Fig. 7. Interaction between Ent3p and Pep12p was dependent on an intact FSD motif in Pep12p. (A) Yeast two-hybrid interactions were determined in L40 cells expressing the ENTH domain of Ent3p as VP16 activation domain fusion (pKW3) together with fusions of LexA DNA binding domain with the N terminus of wild-type Pep12p (AA 1-200, pBK171), with Pep12p (AA 1-200) carrying a deletion of amino acid residues 19-26 (FSD, pJZ10), or with Pep12p (AA 1-200) with the amino acid substitution F20L (pJZ9), respectively. Growth on selective plates containing 5 mM 3-aminotriazole was analyzed. Expression of VP16 activation domain served as negative control. (B) In vitro pull-down assay of a bacterially expressed protein consisting of the ENTH domain of Ent3p (amino acids 1-172) fused to a C-terminal Strep-tag (pKW5). His₆-Pep12p (amino acids 1-268) with the amino acid substitution F20L (pSK3) or carrying a deletion of amino acid residues 19-26 (FSD, pSK4) bound less Ent3-Strep than wild-type His₆-Pep12p (amino acids 1-268, pFvM135). Some background binding to His₆-Tlg1p (Tlg1p, amino acids 1-137, pFN3) was observed. Immunoblots were stained with antisera against Pep12p (top panel) or Ent3p (bottom panel) and quantified. The ratio of bound Ent3p to wild-type Pep12p was set to one.