Figure 1. End3 binds to the Central Domain of Pan1.

A) Schematics of Pan1, End3, and end3-1 protein. The thick bars above Pan1 and End3 represent the yeast two-hybrid interacting regions. Domains and motifs important for each protein are indicated. EH, Eps15 Homology Domain; LR, Long Repeat; A, Acidic motif; PP, polyproline; 1, End3 Repeat 1; 2, End3 Repeat 2. The green bars represent predicted coiled-coils within Pan1 and End3. The sequence alignment of End3 with end3-1 is shown below the schematics and begins at the final residue of the second EH domain. end3-1 contains a frame shift due to deletion of nucleotide 715 and produces 33 non-native amino acids before termination. The underlined residues are the End3 Repeats at the C-terminus of End3, and the bolded and italicized sequence is the non-native end3-1 protein sequence. B) End3 binds to the coiled-coil region of Pan1. Recombinant protein-binding assay with normalized protein amounts of GST, GST-LR2^360–690, or GST-CC^792–1252 bound to glutathione agarose beads incubated with lysates from bacteria expressing His₆-tagged full length End3. His₆-End lysate input, aliquots of each supernatant (S) and washed pellet (P) were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with anti-His₆ or anti-GST antibodies, as indicated. The percentages of End3 protein bound were calculated from anti-His₆ pellet band intensity relative to the supernatant lane from the same sample.