FIG 3.

Localization of the PAK-binding site(s) in sortilin by yeast two-hybrid (Y2H) analysis. (A) Localization of the PAK-binding site in sortilin by Y2H analysis using the entire cytoplasmic domain (sort-cd) or C-terminally truncated constructs (1–35, 1–27, or 1–20) against the C-terminal kinase domains of PAK1-3. GGA2 is included as a positive control, and plasmids without inserts are used as negative controls. Growing colonies indicate interactions between expressed proteins. (B) Alanine scanning for PAK interaction sites in sortilin by Y2H analysis using a series of sortilin mutants in which two to three consecutive residues were replaced with alanine residues. Taken together, the mutations cover residues 8 to 34 of the sortilin-cd, as indicated. Amino acid residues marked in red affect binding in Y2H assays. (C) Y2H analysis investigating the effect on PAK1-3 binding when Ser¹⁵ in the sortilin-cd is replaced with either Ala (S15A) or Asp (S15D) (mimicking phosphorylation). His, histidine; 3-AT, 3-amino-1,2,4-triazole.