assay, with the deviation being less than 15%.

Effect of Leu3 on UGA4 regulation. (A) β-Galactosidase activity was determined for leu3Δ (SBCY01) cells carrying the UGA4-lacZ fusion gene. Cells were grown in minimal medium and preincubated with 1.3 mM leucine for 30 min before the addition of 0.1 mM GABA (squares) or not preincubated (circles). Then, samples were taken out at the indicated time points. The results shown, expressed in Miller Units, are the means for duplicates within a representative assay, with the deviation being less than 15%. (B) Wild-type cells expressing the Leu3-HA fusion protein (SBCY02) were grown in minimal medium (MM) preincubated for 30 min with 1.3 mM leucine or not preincubated. ChIP assays were carried out using antibodies against the HA epitope. qPCR was performed with specific primers that amplify the UGA4 promoter (F/R UGA4qPCR) (black bars), a region 2.5 kb downstream of the UGA4 promoter (F/R UGA4 UCqPCR) (white bars) (used as a negative control), and the LEU2 promoter (F/R LEU2qPCR) (gray bars) (used as a positive control). ChIP DNA was normalized to input DNA and calculated as a signal-to-noise ratio over an IgG control ChIP. The ΔΔC_T method was used to calculate the fold change of binding to the promoter of interest.

Binding of HA-tagged Uga35/Dal81 and Uga3 to the UGA4 promoter in wild-type and ssy1Δ cells. Wild-type (A and B) and ssy1Δ (C and D) cells expressing the HA-Uga35 (SBCY10 and SBCY24) (A and C) and HA-Uga3 (SBCY13 and SBCY26) (B and D) fusion proteins were grown in minimal medium (MM), preincubated for 30 min with 1.3 mM leucine or not preincubated, and then incubated with 0.1 mM GABA or not incubated. ChIP assays were carried out using antibodies against the HA epitope. qPCR was performed with specific primers that amplify the UGA4 promoter (F/R UGA4qPCR) (black bars) and a region 2.5 kb downstream of the UGA4 promoter (F/R UGA4 UCqPCR) (white bars), used as a negative control. ChIP DNA was normalized to input DNA and calculated as a signal-to-noise ratio over an IgG control ChIP. The ΔΔC_T method was used to calculate the fold change of binding to the promoter of interest.

Binding of HA-tagged Uga35/Dal81 and Uga3 to the UGA4 promoter in leu3Δ cells. leu3Δ cells expressing the HA-Uga3 (SBCY22) (left panel) and HA-Uga35 (SBCY23) (right panel) fusion proteins were grown in minimal medium (MM) and then incubated with 0.1 mM GABA or not incubated. ChIP assays were carried out using antibodies against the HA epitope. qPCR was performed with specific primers that amplify the UGA4 promoter (F/R UGA4qPCR) (black bars) and a region 2.5 kb downstream of the UGA4 promoter (F/R UGA4 UCqPCR) (white bars), used as a negative control. ChIP DNA was normalized to input DNA and calculated as a signal-to-noise ratio over an IgG control ChIP. The ΔΔC_T method was used to calculate the fold change of binding to the promoter of interest.

Effect of leucine on the expression of the UGA4-lacZ and the UAS_GABAmut-lacZ fusion genes in wild-type, leu3Δ, uga35Δ and leu3Δ uga35Δ cells. β-Galactosidase activity was determined for wild-type (23344c), leu3Δ (SBCY01), uga35Δ (SBCY17), and leu3Δ uga35Δ (SBCY20) cells carrying the UGA4-lacZ (left panel) or the UAS_GABAmut-lacZ (right panel) fusion gene. Cells were grown in minimal medium. White bars correspond to untreated cells, black bars correspond to cells treated with 0.1 mM GABA for 60 min, and gray bars correspond to cells treated with 1.3 mM leucine for 30 min and 0.1 mM GABA for 60 min. The results shown, expressed in Miller Units, are the means ± standard deviations for duplicates within a representative assay.

Schematic representation of the molecular events triggered by leucine and GABA affecting UGA4 gene expression. (Adapted from reference with permission of the publisher.)