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. 2016 Jun 13;82(13):3875–3885. doi: 10.1128/AEM.00154-16

FIG 6.

FIG 6

Mutation of the RVxF motif in Reg2 affects its ability to interact with Glc7 and to dephosphorylate Snf1. (A) Alignment of the RVxF motifs of Reg1 and Reg2. The amino acid identities and similarities are shaded black and gray, respectively. The arrows indicate the changes introduced in the Reg2-IF mutant. (B) Reg2-IF does not interact with Glc7 in the two-hybrid system. Transformants of CTY10-5d with plasmids expressing the indicated protein pairs were grown to mid-log phase in selective SC medium lacking histidine and leucine and containing 2% glucose and then shifted for 3 h to an otherwise identical medium containing 0.05% glucose. β-Galactosidase activity was assayed in the permeabilized cells and expressed in Miller units. The values are averages for 4 to 8 transformants. The error bars indicate standard errors. (C) Representative transformants of CTY10-5d expressing GAD-Reg2 (GAD-Reg2, WT) or GAD-Reg2-IF (GAD-Reg2, IF), carrying the corresponding vector (GAD-Reg2, −) and simultaneously expressing LexA-Glc7 (LexA-Glc7, +), or carrying the corresponding vector (LexA-Glc7, −) were grown as for panel B, and expression of the fusion proteins was confirmed by immunoblotting as described in Materials and Methods. (D) Overexpression of wild-type Reg2, but not the Reg2-IF mutant, improves growth of reg1Δ cells. reg1Δ cells of strain KBY247 carrying vector pEG202 (LexA), plasmid pLexA-Reg2 (LexA-Reg2), or plasmid pLexA-Reg2-IF (LexA-Reg2-IF) were streaked on SC medium lacking histidine and containing 2% glucose and grown for 3 days. (E) Overexpression of LexA-Reg2-IF during growth in abundant glucose does not reduce phospho-Thr210-Snf1 levels. reg1Δ cells of strain KBY247 carrying plasmid pLexA-Reg2 (LexA-Reg2, WT), pLexA-Reg2-IF (LexA-Reg2, IF), or the corresponding vector pEG202 (LexA-Reg2, −) were grown to mid-log phase in selective SC medium lacking histidine and containing 2% glucose. The levels of LexA-tagged proteins (LexA-Reg2), phospho-Thr210-Snf1 (P-T210), and total Snf1 protein (Total) were analyzed by immunoblotting. (F) Reg2-IF interacts with Snf1 in the yeast two-hybrid system. Transformants of CTY10-5d with plasmids expressing the indicated protein pairs were grown as for panel B. β-Galactosidase activity was assayed in permeabilized cells and expressed in Miller units. Shown are the results for limiting glucose; the values are averages for 4 to 8 transformants, and the error bars indicate standard errors.