Structure of the Sac1–Vps74 complex. (A) Vps74 (residues 64–341) is yellow with residues in the PtdIns4P binding pocket shown in red. Sac1 (residues 2–449) is blue with the catalytic CX5R(T/S) motif in orange. Unstructured loops are depicted as dotted lines. The homologous positions of mutations that cause CMT4J in the orthologous Sac3/Fig4 are indicated with cyan spheres (Sac1 numbering corresponds to I41T, D48G, and D53Y in Sac3/Fig4; Manford et al., 2010). The complex is tilted back slightly to orient the membrane-binding surface at the bottom toward the reader. (B) Residues 166–176 in Vps74 make extensive contact with Sac1. (C) The surface of Sac1 where Vps74 binds is shown in blue. (D) GST-Sac1 pulldown assays show that Vps74 (L170R and I173R) mutations ablate binding to Sac1, whereas mutations in a second site of contact between Vps74 and Sac1 observed in the crystal do not. (E) Bimolecular fluorescence assay shows that wild-type Vps74 and Sac1 associate in vivo, but Vps74 (L170R and I173R) mutant does not. Bar, 5 µm.
