FIGURE 7.

Clavesins bind to PtdIns(3,5)P₂. A, liposomes composed of 80% phosphatidylcholine, 20% phosphatidylethanolamine (PC/PE) and liposomes in which 10% phosphatidylcholine was substituted for the indicated PtdIns were probed for binding of recombinant clavesin 1 using a surface plasmon resonance biosensor. Binding to PtdIns(3,5)P₂ was most significant. B, a triple point mutation in the Sec14 domain (RFK/AAA mutant) abolished binding, indicating that clavesin 1 binding is specific, although it exhibits a relatively low affinity. C and D, a series of injections with varying concentrations of clavesin-1 were used to calculate a rate constant of 11.3 μm for clavesin 1 binding to PtdIns(3,5)P₂.